CA2261164A1 - Unique associated kaposi's sarcoma virus sequences and uses thereof - Google Patents

Abstract

This invention provides an isolated nucleic acid molecule which encodes Kaposi's Sarcoma-Associated Herpesvirus (KSHV) polypeptides. This invention provides an isolated polypeptide molecule of KSHV. This invention provides an antibody specific to the polypeptide. Antisense and triplex oligonucleotide molecules are also provided. This invention provides a vaccine for Kaposi's Sarcoma (KS). This invention provides methods of vaccination, prophylaxis, diagnosis and treatment of a subject with KS and of detecting expression of a DNA virus associated with Kaposi's sarcoma in a cell.

Description

W 098/04576 PCTrUS97/13346 UNIOUE ASSOCIATED KAPOSI'S SARCOMA VIRUS SEOUENCES AND
USES l~REOF

The invention disclosed herein was made with Government support under a co-opera~ive agreement CCU210852 from the Centers for Disease Control and Prevention, and under National Institutes of Health, National Cancer Institute award CA67391 of the Department of Health and Human Services. Accordingly, the U.S. Government has certain rights in this invention.

Throughout this application, various publications may be referenced by Arabic numerals in brackets. Full citations for these publications may be found at the end of the Detailed Description of the Invention. The disclosures of all publications cited herein are in their entirety hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 BACKGROU~D OF THE IN~JENTION

Kaposi's sarcoma-associated herpesvirus (KSHV) is a new human herpesvirus (HHV8) believed to cause Kaposi's sarcoma (KS) [1,2].

Kaposi's sarcoma is the most common neoplasm occurring in persons with acquired immunodeficiency syndrome (AIDS). Approximately 15-20~ of AIDS patients develop this neoplasm which rarely occurs in immunocompetent individuals. Epidemiologic evidence suggests that AIDS-associated KS (AIDS-KS) has an infectious etiology. Gay and bisexual AIDS patients are approximately twenty times more likely than hemophiliac AIDS patients to develop KS, and KS may be associated with specific sexual practices among gay men wlth AIDS. KS is uncommon among adult AIDS
patients infected through heterosexual or parenteral HIV transmission, or among pediatric AIDS patients infected through vertical HIV transmission. Agents previously suspected of causing KS include cytomegalovirus, hepatitis B virus, human papillomavirus, Epstein-Barr virus (EBV), human herpesvirus 6, human immunodeficiency virus (HIV), and Mycoplasma penetrans. Non-infectious environmental agents, such as nitrite inhalants, also have been proposed to play a role in KS tumorigenesis.
Extensive investigations, however, have not demonstrated an etiologic association between any of these agents and AIDS-KS.

W098/04576 PCTtUS97tl3346 SUMMARY OF THE lNv~NlION

This invention provides an isolated nuclelc acid molecu~e which encodes Kaposi's Sarcoma-Associated Herpesvirus (KSHV) polypeptides. This invention provides an isolated polypeptide molecule of KSHV.
This invention provides an antibody specific to the polypeptide. Antisense and triplex oligonucleotide molecules are also provided. This invention provides a vaccine for Kaposi's Sarcoma (KS). This invention provides methods of vaccination, prophylaxis, diagnosis and treatment of a subject with KS and of detecting expression of a DNA virus associated with Kaposi's sarcoma in a cell.

W O 98t04576 PCTrUS97/13346 BRIEF DESCRIPTION OF THE FIGUn~ES

Ficm re 1:
Annotated long unique region (LUR) and terminal repeat (TR) of the KSHV genome. The orientation of identified ORFs in the LUR are denoted by the direction of arrows, with ORFs similar t_O HVS in dark blue and dis-similar ORFs in light blue.
Seven blocks (numbered) of conserved herpesvirus genes with nonconserved interblock regions (lettered) are shown under the kilobase marker;
the block numbering scheme differs from the original description by Chee (Chee et al., 1990, Curr . Topi cs Mi crobi ol . Immunol ~ 154, 125-16g) The overlapping cosmid (Z prefix) and lambda (L
prefix) clones used to map the KSHV genome are compared to the KS5 lambda phage clone from a KS
lesion and shown below. Features and putative coding regions not specifically designated are shswn above the ORF map. Re~eat re~1ons ~r~
shown as white lines (frnk, vnct, waka/jwka, zppa, moi, mdsk). Putative coding regions and other features (see Experimental Details Section I) not designated as ORFs are shown as solid lines~

Fiqure 2A-2D:
(Fig. 2A) Sequence of terminal repeat unit (TR) demonstrating its high G+C content (SEQ ID
NO:16). Sequences highly similar to conserved herpesvirus pacl sites are underlined with less similar sites to specific pacl and pac2 sequences italicized. (Fig. 2B) Southern blot of DNA from BC-1 (lane 1), BCP-l (lane 2) and a KS lesion (lane 3) digested with NdeII which cuts once in the TR sequence and probed with a plasmid containing the TR sequence. The intense W O 98t04576 PCT~US97/13346 hybridization band at 0.8 kb represents multiple copies of the NdeII-digested single unit TR (Fig.
2C) A schematic representation (Fig. 2C) of genome structures of KSHV in BCP-1 and BC-1 cell lines consistent with the data presented in (Fig.
2B) and (Fig. 2D). TaqI (T) sites flank the TR
regions and Nde II (N) sites are within the TRs.
Lower case tr refers to the deleted truncated TR
unit at the left end of the unique region. DR
represents the duplicated region of the LUR
buried within the TR. (Fig. 2D) Southern blot hybridization with TR probe of DNA from BC-1 (lane 1), BCP-1 (lane 2), a KS lesion (lane 3), and HBL-6 (lane 4) digested with Taq I, which does not cut in the TR. Taq I-digested DNA from both BC-1 (lane 1) and HBL-6 (lane 4j show similar TR hybridization patterns suggesting identical insertion of a unique sequence into the TR region, which sequencing studies demonstrate is a duplicated portion of the LUR (see Experimental Details Section). BCP-1 TR
hybridization (lane 2) shows laddering consistent with a virus population having variable TR region lengths within this cell line due to lytic replication. The absence of TR laddering in KS
leslon DNA (lane 3) suggests that a clonal virus population is present in the tumor.

Figures 3A-3C:
CLUSTAL W alignments of KSHV-encoded polypeptide sequences to corresponding human cell signaling pathway polypeptide sequences. Fig. 3A. Two KSHV
MIP-like polypeptides (vMIP-I and vMIP-II) are compared to human MIP-1~, MIP-1~ and RANTES
(amino acid identity to vMIP-I indicated by black reverse shading, to vMIP-II alone by gray reverse shading, and the C-C dimer motif is italicized).

W098/04576 PCT~S97/13346 Both KSHV MIP genes encode 19 residue N-terminus hydrophobic secretory leader sequences which are relatively poorly conserved (vMIP-I also has a second C-C dimer in the hydrophobic leader sequence without similarity to the chemokine dicysteine motif)O Potential O-linked glycosylation sites for vMIP-I (gapped positlons 22 and 27) are not present in vMIP-II, which has only one predicted potential serine glycosylation site (position 51) not found in vMIP-I. Fig. 3B.
Alignment of the KSHV vIL-6 to human IL-6. Fig.
3C-1 and 3C-2. Alignment of the KSHV vIRF
polypeptide to human ICSBP and ISGF3 with the putat1ve ICS-binding typtophans (W) for ICSBP and ISGF3 in italics.

Fiqures 4A-4F:
Northern hybridization of total RNA extracted from BCP-1 and BC-1 cells with or without 48 hour incubation with TPA and control P3HR1 cells after TPA incubation. All four genes (Fig. 4A, vMIP-I;
Fig. 4B, vMIP-II, Fig. 4C, vIL-6; Fig. 4D, vIRF) are TPA inducible but constitutive, noninduced expression of vIL-6 (Fig. 4C) and vIRF (Fig. 4D) is also evident for BCP-1 and BC-1 and of vMIP-I
for BCP-1 (Fig. 4A). Representative hybridizations to a human ~-actin probe (Figs.
4E-4F) demonstrate comparable loading of RNA for cell preparations.
Fiqures 5A-5B:
Fig. 5A. Immunoblot of rabbit antipeptide antibodies generated from amino acid sequences of vIL-6, THYSPPKFDR (SEQ ID NO:2) and PDVTPDVHDR
~SEQ ID N0:3), against cell lysates of BCP-1, BC-1, P3HR1 cell lines with and without TPA
induction (lanes 1-6), 1 ~g human rIL-6 (lane 7), W 098/04576 PCTrUS97/13346 and concentrated COS7 rvIL-6 and 6-LIv supernatants (lanes 8-9). Anti-vIL-6 antibodies specifically recognize the viral IL-6 polypeptide in both recombinant supernatants and cell lines but not human IL-6. The BCP-l cell line constitutively expresses low levels of vIL-6 whereas polypeptide expression increases on TPA
treatment for both BC-1 (KSHV and EBV coinfected) and BCP-1 (KSHV infection alone) indicating lytic phase expression. Preimmune sera from immunized rabbits did not react on immunoblotting to any of the preparations. Fig. 5B. Anti-huIL-6 monoclonal antibodies do not cross-react with cell-associated or recombinant vIL-6 preparations.

Figure 6:
Dose-response curves for 3H-thymidine uptake in IL-6-dependent B9 mouse plasmacytoma cells with serial dilutions of rhuIL-6 (filled squares) and COS7 supernatants of rvIL-6 (filled circles), r6-LIv (open squares) or control LacZ (open circles) pMET7 transfections. Undiluted rvIL-6 supernatants from this transfection lot show similar B9 proliferation activity to huIL-6 ~0.02 ng/ml whereas the reverse construct (r6-LIv) and the LacZ control show no increased ability to induce B9 proliferation. Concentrated supernatants at greater than 1:1 dilution may have increased activity due to concentration of COS7 conditioning factors.

Fiqures 7A-7F:
Rabbit anti-vIL-6 peptide antibody reactivity localized using goat-antirabbit immunoglobulin-peroxidase conjugate (brown) with hematoxylin counterstaining (blue) at X100 magnification W O 98/04~76 PCT~US97/13346 demonstrates vIL-6 production in ~oth KSHV-infected cell lines and tissues. The KSHV-infected cell line BCP-1 (Fig. 7A), but not the control EBV-infected cell line P3HR1 (Fig.
7B), shows prominent cytoplasmic vIL-6 localization. (Fig. 7C) Cytoplasmic localization of vIL-6 in spindle-shaped cells from an AIDS-KS
lesion. Of eight KS lesions, only one had readily identifiable vIL-6 staining of a subpopulation of cells. In contrast, the majority of pelleted lymphoma cells from a nonAIDS, EBV-negative PEL have intense vIL-6 staining (Fig. 7E). NQ ;rnrmlnostaining is present in control angiosarcoma (Fig. 7D) or multiple myeloma tissues (Fig. 7F~.

Fi~ures 8A-8D.
Double antibody labeling of anti-vIL-6 and cell surface antigens. Examples of both CD34 and CD20 colocalization with vIL-6 were found in a KS
lesion. Fig. 8A. CD34 (red) and vIL-6 colocalize (blue) in a KS spindle cell (arrow). Purple coloration is due to overlapping chromagen staining (lOOX). Fig. 8B. CD45 common leukocyte antigen staining (blue, arrow) on vIL-6 (red) expressing Kaposi's sarcoma cells ~lOOX). Fig.
8C. Low power magnification (20X) demonstrating numerous vIL-6 producing hematopoietic cells (red) in a lymph node from a patient with KS.
Arrows only indicate the most prominently staining cells; nuclei counterstained with hematoxylin. Fig. 8D. Colocalization of CD20 (brown, arrows) with vIL-6 (red) in an AIDS-KS
patient's lymph node (lOOX).
Fiqure 9:

W098/04576 PCT~S97/13346 Quantification of CCC/CD4 cell infection by primary NSI SFl62 and M23 HIV-1 strains and HIV-2 strain ROD/B in the presence or absence of vMIP-I. CCC/CD4 cells were transiently cotransfected with CCR5 alone, CCR5 plus empty pMET7 vector, CCR5 plus vMIP-I ln pMET7 vector, or CCR5 plus the reverse orientation I-PIMv. The results after 72 hours of incubation with each retrovirus are expressed as a percentage of the foci forming units for cells transfected with CCR5 alone. The forward vMIP-I construct inhibited NSI HIV-1 replication but not HIV-2 replication while the reverse I-PIMv construct had no effect on replication of any of the retroviruses.

W O 98/04576 PCT~US97/13346 DETAILED DESCRIPTION OF THE lNV~N-llON

Definitions The following standard abbreviations are used throughout the specification to indicate specific nucleotides:

C=cytosine A=adenosine lG T=thymidine G=guanosine The term "nucleic acid", as used herein, refers to either DNA or RNA, including complementary DN~ (cDNA), genomlc DNA and messenger RNA (mRNA). As used herein, "genomic" means both coding and non-coding regions of the isolated nucleic acid molecule. "Nucleic acid sequence" refers to a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes both self-replicating plasmids, infectious polymers of DNA
or RNA and nonfunctional DNA or RNA.

The term ~polypeptide~, as used herein, refers to either the full length gene product encoded by the nucleic acid, or portions thereof. Thus, I'polypeptide'' includes not only the full-length protein, but also partial-length fragments, lncluding peptides less than fifty amino acid residues in length.
The term "SSC" refers to a citrate-saline solution of 0.15 M sodium chloride and 20 mM sodium citrate.
Solutions are often expressed as multiples or fractions of this concentration. For example, 6XSSC
refers tc a solution having a sodium chloride and sodium citrate concentration of 6 times this amount or 0.9 M sodium chloride and 120 mM sodium citrate.

W 098/04576 PCTrUS97/13346 0.2XSS~ refers to a solution 0.2 times the SSC
concentratlon or 0.03 M sodium chloride and 4 mM
sodium citrate.

The phrase "selectively hybridizing to" and the phrase "specific h~bridization" describe a nucleic acid probe that hybridizes, duplexes or binds only to a particular target DNA or RNA sequence when the target sequences are present in a preparation of total cellular DNA or RNA. By selectively hybridizing it is meant that a probe binds to a given target in a manner that is detectable in a different manner from non-target sequence under high stringency conditions of hybridization.
"Complementary" or "target" nucleic acid sequences refer to those nucleic acid sequences which selectively hybridize to a nucleic acid probe. Proper annealing conditions depend, for example, upon a probe's length, base composition, and the number of mismatches and their position on the probe, and must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, Vols. 1-3 or Ausubel~ F., et al.
(1987) Current Protocols in Molecular Biology, New York.

The phrase "nucleic acid molecule encoding" refers to a nucleic acid molecule which directs the expression of a specific polypeptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed lnto RNA, the complementary DNA strand, and the RNA sequence that is translated into protein.
The nucleic acid molecule includes both the full length nucleic acid sequence as well as non-full length sequences. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.

A nucleic acid probe is "specific" for a target organism of interest if it includes a nucleotide sequence which when detected is determinative of the presence of the organism in the presence of a heterogeneous population of proteins and other biologics. A specific nucleic acid probe is targeted to that portion of the sequence which is determinative of the organism and will not hybridize to other sequences, especially those of the host r where a pathogen is being detected.

The phrase "expression cassette~, refers to nucleotide sequences which are capable of affecting expression of a structural gene in hosts compatible with such sequences. Such cassettes include at least promoters and optionally, transcription termination signals.
Additional factors necessary or helpful in effecting expression may also be used as described herein.

The term "operably linked" as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.

The term ~vector", refers to viral expression systems/
autonomous self-replicating clrcular DNA (plasmids), and includes both expression and nonexpression plasmids. Where a recombinant microorganism or cell culture is described as hosting an "expression vector," this includes both extrachromosomal circular DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by W O 98/04576 PCT~US97/13346 a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.

The term "plasmid" refers to an autonomous circular DNA molecule capable of replication in a cell, and includes both the expression and nonexpression types.
Where a recombinant microorganism or cell culture is described as hosting an "expression plasmidll, this includes latent viral DNA integrated into the host chromosome(s). Where a plasmid is being maintained by a host cell, the plasmid is either being stably replicated by the cells during mitosis as an autonomous structure or is incorporated within the host's genome.

The phrase "recombinant protein" or "recombinantly produced protein" refers to a polypeptide produced using non-native cells. The cells produce the protein because they have been genetically altered by the introduction of the appropriate nucleic acid sequence.

The followlng terms are used to describe the sequence relatlonshlps between two or more nucleic acid molecules: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing or may comprise a complete cDNA or gene sequence.
optimal alignment of sequences in a comparison window may be conducted by the algorithm of Smith and W 098/04576 PCTrUS97/13346 Waterman (1981) Adv Appl ~ MathA 2:482, by the algorithm of Needleman and Wunsch (1970) ~. Mol. Biol.
48:443, by the search-for-similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444, or by computerized implementations of these algorithms ~GAP, BESTFIT, FASTA, and TFASTA in GCG, the Wisconsin Genetics Software Package Release 8.0, Genetics Computer Group, 575 Science Dr.~ Madison, WI).

As applied to polypeptides, the terms "substantial identity~' or "substantial sequence identity" mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap which share at least 90 percent sequence identity, preferably at least 95 percent sequence identity, more preferably at least 99 percent sequence identity or more.

"Percentage amino acid identity" or "percentage amino acid sequence identity" refers to a comparison of the amino acids of two polypeptides which, when optimally aligned, have approximately the designated percentage of the same amino acids. For example, "95~ amino acid identity" refers to a comparison of the amino acids of two polypeptides which when optimally aligned have 95~
amino acid identity. Preferablyl residue positions which are not identical differ by conservative amino acid substitutions. For example, the substitution of amino acids having similar chemical properties, such as charge or polarity, are not likely to effect the properties of a protein. Examples include glutamine for asparagine or glutamic acid for aspartic acid.

The phrase "substantially purified" or "isolated" when referring to a herpesvirus polypeptide, means a chemical composition which is essentially free of other cellular components. It is preferably in a W O 98/04576 PCT~US97113346 homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. Generally, a substantially purified or isolated protein will comprise more than 80~ of all macromolecular species present in the preparation. Preferably, the protein is purified to represent greater than 90~ of all macromolecular species present More preferably the protein is purified to greater than 95~, and most preferably the protein is purified to essential homogeneity, wherein other macromolecular species are not detected by conventional techniques.

The phrase "specifically binds to an antibody" or ~specifically immunoreactive with", when referring to a polypeptide, refers to a binding reaction which is determinative of the presence of the KSHV polypeptide of the invention in the presence of a heterogeneous population of polypeptides and other biologics including viruses other than KSHV. Thus, under designated immunoassay conditionsl the specified antibodies bind to the KSHV antigen and do not bind in a significant amount to other antigens present in the sample.

"Specific binding" to an antibody under such conditions may require an antibody that is selected for its specificity for a particular antigen. For example, antibodies raised to KSHV antigens described herein can be selected to obtain antibodies specifically immunoreactive with KSHV polypeptides and not with other polypeptides.

"Biological sample" as used herein refers to any sample obtalned from a living organlsm or from an organism that has died. Examples of biological samples include body fluids and tissue specimens.

It will be readily understood by those skilled in the art and it is intended here, that when reference is made to particular sequence listings, such reference includes sequences which substantially correspond to the listing and it's complement, including allowances for minor sequencing errors, single base changes, deletions, substitutions and the like, such that any such sequence variation corresponds to the nucleic acid sequence of the pathogenic organism or disease marker to which the relevant sequence listing relates.

I. Nucleic Acid Molecule from KSHV

This invention provides an isolated nucleic acid molecule which encodes a Kaposi's sarcoma-assoclated herpesvirus (KSHV) polypeptide.

In one embodiment, the isolated nucleic acid molecule which encodes a KSHV polypeptide has the nucleotide sequence as set forth in GenBank Accession Number U75698 and the start and stop codons set forth in Table 1. In another embodiment, the isolated nucleic acid molecule which encodes a KSHV polypeptide has the amino acid sequence defined by the translation of the nucleotide sequence set forth in GenBank Accession Number U75698 and the start and stop codons set forth in Table 1.

In one embodiment, the isolated nucleic acid molecule for a KSHV polypeptide has the 5' untranslated sequence as set forth in GenBank Accession Number U75698 upstream of the ATG start codon. In another W098/04576 PCT~S97/13346 embodiment, the isolated nucleic acid molecule for a KSHV polypeptide has the 3' untranslated sequence as set forth in GenBank Accession Number U75698 downstream of the stop codon.

In one embodiment the isolated nucleic acid molecule - is genomic DNA. In another embodiment the isolated nucleic acid molecule is cDNA. In another embodiment RNA is derived from the isolated nucleic acid molecule or is capable of hybridizing with the isolated nucleic acid molecule.

Further, the nucleic acid molecule above may be associated with lymphoproliferative diseases including, but not limited to: Hodgkin's disease, non-Hodgkin's lymphoma, lymphatic leukemia, lymphosarcoma, splenomegaly, reticular cell sarcoma, Sezary's syndrome, mycosis fungoides, central nervous system lymphoma, AIDS related central nervous system lymphoma, post- transplant lymphoproliferative disorders, and Burkitt's lymphoma. A lympho-proliferative disorder is characterized as being the uncontrolled clonal or polyclonal expansion of lymphocytes involving lymph nodes, lymphoid tissue and other organs.

A. Isolation and Propaqation of KSHV

KSHV can be propagated in vi tro . For example, techniques for growing herpesviruses have been described by Ablashi et al. in Virology 184, 545-552.
Briefly, PHA stimulated cord blood mononuclear cells, macrophage, neuronal, or glial cell lines are cocultivated with cerebrospina~ fluid, plasma, peripheral blood leukocytes, or tissue extracts containing viral infected cells or purified virus.
The recipient cells are treated with 5 ~g/ml polybrene W O 98/04576 PCTnUS97/13346 for 2 hours at 37~ C prior to infection. Infected cells are observed by demonstrating morphological changes, as well as being viral antigen positive.

For KSHV isolation, the virus is either harvested directly from cell culture fluid by centrifugation, or the infected cells are harvested, homogeni2ed or lysed and the virus is separated from cellular debris and purified by standard methods of isopycnic sucrose density gradient centrifugation.

One skilled in the art may isolate and propagate KSHV
employing the following protocol. Long- term establishment of a B lymphoid cell line infected with KSHV (e.g., RCC-1, HBL-6 or BCBL-1) is accomplished using body-cavity based lymphomas and standard techniques (Glick, 1980, Fundamentals of Human Lymphoid Culture, Marcel Dekker, New York; Knowles et al., 1989~ Blood 73, 792-798; Metcalf, 1984, Clonal Culture of Hematopoeitic Cells: Techni~ues and Applications, Elsevier, New York).

Fresh lymphoma tissue containing viable infected cells is filtered to form a single cell suspension. The cells are separated by Ficoll-Plaque centrifugation and lymphocyte layer is removed. The lymphocytes are then placed at >lx106 cells/ml into standard lymphocyte tissue culture medium, such as RPMI 1640 supplemented with 10~ fetal calf serum. Immortalized lymphocytes 3~ containing KSHV are indefinitely grown in the culture media while non-immortalized cells die during course of prolonged cultivation.

Further, KSHV may be propagated in a new cell line by removing media supernatant containing the virus from a continuously-infected cell line at a concentration of ~1x106 cells/ml. The media is centrifuged at 2000xg W O 98/04~76 PCTrUS97/13346 for 10 minutes and filtered through a 0.45u filter to remove cells~ The media is applied in a 1:1 volume with cells growing at ~lx106 cells/ml for 48 hours.
The cells are washed, pelleted and placed in fresh culture medium, then tested for KSHV after 14 days.

KSHV may be isolated from a cell line in the following manner. An infected cell line is lysed using standard methods, such as hyposmotic shock or Dounce homogenization or using repeated cycles of freezing and thawing in a small volume (~3 ml), and pelleted at 2000xg for 10 minutes. The supernatant is removed and centrifuged again at 10,000xg for 15 minutes to remove nuclei and organelles. The resulting low-speed, cell-free supernatant is filtered through a 0.45~ filter and centrifuged at 100,000xg for 1 hour to pellet the virus. The virus can then be washed and re-pelleted.
The DNA is extracted from the viral pellet by standard techniques (e.g., phenol/ chloroform~ and tested for the presence of KSHV by Southern blotting and/or PCR
using the spe-~ific probes described above.

For banding whole virion, the low-speed cell-free supernatant is adjusted to contain 7~ PEG-8000. The PEG-supernatant is spun at 10,000 xg for 30 min. The supernatant is poured off and the pellet collected and resuspended in a small volume (1-2 ml) of virus buffer (VB, O.1 M NaCl, 0.01 M Tris, pH 7.5~. The virion are isolated by centrifugation at 25,000 rpm in a 10-50~
sucrose gradient made with VB. One ml fractions of the gradient are obtained by standard techniques (e.g., using a fractionator) and each fraction is tested by dot blotting using specific hybridizing probes to determine the gradient fraction containing the purified virus (preparation of the fraction is needed in order to detect the presence of the virus, i.e., standard DNA extraction).

The method for isolating the KSHV genome is basea on Pellicer et al. ,1978, Cell 14, 133-141 and Gibson and Rolzmann, 1972, J. Virol. lOi 1044-52.

A final method for isolating the KSHV genome is clamped homogeneous electric field (CHEF) gel electrophoresis. Agarose plugs are prepared by resuspending cells infected with KSHV in 1~ LMP
agarose (Biorad) and 0.9~ NaC1 at 42~C to a final concentration of 2.5 x 107 cells/ml. Solidified agarose plugs are transferred into lysis buffer (0.5M
EDTA pH 8.0, 1~ sarcosyl, proteinase K at 1 mg/ml final concentration) and incubated for 24 hours.
Approximately 107 cells are loaded in each lane Gels are run at a gradient of 6.0 V/cm with a run time of 28 h on a CHEF Mapper XA pulsed field gel electrophoresis apparatus ~Biorad), Southern blotted and hybridized to KS631Bam, KS330Bam and an EBV
terminal repeat sequence.
To make a new cell line infected with KSHV, already-infected cells are co-cultivated with a Raji cell line separated by a 0.45~ filter. Approximately, 1-2 x 106 already-infected BCBL-1 and 2X106 Raji cells are co-cultivated for 2-20 days in supplemented RPMI alone or with 20 ng/ml 12-O-tetradecanoyl phorbol-13-acetate (TPA). After 2-20 days co-cultivation, Raji cells are removed, washed and placed in supplemented RPMI 1640 media. A Raji culture co-cultivated with BCBL-1 in 20 ng/ml TPA for 2 days survived and has been kept in continuous suspension culture for >10 weeks. This cell line, designated RCC-1 (Raji Co-Culture, No.1) remains PCR positive for the KSHV sequence after multiple passages. RCC-1 cells periodlcally undergo rapid cytolysis suggestive of lytic reproduction of KSHV. Thus, RCC-1 is a Raji cell line newly-infected with KSHV.

W 098/04576 PCTrUS97/13346 RCC-1 and RCC_12F~ were deposited on October 19~ 1994 under ATCC Accession No. CRL 11734 and CRL 11735, respectively, pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A.
HBL-6 was deposited (as BHL-6~ on November 18, 1994 under ATCC Accession No. CRL 11762 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A.
B. Hybridization Probes of KSHV

This invention provides a nucleic acid molecule of at least 14 nucleotides capable of specifically hybridizing with the isolated nucleic acid molecule as set forth in GenBank Accession Numbers U75698, U75699, U75700.

In one embodiment the nucleic acid molecule set forth in GenBank Accession Number U75698 comprises the long unique region (LUR) encoding KSHV polypeptides. In another embodiment the nucleic acid molecule set forth in GenBank Accession Number U75699 comprises the prototypical terminal repeat (TR). In another embodiment the nucleic acid molecule set forth in GenBank Accession Number U75700 comprises the incomplete terminal repeat (ITR) In one embodiment the molecule is 8 to 36 nucleotides.
In another embodiment the molecule is 12 to 25 nucleotides. In another embodiment the molecule is 14 nucleotides.

WO98/04576 PCT~S97tl3346 In one embodiment the molecule is DNA. In another embodiment the molecule is RNA.

In one embodiment the TR molecule contains cis-active elements required for DNA replication and packaging.
In another embodiment the TR molecule is contained in a gene-cloning vector. Tn another embodiment the TR
molecule is contained in a gene-therapy vector. In another embodiment the gene-therapy vector is expressed in lymphoid cells. In another embodiment, the TR comprises a molecular marker for determining the clonality of a tumor. In another embodiment, the marker provides a defining feature of the natural history of a tumor in a diagnostic assay.
This invention provides a B-lymphotrophic DNA vector comprising a plasmid or other self-replicable DNA
molecule containing the 801 bp KSHV TR or a portion thereof.

High stringency hybridization conditions are selected at about 5~C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The lm iS the temperature (under defined ionic strength and pH) at which 50~ of the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60~C. As other factors may significantly affect the stringency of hybridization, including, among others, base composition and size of the complementary strands, the presence of organic solvents, i.e. salt or formamide concentration, and the extent of base mlsmatching, the combination of parameters is more important than the absolute measure of any one. For example, high stringency may be attained by overnight hybridization at about 68~C in a 6X SSC solution, washing at room W 098/04576 PCT~US97/13346 temperature with 6X SSC solution, followed by washing at about 68~C in a 0.6X SSC solution.

Hybridization with moderate stringency may be attained for example by: 1) filter pre-hybridizing and hybridizlng with a solution of 3X SSC, 50~ formamide, O.1M Tris buffer at pH 7.5, 5X Denhardt's solution;

2.) pre-hybridization at 37~C for 4 hours; 3) hybridization at 37~~ with amount of labeled probe equal to 3,000,000 cpm total for 16 hours; 4) wash in x SSC and 0.1~ SDS solution; 5) wash 4X for 1 mlnute each at room temperature in 4X SSC at 60~C for 30 minutes each; and 6) dry and expose to film.

Nucleic acid probe technology is well known to those skilled in the art who readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe.
DNA probe molecules may be produced by insertion of a DNA molecule having the full-length or a fragment of the isolated nucleic acid molecule of the DNA virus into suitable vectors, such as plasmids or bacteriophages, followed by transforming into suitable bacterial host cells, replication in the transformed bacterial host cells and harvesting of the DNA probes, using methods well known in the art. Alternatlvely, probes may be generated chemically from DNA
synthesizers.
RNA probes may be generated by inserting the full length or a fragment of the isolated nucleic acid molecule of the DNA virus downstream of a bacteriophage promoter such as T3, T7 or SP6. ~arge amounts of RNA probe may be produced by incubating the labeled nucleotides with a linearized isolated nucleic acid molecule of the DNA virus or its fragment where W O 98/04576 PCTrUS97/13346 it contains an upstream promoter in the presence of the approprlate RNA polymerase~

As defined herein nucleic acid probes may be DNA or RNA fragments. DNA fragments can be prepared, for example, by digesting plasmid DNA, or by use of PCR, or synthesized by either the phosphoramidite method described by Beaucage and Carruthers, 1981, Tetrahedron Lett. 22, 1859-1862 or by the triester method according to Matteucci et al., 1981, Am. Chem.
Soc. 103:3185. A double stranded fragment may then be obtained, if desired, by annealing the chemically synthesized single strands together under appropriate conditions or by synthesizing the complementary strand using DNA polymerase with an appropriate primer sequence. Where a specific sequence for a nucleic acid probe is given, it is understood that the complementary strand is also identified and included.
The complementary strand will work equally well in situations where the target is a double-stranded nucleic acid. It is also understood that when a specific sequence is identified for use a nucleic probe, a subsequence of the listed sequence which is base pairs (bp) or more in length is also encompassed for use as a probe.

The nucleic acid molecules of the subject invention also include molecules coding for polypeptide analogs, fragments or derivatives of antigenic polypeptides which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the polypeptide, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs where in one or more amino acid residues is added to a terminal or medial portion of the W 098/04576 PCT~US97/13346 polypeptides) and which share some or all properties of naturally-occurring forms. These molecules include: the incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial r terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors.

C. PolYPeptides of KSHV and Antibodies (Ab's) Thereto This invention provides an isolated KSHV polypeptide, one from the list as set forth in Table 1 and below.
This inventio~l provides the isolated KSHV polypeptide comprising viral macrophage inflammatory protein III
(vMIP-III). In one embodiment, vMIP-III comprises an orphan cytokine. In another embodiment, vMIP-III is encoded by nucleotides 22,529-22,185. In another embodiment, VMIP-III comprises an anti-inflammatory drug. In a preferred embodiment, the drug is useful in treatment of an autoimmune disorder. In the most preferred embodiment, the drug is useful in treatment of rheumatoid arthritis.

This invention provides the isolated KSHV polypeptide comprising dihydrofolate reductase (DHFR) encoded by ORF 2. In one embodiment, DHFR participates in KSHV

3~ nucleotide synthesis. In another embodiment, DHFR
comprises an enzyme essential for viral replication, inhibition of which prevents virus production. In another embodiment, DHFR comprises a subunit vaccine.
In another embodiment, DHFR comprises an antigen for immunologic assays.

In another embodiment, DHFR has the amino acid sequence as set forth in SEQ ID NO:1 In another embodiment t KSHV DHFR is inhibited by a sulfa drug known to inhibit bacterial DHFR. In a preferred embodiment, KSHV DHFR is inhibited by methotrexate or a derivative thereof known to inhibit mammalian DHFR. In another embodiment, the sulfa drug, methotrexate or a derivative thereof is selective among the human herpesviruses for inhibition of KSHV.

This invention provides the isolated KSHV polypeptide comprising thymidylate synthase (TS) encoded by ORF
70. In one embodiment, TS participates in KSHV
nucleotide metabolism. In another embodiment, TS
comprises an enzyme essential for viral replication, inhibition of which prevents virus production. In another embodiment, TS comprises a subunit vaccine.
In another embodiment, TS comprises an antigen for immunologic assays.

This invention provides the isolated KSHV polypeptide comprising DNA polymerase encoded by ORF 9. In one embodiment, DNA polymerase comprise an enzyme essent1al for viral replication, inhibition of which prevents virus production. In another embodiment, DNA
polymerase comprises a subunit vaccine. In another embodiment, DNA polymerase comprises an antigen for immunologic assays.

This lnvention provides the isolated KSHV polypeptide comprising alkaline exonuclease encoded by ORF 37. In one embodiment, alkaline exonuclease packages KSHV DNA
into the virus particle. In another embodiment, alkallne exonuclease comprises an enzyme essential for viral replication, inhibition of which prevents virus W 098/04576 PCT~US97/13346 production. In another embodiment~ alkaline exonuclease comprises a subunlt vaccine~ In another embodiment, alkaline exonuclease comprises an antigen for immunologic assays.

This invention provides the isolated KSHV polypeptide comprising helicase-primase, subunits 1, 2 and 3 encoded by ORFs 40, 41 and 44, respectively. In one embodiment, helicase-primase comprises an enzyme activity essential for viral DNA replication. In another embodiment, helicase-primase is inhibited by nucleotide analogs. In another embodiment, helicase-primase is inhibited by known antiviral drugs. In another embodiment, inhibition of helicase-primase prevents KSHV replication.

This invention provides the isolated KSHV polypeptide comprising uracil DNA glycosylase (UDG) encoded by ORF

4 6. In one embodiment, uracil DNA glycosylase comprises an enzyme essential for KSHV DNA repair during DNA replication. In another embodiment, uracil DNA glycosylase is inhibited by known antiviral drugs.
In another embodiment, uracil DNA glycosylase comprises a subunit vaccine. In another embodiment, uracil DNA glycosylase comprises an antigen for immunologic assays.

This invention provides the isolated KSHV polypeptide comprising single-stranded DNA binding protein (SSBP) encoded by ORF 06. In one embodiment, SSBP comprises an enzyme essential for KSHV DNA replication. In another embodiment, SSBP is inhibited by known antiviral drugs. In another embodiment, SSBP
increases the processivity of polymerase reactions such as in the conventional PCR method for DNA
amplification.

CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 This invention provides the isolated KSHV polypeptide comprising viral protein kinase encoded by ORF 36. In another embodiment, viral protein kinase comprises an antigen for immunologic assays. In another embodiment, viral protein kinase comprises a subunit vaccine.

This invention provides the isolated KSHV polypeptide comprising lytic cycle transactivator protein (LCTP) encoded by ORF 50. In one embodiment, LCTP is required for activation of productive infection from the latent state. In another embodimentl LCTP is inhibited by known antiviral drugs. In another embodiment, prevention of LCTP expression maintains the virus in a latent state unable to replicate.

This invention provides the isolated KSHV polypeptide comprising ribonucleotide reductase, a two-subunit enzyme in which the small and large subunits are encoded by ORF 60 and ORF 61, respectively. In another embodiment, ribonucleotide reductase catalyzes conversion of ribonucleotides into deoxyribonucleotides for DNA replication. In another embodiment, ribonucleotide reductase is inhibited by known antiviral drugs in terminally differentiated cells not expressing cellular ribonucleotide reductase. In another embodiment, ribonucleotide reductase comprises an antigen for immunologic assays.
In another embodiment, ribonucleotide reductase comprises a subunit vaccine. In another embodiment, ribonucleotide reductase comprises a transforming agent for establishment of immortalized cell lines.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF K1.

W098/04576 PCT~S97/13346 This invention provides the isolated KSHV polypeptide comprising complement-binding protein (v-CBP; CCP) encoded by ORF 4.

This invention provides the isolated KSHV polypeptide compris1ng transport protein encoded by ORF 7.

This învention provides the isolated KSHV polypeptide comprising glycoprotein B encoded by ORF 8.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF l0.

This invention provides the isolated KSHV polypeptide comprislng the protein encoded by ORF 11.

This invention provides the isolated KSHV polypeptide comprising viral interleukin 6 (vIL-6) encoded by ORF
K2. In one embodiment, antibodies selectively recognizing vIL-6 allow differentiation among lymphomas.

This invention provides the isolated KSHV polypeptide comprislng BHV4-IEl I encoded by ORF K3.
This invention provides the isolated KSHV polypeptide comprising vMIP-II encoded by ORF K4. In one embodiment, vMIP-II comprises an anti-inflammatory drug. In a preferred embodiment, the drug is useful in treatment of an autoimmune disorder. In the most preferred embodiment, the drug is useful in treatment of rheumatoid arthritis.

This invention provides the isolated KSHV polypeptide comprising BHV4-IEl II encoded by ORF K5.

W O 98/04576 PCTrUS97/13346 This lnvention provides the isolated KSHV polypeptide comprlsing vMIP-I encoded by ORF K6~ In one embodiment, vMIP-I compr'ses an anti-inflammatory drug. In a preferred embodiment, the drug is useful in treatment of an autoimmune disorder. In the most pre~erred embodiment, the drug is useful in treatment of rheumatoid arthritis.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF K7.

This invention provides the isolated KSHV polypeptide comprising Bc1-2 encoded by ORF 16.

This lnvention provides the isolated KSHV polypeptide comprising capsid protein I encoded by ORF 17.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 18.
This invention provides the isolated KSHV polypeptide comprising tegument protein I encoded by ORF 19.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 20.

This invention provides the isolated KSHV polypeptide comprising thymidine kinase encoded by ORF 21.

This invention provides the isolated KSHV polypeptide comprising glycoprotein H encoded by ORF 22.

In one embodiment, the isolated KSHV polypeptide comprises the protein encoded by ORF 23.
This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 24.

W098/04576 PCT~S97/13346 This invention provides the isolated KSHV polypeptide comprising major capsid protein encoded by ORF 25.

Thls invention provides the isolated KSHV polypeptide comprising capsid protein II encoded by ORF 26.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 27.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 28.

This invention provides the isolated KSHV polypeptide comprising packaging protein II encoded by ORF 29b.
This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 30 This lnvention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 31.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 32.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 33.

This invention provides the isolated KSHV polypeptide comprising packaging protein I encoded by ORF 29a.
This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 34.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 35.

W O 98/04576 PCTrUS97113346 This invention provides the isolated KSHV polypep~ide comprising the protein encoded by ORF 38.

This invention provides the isolated KSHV polypeptide comprising glycoprotein ~ encoded by ORF 39.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 42.

This invention provides the isolated KSHV polypeptide comprising capsid protein III encoded by ORF 43.

This invention provides the isolated KSHV polypeptide comprising virion assembly protein encoded by ORF 45 This invention provides the isolated KSHV polypeptide comprising glycoprotein L encoded by ORF 47.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 48.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 49.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF K8.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 52.
This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 53.

This invention provides the isolated KSHV polypeptide comprising dUTPase encoded by ORF 54.

W 098/04576 PCTrUS97/13346 This invention provides the isolated KSHV polypeptide comprlsing the protein encoded by ORF 55~

This invention provides the isolated KSHV polypeptide comprising DNA replication protein I encoded by ORF
56.

This invention provides the isolated KSHV polypeptide comprising immediate early protein II (IEP-II) encoded by ORF 57.

This invention provides the isolated KSHV polypeptide comprising viral interferon regulatory factor (vIRF1; ICSBP) encoded by ORF K9. In one embodiment, vIRF1 is a transforming polypeptide.

This lnvention provides the isolated KSHV polypeptide comprising the protein encoded by ORF K10.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF K11.

This invention provides the isolated KSHV polypeptide comprising phosphoprotein encoded by ORF 58.
This invention provides the isolated KSHV polypeptide comprising DNA replication protein II encoded by ORF
59.

This invention provides the isolated KSHV polypeptide comprising assembly/DNA maturation protein encoded by ORF 62.

This invention provides the isolated KSHV polypeptide comprising tegument protein II encoded by ORF 63.

W 098/04576 PCTrUS97/13346 This invention provides the isolated KSHV polypeptide comprislng tegument protein III encoded by ORF 64.

This invention provides the isolated KSHV polypeptide comprising capsid protein IV encoded by ORF 65.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 66~

This invention provides the isolated KSHV polypeptide comprising tegument protein IV encoded by ORF 67.

This invention provides the isolated KSHV polypeptide comprising glycoprotein encoded by ORF 68.
This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF 69.

This invention provides the isolated KSHV polypeptide comprising Kaposin encoded by ORF K12.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF K13.

This invention provides the isolated KSHV polypeptide comprising cyclin D encoded by ORF 72.

This invention provides the isolated KSHV polypeptide comprising immediate-early protein (IEP) encoded by ORF 73.

This invention provides the isolated KSHV polypeptide comprising OX-2 encoded by ORF K14.

This invention provides the isolated KSHV polypeptide comprising G-protein coupled receptor encoded by ORF
74.

W O 98/04576 PCT~US97/13346 3~
This invention provides the isolated KSHV polypeptide comprising tegument protein/FGARAT encoded by ORF 75.

This invention provides the isolated KSHV polypeptide comprising the protein encoded by ORF K15.

This invention provides the isolated KSHV polypeptide comprising viral interferon regulatory factor 2 (vIRF2) encoded by nucleotides 88,910-88,410.
This invention provides the isolated KSHV polypeptide comprising viral interferon regulatory factor 3 (vIRF3) encoded by nucleotides 90,541-89,600.

This invention provides the isolated KSHV polypeptide comprising viral interferon regulatory factor 4 (vIRF4) encoded by nucleotides 94,127-93,636.

This invention provides the isolated KSHV polypeptide comprising a precursor of secreted glycoprotein X (gX) encoded by nucleotides 90,173-90~643.

This invention provides the isolated KSHV polypeptide comprising protein Tl.1 (nut-1) encoded by nucleotides 28,661-29,741.

Further, the isolated polypeptide may be linked to a second polypeptide to form a fusion protein by linking the isolated nucleic acid molecule to a second nucleic acid molecule and expression in a suitable host cell.
In one embodiment the second nucleic acid molecule encodes beta-galactosidase. Other nucleic acid molecules which are used to form a fusion protein are known to those skilled in the art.

W 098/04576 PCT~US97/13346 36 This invention provides an antibody which specifically binds to the polypeptide encoded by the isolated nucleic acid molecule. In one embodiment the antibody is a monoclonal antibody~ In another embodiment the antibody recognizes an epitope of the KSHV
polypeptide. In another embodiment the antibody is a polyclonal antibody. In another embodiment the antibody recognizes more than one epitope of the KSHV
polypeptide. In another embodiment the antibody is an anti-idiotypic antibody.

An antibody, polypeptide or isolated nucleic acid molecule may be labeled with a detectable marker including, but not limited to: a radioactive label, or a colorimetric, a luminescent, or a fluorescent marker, or gold. Radioactive labels include, but are not limited to 3H, 14C, 32 p, 33 p; 35 S 36 Cl 51 Cr 57 CO
59Co, 59Fe, 90Y, 125I, 131I, and 136Re. Fluorescent markers include, but are not limited to: fluorescein, rhodamine and auramine. Colorimetric markers include, but are not limited to: biotin, and digoxigenin.
Methods of producing the polyclonal or monoclonal antibody are known to those of ordinary skill in the art.
Further, the antibody, polypeptide or nucleic acid molecule may be detected by a second antibody which may be linked to an enzyme, such as alkaline phosphatase or horseradish peroxidase. Other enzymes which may be employed are well known to one of ordinary skill in the art.

This invention provides a method of producing a polypeptide encoded by the isolated nucleic acid molecule, which comprises growing a host-vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so W O 98/04576 PCT~US97/13346 produced. Suitable host cells include bacteria, yeast, filamentous fungal, plant, insect and mammalian cells. Host-vector systems for producing and recovering a polypeptide are well known to those skilled in the art and include, but are not limited to, E. coli and pMAL (New England Biolabs), the Sf9 insect cell-baculovlrus expression system, and mammalian cells (such as HeLa, COSJ NIH 3T3 and HEK293) transfected with a m~mm~l ian expression vector by Lipofectin (Gibco-BRL) or calcium phosphate precipitation or other methods to achieve vector entry into the cell. Those of skill in the art are knowledgeable in the numerous expression systems available for expression of KSHV polypeptide.
This invention provides a method to select specific regions on the polypeptide encoded by the isolated nucleic acid molecule of the DNA virus to generate antibodies. Amino acid sequences may be analyzed by methods well known to those skilled in the art to determine whether they produce hydrophobic or hydrophilic regions in the polypeptides which they build In the case of a cell membrane polypeptide, hydrophobic regions are well known to form the part of the polypeptide that ls inserted into the lipid bilayer of the cell membrane, while hydrophilic regions are located on the cell surface, in an aqueous environment. Usually, the hydrophilic regions will be more immunogenic than the hydrophobic regions.
Therefore the hydrophilic amino acid sequences may be selected and used to generate antibodies specific to polypeptide encoded by the isolated nucleic acid molecule encoding the DNA virus. The selected peptides may be prepared using commercially available - 35 machines. As an alternative, nucleic acid may be cloned and expressed and the resulting polypeptide recovered and used as an immunogen.

W 098/04576 PCTrUS97/}3346 Polyclonal antibodies against the polypeptide may be produced by lmmunizing animals using a selected KSHV
polypeptide. Monoclonal antibodies are prepared using hybridoma technology by fusing antibody producing B
cells from immunized animals with myeloma cells and selecting the resulting hybridoma cell line producing the desired antibody, as described further below.

W O9B/04576 PCTrUS97/13346 II. Immunoassays The antibodies raised against KSHV polypeptide antigens may be detectably labeled, utilizing conventional labelling techniques well-known to the art, as described above.

In addition, enzymes may be used as labels. Suitable enzymes include alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase and peroxidase. Two principal types of enzyme immunoassay are the enzyme-linked immunosorbent assay (ELISA)~ and the homogeneous enzyme immunoassay, also known as enzyme-multiplied immunoassay (EMIT, Syva Corporation, Palo Alto, CA).
In the ELISA system, separation may be achieved, for example, by the use of antibodies coupled to a solid phase. The EMIT system depends on deactivation of the enzyme in the tracer-antibody complex; activity is thus measured without the need for a separation step.

Additionally, chemiluminescent compounds may be used as labels. Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.
Similarly, bioluminescent compounds may be utilized for labelling, the bioluminescent compounds including luciferin, luciferase, and aequorin.

3~ A descrlption of a radioimmunoassay (RIA) may be found in: Laboratory Techniques in Biochemistry and Molec~lar Biology (1978) North Holland Publishing Company, New York, with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by T. Chard. A description of general immunometric assays of various types can be W 098/04576 PCT~US97tl3346 found in the following U.S. Pat. Nos. 4,376,110 (David et al.) or 4,098,876 (Piasio).

A. Assavs for KSHV Polypeptide Antiqens One can use immunoassays to detect the virus, its components, or antibodies thereto. A general overview of the applicable technology ls in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publication, New York.

In one embodiment, antibodies to KSHV polypeptide antigens can be used. In brief, to produce antibodies, the polypeptide being targeted is expressed and purified. The product is injected into a mammal capable of producing antibodies. Either polyclonal or monoclonal antibodies (including recombinant antibodies) specific for the gene product can be used in various immunoassays. Such assays include competitive immunoassays, radioimmunoassays, Western blots, ELISA, indirect immunofluorescent assays and the like. For competitive immunoassays, see Harlow and Lane at pages 567-573 and 584-589.

Monoclonal antibodies or recombinant antibodies may be obtained by techniques familiar to those skilled in the art. Briefly, spleen cells or other lymphocytes from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, 1976, Eur. J. Immunol. 6, 511-519). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies W098/04576 PCT~S97/13346 produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host~ Newer techniques using recombinant phage antibody expression systems can also be used to generate monoclonal antibodies. See! for example: McCafferty et al. (1990) Nature 348, 552:
Hoogenboom et al. (1991) Nuc~ Acids Res. 19, 4133; and Marks et al. (1991) J. Mol Biol. 222, 581-597.

Methods for characterizing naturally processed peptides bound to MHC (major histocompatibility complex) I molecules can be used. See Falk et al. !
1991, Nature 351, 290 and PCT publication Nc. WO
92/21033 published November 26~ 1992. Typically, these methods involve isolation of MHC class molecules by immunoprecipitation or affinity chromatography from an appropriate cell or cell line.
Other methods involve direct amino acid sequencing of the more abundant peptides in various HP~C fractions by known automatic sequencing of peptides eluted from Class I molecules of the B cell type (Jardetzkey et al., 1991, Nature 353, 326), and of the human MHC
class I molecule, HLA-A2.1 type by mass spectrometry (Hunt et al., 1991, Eur. J. Immunol. 21, 2963-2970).
See also, Rotzschke and Falk, 1991, Immunol. Today 12, 447, for a general review of the characterization of naturally processed peptides in MHC class I. Further, Marloes et al., 1991, Eur. J. Immunol. 21, 2963-2970, describe how class I binding motifs can be applied to the identification of potential viral immunogenic peptides in vitro.

The polypeptides described herein produced by recombinant technology may be purified by standard technlques well known to those of skill in the art.
Recombinantly produced viral polypeptides can be directly expressed or expressed as a fusion protein.

.

W O 98/04576 PCTrUS97/13346 The protein is then purifled by a combination of cell lysis ~e.g., sonication) and affinity chromatography.
For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired peptide.

The polypeptides may be purified to substantia~ purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, Scopes, 1982, Protein Purification:
Principles and Practice, Springer-Verlag, New York.

B. Assays for Antibodies Specifically Bindinq To KSHV Polypeptides Antibodies reactive with polypeptide antigens of KSHV
can also be measured by a variety of immunoassay methods that are similar to the procedures described above for measurement of antigens. For a review of immunological and immunoassay procedures applicable to the measurement of antibodies by immunoassay techniques, see Basic and Clinical Immunology, 7th Edition, Stites and Terr! Eds., and Harlow and ~ane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor, New York.

In brief, immunoassays to measure antibodies reactive with polypeptide antigens of KSHV can be either competitive or noncompetitive binding assays. In competitive binding assays, the sample analyte competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface.
Preferably the capture agent is a purified recombinant human herpesvirus polypeptide produced as described above. Other sources of human herpesvirus polypeptides, including isolated or partlally purified naturally occurring polypeptide, may also be used.

Noncompetitive assays are typically sandwich assays, i~ which the sample analyte is bound between two analyte-specific binding reagents. One of the binding ~ agents is used as a capture agent and is bound to a solid surface. The second binding agent is labeled and is used to measure or detect the resultant complex by visual or instrument means. A number of combinations of capture agent and labeled binding agent can be used. ~ variety of different immunoassay formats, separation techniques and labels can also be used similar to those described above for the measurement of KSHV polypeptide antigens.

Hemagglutination Inhibition ~HI) and Complement Fixation (CF) are two laboratory tests that can be used to detect infection with human herpesvirus by ~esting for the presence of antibodies against the virus or antigens of the virus.

Serological methods can also be useful when one wishes to detect antibody to a specific viral variant. For example, one may wish to see how well a vaccine recipient has responded to a new preparation by assay of patient sera.

W 098/04576 PCTrUS97/13346 IIA. Vector, Cell Line and Transqenic Mammal This invention provides a replicable vector containing the isolated nucleic acid molecule encoding a KSHV
polypeptide. The vector includes, but is not limited to: a plasmid, cosmid, A phage or yeast artificial chromosome (YAC) which contains the isolated nucleic acid molecule.

To obtain the vector, for example, insert and vector DNA can both be exposed to a restriction enzyme to create complementary ends on both molecules which base pair with each other and are then ligated together with DNA ligase. Alternatively, linkers can be ligated to the insert DNA which correspond to a restriction site in the vector DNA~ which is then digested with the restriction enzyme which cuts at that site. Other means are available and well-known to those skilled in the art.
This invention provides a host cell containing the vector. Suitable host cells include, but are not limited to, bacteria (such as E. coli), yeast, fungi, plant, insect and mammalian cells. Suitable animal cells include, but are not limited to Vero cells, HeLa cells, Cos cells, CV1 cells and various primary mammalian cells.

This invention provides a transgenic nonhuman m~mm~l which comprises the isolated nucleic acid molecule introduced into the mammal at an embryonic stage.
Methods of producing a transgenic nonhuman mammal are known to those skilled in the art.

W O 98/04576 PCTrUS97/13346 4~
III. Diaqnostic Assays for KS

This invention embraces diagnostic test kits for detecting the presence of KSHV in biological samples, such as skin samples or samples of other affected tissue, comprising a container containing a nucleic acid sequence specific for a KSHV polypeptide and instructional material for performing the test. A
container containing nucleic acid primers to any one of such sequences is optionally included.

This invention further embraces diagnostic test kits for detecting the presence of KSHV in biological samples, such as serum or solid tissue samples, comprising a container containing antibodies to a KSHV
polypeptide, and instructional material for performing the test. Alternatively, inactivated viral particles or polypeptides derived from the human herpesvirus may be used in a diagnostic test klt to detect antibodies specific for a KSHV polypeptide.

A. Nucleic Acid Assays This invention provides a method of diagnosing Kaposi's sarcoma in a subject which comprises: (a) obtaining a nucleic acid molecule from a tumor lesion or a suitable bodily fluid of the subject; (b) contacting the nucleic acid molecule with a labeled nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with the isolated nucleic acid molecule of KSHV under hybridizing conditions; and (c) determining the presence of the nucleic acid molecule hybridized, the presence of which is indicative of Kaposi's sarcoma in the subjectr thereby diagnosing Kaposi's sarcoma in the subject.

In one embodiment the nucleic acid molecule from the tumor lesion is amplified before step (b). In another embodiment the polymerase chain reaction (PCR, is employed to amplify the nucleic acid molecule.
Methods of amplifying nucleic acid molecules are known to those skilled in the art.

A person of ordinary skill in the art will be able to obtain appropriate nucleic acid sample for diagnosing Kaposi's sarcoma in the subject. The DNA sample obtained by the above described method may be c~eaved by restriction enzyme before analysis, a technique well-known in the art.

In the above described methods, a size fractionation may be employed which is effected by a pol~acrylamide gel. In one embodiment, the size fractionation is effected by an agarose gel. Further, transferring the nucleic acid fragments into a solid matrix may be employed before a hybridization step. One example of such solid matrix is nitrocellulose paper.

This invention provides a method of detecting expression of a KSHV gene in a cell which comprises obtaining mRNA from the cell, contacting the mRNA
with a labeled nucleic acid molecule of KSHV under hybridizing conditions, determining the presence of mRNA hybridized to the molecule, thereby detecting expression of the KSHV gene. In one embodiment cDNA
is prepared from the mRNA obtained from the cell and used to detect KSHV expression.

Accepted means for conducting hybridization assays are known and genera~ overviews of the technology can be had from a review of: Nucleic Acid Hybridization: A
Practical Approach (1985) Hames and Higgins, Eds., IRL
Press; Hybridization of Nucleic Acids Immobilized on CA 0226ll64 l999-0l-22 Solid Supports, Meinkoth and Wahl; Analytlcal Biochemistry (1984) 238, 267-284 and Innis et al., PC~
Protocols (1990) Academic Press, San Diego.

Target-specific probes may be used in the nucleic acid hybridization diagnostic assays for KS. The probes ~ are specific for or complementary to the target of interest. Fo~ precise allelic differentiations, the probes should be about 14 nucleotides long and preferably about 20-30 nucleotides. For more general detection of KSHV, nucleic acid probes are about 50 to 1000 nucleotides, most preferably about 200 to 400 nucleotides.

A specific nucleic acid probe can be RNA, DNA, oligonucleotide, or their analogs. The probes may be single or double stranded nucleic acid molecules. The probes of the invention may be synthesized enzymatically~ using methods well known in the art (e.g., nick translation, primer extension, reverse transcription, the polymerase chain reactiont and others) or chemically (e . g., by methods described by Beaucage and Carruthers or Matteucci et al., supra).

The probe must be of sufficient length to be able to form a stable duplex with its target nucleic acid in the sample, i. e., at least about 14 nucleotides, and may be longer (e.g., at least about 50 or 100 bases in length). Often the probe will be more than about 100 bases in length. For example, when probe is prepared by nick-translation of DNA in the presence of labeled nucleotides the average probe length may be about 100-600 bases.

3 5 For discussions of nucleic acid probe design and annealing conditions see, for example, Ausubel et al., supra; Berger and Kimmel, Eds ., Methods in Enzymology Vol. 152~ ~1987) Academic Press, New York; or Hybridization with Nucleic Acid Probes, pp 495-524!
(1993) Elsevier, Amsterdam.

Usually, at least a part of the probe will have considerable sequence identity with the target nucleic acid. Although the extent of the sequence identity required for specific hybridization will depend on the length of the probe and the hybridization conditions, the probe will usually have at least 70~ identity to the target nucleic acid, more usually at least 80~
identity, still more usually at least 90~ identity and most usually at least 95~ or 100~ identity.

The following stringent hybridization and washing conditions will be adequate to distinguish a specific probe (e.g., a fluorescently labeled nucleic acid probe) from a probe that is not specific: incubation of the probe with the sample for 12 hours at 37~C in a solution containing denatured probe, 50~ formamide, 2X SSC, and 0.1~ (w/v) dextran sulfate, followed by washing in lX SSC at 70~C for 5 minutes; 2X SSC at 37~C for 5 minutes; 0.2X SSC at room temperature for 5 minutes, and H2O at room temperature for 5 minutes.
Those of skill are aware that it will often be advantageous in nucleic acid hybridizations (i. e., in situ, Southern, or Northern) to include detergents ( e . g., sodium dodecyl sulfate), chelating agents (e.g., EDTA) or other reagents (e.g., buffers, Denhardt's solution, dextran sulfate) in the hybridization or wash solutions. To evaluate specificity, probes can be tested on host cells containing KSHV and compared with the results from cells containing non-KSHV virus.
It wil~ be apparent to those of ordinary skill in the art that a convenient method for determining whether a probe is specific for a KSHV nucleic acid molecule utilizes a Southern blot (or Dot blot) using DNA
prepared from the virus. Briefly, to identify a target-specific probe, DNA is isolated from the virus.
Test DNA! either viral or cellular, is transferred to a solid (e.g., charged nylon) matrix. The probes are labeled by conventional methods. Following denaturation and/or prehybridization steps known in the ar~, the probe is hybridized to the immobilized DNAs under stringent conditions, such as defined above.

It is further appreciated that in determining probe specificity and in utilizing the method of this invention to detect KSHV, a certain amount of background signal is typical and can easily be distinguished by one of skill from a specific signal.
Two-fold signal over background is acceptable.

A preferred method for detecting the KSHV polypeptide is the use of PCR and/or dot blot hybridization.
Other methods to test for the presence or absence of KSHV for detection or prognosis, or risk assessment for KS includes Southern transfers, solution hybridization or non-radioactive detection systems, all of which are well known to those of skill in the art. Hybridization is carried out using probes.
Visualization of the hybridized portions allows the qualitative determination of the presence or absence of the causal agent.

Similarly, a Northern transfer or reverse transcriptase PCR may be used for the detection of KSHV messenger RNA in a sample. These procedures are also well known in the art. See Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual ~2nd ed.), Cold Spring Harbor Laboratory, Vols. 1-3.

W 098/04576 PCTrUS97/13346 An alternative means for determining the presence of the human herpesvirus is in situ hybridization, or more recently, in si tu polymerase chain reaction. In situ PCR is described in Neuvo et al. ~1993) Intracellular localization of PCR-amplified hepatitis C DNA, in American Journal of Surgical Pathology 17(7), 683-690; Bagasra et al. (1992) Detection of HIV-1 provirus in mononuclear cells by in situ PCR, in New England Journal of Medicine 326(21),1385-1391;
and Heniford et al.(1993) Variation in cellular EGF
receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction, in Nucleic Acids Research 21, 3159-3166. In situ hybridization assays are well known and are generally described in Methods Enzymol. Vol. 152, (1987) Berger and Kimmel, Eds., Academic Press, New York. In an in situ hybridization, cells are fixed to a solid support, typically a glass slide. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of target-specific probes that are labeled. The probes are preferably labeled with radioisotopes or fluorescent reporters.

The above-described probes are also useful for ln si tu hybridization or in order to locate tissues which express the gene, or for other hybridization assays for the presence of the gene or its mRNA in various biological tissues. In si tu hybridization is a sensitive localization method which is not dependent on expression of polypeptide antigens or native versus denatured conditions.

Synthetic oligonucleotide (oligo) probes and riboprobes made from KSHV phagemids or plasmids are also provided. Successful hybridization conditions in tissue sections is readily transferrable from one probe to another. Commercially-synthesized oligonucleo~ide probes are prepared using the nucleotide sequence of the identified gene. These probes are chosen for length (45-65 mers,~ high G-C
content ~50-70~) and are screened for uniqueness against other viral sequences in GenBank.

~ Oligos are 3'end-labeled with [~-35S] dATP to specific activlties in the range of 1 x 101~ dpm/~g using terminal deoxynucleotidyl transferase. Unincorporated labeled nucleotides are removed from the oligo probe by centrifugation through a Sephadex G-25 column or by elution from a Waters Sep Pak C-18 column.

KS tissue embedded in OCT compound and snap frozen in freezing isopentane cooled with dry ice is cut at 6 ~m intervals and thawed onto 3-aminopropyltriethoxysilane treated slides and allowed to air dry. The slides are then fixed in 4~ freshly prepared paraformaldehyde and rinsed ln water. Formalin-fixed, paraffin embedded KS
tissues cut at 6 ~m and baked onto glass slides can also be used. These sections are then deparaffinized in xylenes and rehydrated through graded alcohols.
Prehybridization in 20mM Tris pH 7.5, 0.02~ Denhardt's solution, 10~ dextran sulfate for 30 min at 37~C is followed by hybridization overnight in a solution of 50~ formamide (v/v), 10~ dextran sulfate (w/v), 20mM
sodium phosphate (pH 7.4), 3X SSC, lX Denhardt's solution, 100 ~g/ml salmon sperm DNA, 125 ~g/ml yeast tRNA and the oligo probe (106 cpm/ml) at ~2 C
overnight. The slides are washed twice with 3X SSC
and twice with lX SSC for 15 minutes each at room temperature and visualized by autoradiography.
Briefly~ sections are dehydrated through graded alcohols containing 0.3M ammonium acetate, and air dried. The slides are dipped in Kodak NTB2 emulsion, exposed for days to weeks, developed, and counterstained with hematoxylin and eosin (H&E).

.,, . ~ . ~ ,. .. . . . .

W098/04S76 PCT~S97/13346 Alternative lmmunohistochemical protocols may be employed which are well known to those skilled in the art.

B. Immunologic Assays This invention provides a method of diagnosing Kaposi's sarcoma in a subject, which comprises (a) obtaining a suitable bodily fluid sample from the subject, (b) contacting the suitable bodily fluid of the subject to a support having already bound thereto an antibody recognizing the KSHV polypeptide, so as to bind the antibody to a specific KSHV polypeptide antigen, (c) removing unbound bodily fluid from the support, and (d) determining the level of the antibody bound by the antigen, thereby diagnosing Kaposi's sarcoma.

This invention provides a method of diagnosing Kaposi's sarcoma in a subject, which comprises (a) obtaining a suitable bodily fluid sample from the subject, (b) contacting the suitable bodily fluid of the subject to a support having already bound thereto the KSHV polypeptide antigen, so as to bind the antigen to a specific Kaposi's sarcoma antibody, (c) removing unbound bodily fluid from the support, and (d) determining the level of the antigen bound by the Kaposi's sarcoma antibody, thereby diagnosing Kaposi's sarcoma.
The suitable bodily fluid sample is any bodily fluid sample which would contain Kaposi's sarcoma antibody, antigen or fragments thereof. A suitable bodily fluid includes, but is not limited to: serum~ plasma, cerebrospinal fluid, lymphocytes, urine, transudates, or exudates. In the preferred embodiment, the suitable bodily fluid sample is serum or plasma. In W098/04576 PCTrUS97/13346 addition, the sample may be cells from bone marrow, or a supernatant from a cell culture. Methods of obtaining a suitable bodily fluid sample from a subject are known to those skilled in the art~
Methods of determining the level of antibody or antigen include, but are not limited to: ELISA, IFA, and Western blotting. Other methods are known to those skilled in the art. Further, a subject infected with KSHV may be diagnosed as infected with the above-described methods.

The detection of KSHV and the detection of virus-associated KS are essentially identical processes.
The basic principle is to detect the virus using specific ligands that bind to the virus but not to other polypeptides or nucleic acids in a normal human cell or its environs. The ligands can be nucleic acid molecules, polypeptides or antibodies. The ligands can be naturally-occurring or genetically or physically modified, such as nucleic acids with non-natural nucleotide bases or antibody derivatives, .e., Fab or chimeric antibodies. Serological tests for detection of antibodies to the virus present in subject sera may also be performed by using the KSHV
polypeptide as an antigen, as described herein.

Samples can be taken from patients with KS or from patients at risk for KS, such as AIDS patients.
Typically the samples are taken from blood (cells, serum and/or plasma) or from solid tissue samples such as skin lesions. The most accurate diagnosis for KS
will occur if elevated titers of the virus are detected in the blood or in involved lesions. KS may also be indicated if antibodies to the virus are detected and if other diagnostic factors for KS are present.

............

W 098/04576 PCT~US97/13346 See Immunoassays above for more details on the immunoreagents of the invention for use in diagnostic assays for KS.

IV. Treatment of Human HerPesvirus-Induced KS

This lnvention provides a method for treating a subject with Kaposi's sarcoma ~KS) comprising adminlstering to the subject having KS a pharmaceutically effective amount of an antiviral agent in a pharmaceutically acceptable carrier, wherein the agent is effective to treat the subject with KSHV.

Further, this invention provides a method of prophylaxis or treatment for Kaposi's sarcoma (KS) by administering to a patient at risk for KS, an antibody that binds to KSHV in a pharmaceutically acceptable carrier .
This invention provides a method of treating a subject with Kaposi's sarcoma comprising administering to the subject an effective amount of an antisense molecule capable of hybridizing to the isolated DNA molecule of KSHV under conditions such that the antisense molecule selectively enters a KS tumor cell of the subject, so as to treat the subject.

A. Nucleic Acid Therapeutics This invention provides an antisense molecule capable of hybridizing to the 1solated nucleic acid molecule of KSHV. In one embodiment the antisense molecule is DNA. In another embodiment the antisense molecule is RNA. In another embodiment, the antisense molecule is a nucleic acid derivative (e.g., DNA or RNA with a protein backbone).

The present invention extends to the preparation of antisense nucleic acids and ribozymes that may be used to interfere with the expression of a polypeptide either by masking the mRNA with an antisense nucleic acid or cleaving it with a ribozyme, respectively.

This invention provides inhibitory nucleic acid therapeutics which can inhibit the activity of herpesviruses in patients with KS by binding to the isolated nucleic acid molecule of KSHV. Inhibitory nucleic acids may be single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex or triplex is formed. These nucleic acids are often termed "antisense" because they are usually complementary to the sense or coding strand of the gene, although recently approaches for use of "sense"
nucleic acids have also been developed. The ter~
"inhibitory nucleic acids" as used herein, refers to both "sense" and "antisense" nucleic acids.

By binding to the target nucleic acid, the inhibitory nucleic acid can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking DNA transcription, processing or poly(A) addition to mRNA, DNA replication, translation, or W 098/04576 PCTrUS97/13346 promoting inhibitory mechanisms of the cells, such as promoting RNA degradation. Inhibitory nucleic acid methods therefore encompass a number of different approaches to altering expression of herpesvirus genes. These different types of inhibitory nucleic acid technology are described in Helene and Toulme (1990) Biochim. Biophys. Acta. 1049, 99-125, which is referred to hereinafter as "Helene and Toulme."

In brief, inhibitory nucleic acid therapy approaches can be classified into those that target DNA
sequences, those that target RNA sequences (including pre-mRNA and mRNA), those that target proteins (sense strand approaches), and those that cause cleavage or chemical modification of the target nucleic acids.

Approaches targeting DNA fall into several categories.
Nucleic acids can be designed to bind to the major groove of the duplex DNA to form a triple helical or "triplex" structure. Alternatively, inhibitory nucleic acids are designed to bind to regions of single stranded DNA resulting from the opening of the duplex DNA during replication or transcription.

More commonly, inhibitory nucleic acids are designed to bind to mRNA or mRNA precursors. Inhibitory nucleic acids are used to prevent maturation of pre-mRNA. Inhibitory nucleic acids may be designed to interfere with RNA processing, splicing or translation.

The inhibitory nucleic acids can be targeted to mRNA.
In this approach, the inhibitory nucleic acids are designed to specifically block translation of the encoded protein. Using this approach, the inhibitory nucleic acid can be used to selectively suppress certain cellular functions by inhibition of WO 98t04576 PCTrUS97/13346 translation of mRNA encoding critical proteins. For example, an inhibitory nucleic acid complementary to regions of c-myc mRNA inhibits c-myc protein expression in a human promyelocytic leukemia cell line, HL60, which overexpresses the c-myc proto-oncogene. See Wickstrom et al. (1988) PNAS 85, 1028-1032 and Harel-Bellan et al. (1988) Exp. Med. 168, 2309-2318. As described in Helene and Toulme, inhibitory nucleic acids targeting mRNA have been shown to work by several different mechanisms to inhibit translation of the encoded protein(s).

The inhibitory nucleic acids introduced into the cell can also encompass the "sense" strand of the gene or mRNA to trap or compete for the enzymes or binding proteins involved in mRNA translation, as described in Helene and Toulme.

Lastly, the inhibitory nucleic acids can be used to induce chemica~ inactivation or cleavage of the target genes or mRNA. Chemical inactivation can occur by the induction of crosslinks between the inhibitory nucleic acid and the target nucleic acid within the cell.
Other chemical modifications of the target nucleic acids induced by appropriately derivatized inhibitory nucleic acids may also be used.

Cleavage, and therefore inactivation, of the target nucleic acids may be effected by attaching a substituent to the inhibitory nucleic acid which can be activated to induce cleavage reactions. The substituent can be one that affects either chemical, or enzymatic cleavage. Alternatively, cleavage can be induced by the use of ribozymes or catalytic RNA.
In this approach, the inhibitory nucleic acids would comprise either naturally occurring RNA (ribozymes) or synthetic nucleic acids with catalytic activity.

The targeting of inhibitory nucleic acids to specific cells of the immune ~ystem by conjugation with targeting moieties binding receptors on the surface of these cells can be used for all of the above forms of inhibitory nucleic acid therapy. Thi~ inventlon encompasses all of the forms of inhibitory nucleic acid therapy as described above and as described in Helene and Toulme.

An example of an antiherpes virus inhibitory nucleic acid is ISIS 2922 (ISIS Pharmaceuticals) which has activity against CMV ( see Biotechnology News 14:5).

A problem associated with inhibitory nucleic acid therapy is the effective delivery of the inhibitory nucleic acid to the target cell in vivo and the subsequent internalization of the inhibitory nucleic acid by that cell. This can be accomplished by linking the inhibitory nucleic acid to a targeting moiety to form a conjugate that binds to a specific receptor on the surface of the target infected cell, and which is internalized after binding.

B. Antiviral Aqents The use of combinations of antiviral drugs and sequential treatments are useful for treatment of herpesvirus infections and will also be useful for the treatment of herpesvirus-induced KS. For example, Snoeck et al. (1992) Eur. J. Clin. Micro. Infect. Dis.
11, 1144-1155, found additive or synergistic effects against CMV when combining antiherpes drugs (e.g., combinations of zidovudine [3'-azido-3'-deoxythymidine, AZT] with HPMPC, ganciclovir, foscarnet or acyclovir or of HPMPC with other antivirals). Similarly, in treatment of cytomegalovirus retinitis, induction with ganciclovir W O 98/04576 PCTrUS97/13346 followed by maintenance with foscarnet has been suggested as a way to maximize efficacy while minimizing the adverse side effects of either treatment alone. An anti-herpetic composition that contains acyclovir and, e.g., 2-acetylpyridine-5-((2-pyridylamino)thiocarbonyl)-thiocarbonohydrazone is described in U.S. Pat. 5l175,165 (assigned to Burroughs Wellcome Co.). Combinations of TS-inhibitors and viral TK-inhibitors in antiherpetic medicines are disclosed in U.S. Pat. 5,137,724, assigned to Stichting Rega VZW. A synergistic inhibitory effect on EBV replication using certain ratios of combinations of HPMPC with AZT was reported by Lin et al. (1991) Antimicrob Agents Chemother 35:2440-3.

U.S. Patent Nos. 5,164,395 and 5,021,437 (Blumenkopf;
Burroughs Wellcome) describe the use of a ribonucleotide reductase inhibitor (an acetylpyridine derivative) for treatment of herpes infections, including the use of the acetylpyridine derivative in combination with acyclovir. U.S. Patent No. 5,137,724 (Balzari et al. (1990) Mol. Pharm. 37,402-7) describes the use of thymidylate synthase inhibitors ( e.g., 5-fluoro-uracil and 5-fluro-2~-deoxyuridine) in combination with compounds having viral thymidine kinase inhibit1ng activity.

With the discovery of a disease causal agent for KS
now identified, effective therapeutic or prophylactic protocols to~alleviate or prevent the symptoms of herpes virus-associated KS can be formulated. Due to the viral nature of the disease, antiviral agents have application here for treatment, such as interferons, nucleoside analogues, ribavirin, amantadine, and pyrophosphate analogues of phosphonoacetic acid (foscarnet) (reviewed in Gorbach et al., 1992, W098/04576 PCT~S97/13346 Infectious Disease ~h.35, 289, W.B Saunders, Philadelphia, Pennsylvania) and the like~
Immunological therapy will also be effective in many cases to manage and alleviate symptoms caused by the disease agents described here~ Antiviral agents include agents or compositions that directly bind to viral products and interfere with disease progress;
and, excludes agents that do not impact directly on viral multipllcation or viral titer. Antlviral agents do not include immunoregulatory agents that do not directly affect viral titer or bind to viral products.
Antiviral agents are effective if they inactivate the virus, otherwise inhibit its infectivity or multiplication, or alleviate the symptoms of KS.
The antiherpesvirus agents that will be useful for treating virus-induced KS can be grouped into broad classes based on their presumed modes of action.
These classes include agents that act (l) by inhibition of viral DNA polymerase, (2) by targeting other viral enzymes and proteins, (3) by miscellaneous or incompletely understood mechanisms, or (4) by binding a target nucleic acid (i.e.~ inhibitory nucleic acid therapeutics, supra). Antiviral agents may also be used in combination (i.e., together or sequentially) to achieve synergistic or additive effects or other benefits.

Although it is convenient to group antiviral agents by their supposed mechanism of action the applicants do not intend to be bound by any part~cular mechanism of antiviral action. Moreover, it will be understood by those of skill that an agent may act on more than one target in a virus or virus-infected cell or through more than one mechanism.

i) Inhibitors of DNA Polymerase W 098t04576 PCTAJS97/13346 61 Many an~iherpesvirus agents in clinical use or in development today are nucleoside analogs believed to act through inhibition of viral DNA replication, especially through inhibition of viral DNA polymerase.
These nucleoside analogs act as alternative substrates for the viral DNA polymerase or as competitive inhibitors of DNA polymerase substrates~ Usually these agents are preferentially phosphorylated by viral thymidine kinase (TK), if one is present, and~or have higher affinity for viral DNA polymerase than for the cellular DNA polymerases, resulting in selective antiviral activity. Where a nucleoside analogue is incorporated into the viral DNA, viral activity or reproduction may be affected in a variety of ways.
For example, the analogue may act as a chain terminator, cause increased lability ( e . g., susceptibility to breakage) of analogue-containing DNA, and/or impair the ability of the substituted DNA
to act as template for transcription or replication ~see, e.g., Balzarini et al., supra).

It will be known to one of skill that, like many drugs, many of the agents useful for treatment of herpes virus infections are modified (i.e., "activa~ed") by the host, host cell, or virus-infected host cell metabolic enzymes. For example, acyclovir is triphosphorylated to its active form, with the first phosphorylation being carried out by the herpes virus thymidine kinase, when present. Other examples are the reported conversion of the compound HOE 602 to ganciclovir in a three-step metabolic pathway (Winkler et al., 1990, Antiviral ~esearch 14, 61-74) and the phosphorylation of ganciclovir to its active form by, e. g., a CMV nucleotide kinase. It will be apparent to one of skill that the specific metabolic capabilities of a virus can affect the sensitivity of that virus to specific drugs, and is one factor in the choice of an W O 98/04576 PCTrUS97/13346 antiviral drug. The mechanism of action of certain anti-herpesvirus agents is discussed in De Clercq (1993, Antimicrobial Chemotherapy 32, Suppl. Al 121-132) and in other references cited supra and infra.

Anti-herpesvirus medications suitable for treating viral induced KS include, but are not limited to, nucleoside analogs including acyclic nucleoside phosphonate analogs ( e . g., phosphonyl-methoxyalkylpurines and -pyrimidines), and cyclic nucleoside analogs. These include drugs such as:
vidarabine (9-~-D-arabinofuranosyladenine; adenine arabinoside, ara-A, Vira-A, Parke-Davis); 1-~-D-arabinofuranosyluracil (ara-U); 1-~-D-arabinofuranosyl-cytosine (ara-C); HPMPC [(S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine ( e . g., GS
504, Gilead Science)] and its cyclic form (cHPMPC);
HPMPA [(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine] and its cyclic form (cHPMPA); (S)-HPMPDAP
[(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine]i PMEDAP [~-(2-phosphonyl-methoxyethyl)-2,6-diaminopurine]i HOE 602 [2-amino-9-(1,3-bis(isopropoxy)-2-propoxymethyl)purine]; PMEA [9-~2-phosphonylmethoxyethyl)adenine]; bromovinyl-deoxyuridine (Burns and Sandford, 1990, ~. Infect.
Dis. 162:634-7), 1-~-D-arabinofuranosyl-E-5-(2-bromovinyl)-uridine or -2'-deoxyuridine; BVaraU (1-~-D-arabinofuranosyl-E-5-(2-bromovinyl)-uracil, brovavir, Bristol-Myers Squibb, Yamsa Shoyu); BVDU
[(E)-5-(2-bromovinyl~-2'-deoxyuridine, brivudin, e.g., Helpin] and its carbocyclic analogue (in which the sugar moiety is replaced by a cyclopentane ring); IVDU
[(E)-5-(2-iodovinyl)-2'-deoxyuridine] and its car~ocyclic analogue, C-IVDU (Balzarini et al ., supra); and 5-mercutithio analogs of 2'-deoxyuridine (Holliday and Williams, 1992, An timi crob . Agen ts Chemother. 36, 1935)i acyclovir [9-([2-W O 981~4576 PCT~US97/13346 hydroxyethoxylmethyl)guanine; e.g., Zovirax (Burroughs Wellcome)], penciclovir (9-[4-hydroxy-2-(hydroxymethyl)butyl~-guanine)i ganciclovir [(9-[1,3-dihydroxy-2 propoxymethyl]-guanine) e.g., Cymevene, Cytovene (Syntex), DHPG (Stals et al., 1993, Antimicrobial Agents Chemother. 37, 218-223;
isopropylether derivatives of ganciclovir (see, e.g., Winkelmann et al., 1988, Drug Res. 381 1545-1548);
cygalovir; famciclovir [2-amino-9-(4-acetoxy-3-(acetoxymethyl)but-l-yl)purine (Smithkline Beecham)];
valacyclovir (Burroughs Wellcome); desciclovir ~(2-amino-9-(2-ethoxymethyl)purine)] and 2-amino-9-~2-hydroxyethoxymethyl)-9H-purine, prodrugs of acyclovir]; CDG (carbocyclic 2'-deoxyguanosine); and purine nucleosides with the pentafuranosyl ring replaced by a cyclo butane ring (e.g., cyclobut-A [(+-)-9- El~,2~,3~)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine], cyclobut-G [(+-)-9-[1~,2~,3~)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine], BHCG
[ ( R ) - ( 1 ~ , 2 ~ , 1 ~ ) - 9 - ( 2 , 3 -bis(hydroxymethyl)cyclobutyl]guanine], and an active isomer of racemic BHCG, SQ 34,514 [lR-1~,2~,3~)-2-amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-6H-purin-6-one (see, Braitman et al., 1991, Antimicrob. Agents and Chemotherapy 35, 1464-1468). Certain of these antiherpesviral agents are discussed in Gorach et al., 1992, Infectious Disease Ch.35, 289, W.B. Saunders, Philadelphia; Saunders et al., 1990, J. Acquir. Immune Defic. Syndr. 3, 571; Yamanaka et al., 1991, Mol.
Pharmacol. 40, 446; and Greenspan et al., 1990, J.
Acguir~ Immune Defic. Syndr. 3, 571.

Triciribine and triciribine monophosphate are potent inhibitors against herpes viruses. (Ickes et al., 1994, Antiviral Research 23, Seventh International Conf. on Antiviral Research, Abstract No. 122, Supp.
1.), HIV-l and HIV-2 (Kucera et al., 1993, AIDS Res.

CA 0226ll64 l999-0l-22 Human Retrovlruses 9, 307-314? and are additional nucleoside analogs that may be used to treat KS. An exemplary protocol for these agents is an intravenous injection of about 0. 35 mg/meter2 (0.7 mg/kg) once weekly or every other week for at least two doses, preferably up to about four to eight weeks~

Acyclovir and ganciclovir are of interest because of their accepted use in clinical settings. Acyclovir, an acyclic analogue of guanine, is phosphorylated by a herpesvirus thymidine kinase and undergoes further phosphorylation to be incorporated as a chain terminator by the viral DNA polymerase during viral replication. It has therapeutic activity against a broad range of herpesviruses, Herpes simplex Types and 2, Varicella- Zoster, Cytomegalovirus, and Epstein-Barr Virus/ and is used to treat disease such as herpes encephalitis, neonatal herpesvirus infections, chickenpox in immunocompromised hosts, herpes zoster recurrences, CMV retinitis, EBV
infections, chronic fatigue syndrome, and hairy leukoplakia in AIDS patients. Exemplary intravenous dosages or oral dosages are 250 mg/kg/m2 body surface area, every 8 hours for 7 days, or maintenance doses of 200-400 mg IV or orally twice a day to suppress recurrence. Ganciclovir has been shown to be more active than acyclovir against some herpesviruses. See, e.g., Oren and Soble, 1991, C7inical Infectious Diseases 14, 741-6. Treatment protocols for ganciclovir are 5 mg/kg twice a day IV or 2.5 mg/kg three times a day for 10-14 days. Maintenance doses are 5-6 mg/kg for 5-7 days.

Also of interest is HPMPC. HPMPC is reported to be more active than either acyclovir or ganciclovir in the chemotherapy and prophylaxis of various HSV-l, W 098/04576 PCT~US97/13346 HSV-2, TK- HSV, VZV or CMV infections in animal models (De Clercq, supra) .

Nucleoside analogs such as BVaraU are potent inhibitors of HSV-1, EBV/ and VZV that have greater activity than acyclovir in animal models of encephalitis. FIAC (fluroidoarbinosyl cytosine) and its related fluroethyl and iodo compounds (e.g., FEAU, FIAU) have potent selective activity against herpesviruses, and HPMPA ((S)-1-([3-hydroxy-2-phosphorylmethoxy]propyl)adenine) has been demonstrated to be more potent against HSV and CMV
than acyclovir or ganciclovir and are of choice in advanced cases of KS. Cladribine ( 2-chlorodeoxyadenosine) is another nucleoside analogue known as a highly specific antilymphocyte agent (i. e., a immunosuppressive drug).

Other useful antiviral agents include: 5-thien-2-yl-2'-deoxyuridine derivatives, e.g., BTDU 15-5(5-bromothlen-2-yl)-2'-deoxyuridine] and CTDU [b-(5-chlorothien-2-yl)-2'-deoxyuridine]; and OXT-A [9- (2-deoxy-2-hydroxymethyl-~-D-erythro-oxetanosyl)adenine]
and OXT-G [9-(2-deoxy-2-hydroxymethyl-~-D-erythro-oxetanosyl)guanine]. Although OXT-G lS believed to act by inhibiting viral DNA synthesis its mechanism of action has not yet been elucidated. These and other compounds are described in Andrei et al., 1992, Eur.
J. Clin. Microbiol. Infect. Dis. 11, 143-51.
Additional antiviral purine derivatives useful in treating herpesvirus infections are disclosed in US
Pat. 5,108,994 (assigned to Beecham Group P.L.C.). 6-Methoxypurine arabinoside (ara-M; Burroughs Wellcome) is a potent inhibitor of varicella-zoster virus, and will be useful for treatment of KS.

CA 0226ll64 l999-0l-22 W O 98/04576 PCTrUS97/13346 Certain thymidine analogs ~e.g.~ idoxuridine (5-ido-2'-deoxyuridine)] and trifluroth~midine) have antiherpes viral activity, but due to their systemic toxicity, are largely used for topical herpesviral infections~ including HSV stromal keratitis and uveitis, and are not preferred here unless other options are ruled out.

Other useful antiviral agents that have demonstrated antiherpes viral activity include foscarnet sodium (trisodium phosphonoformate, PFA, Foscavir (Astra)) and phosphonoacetic acid (PAA). Foscarnet is an inorganic pyrophosphate analogue that acts by competitively blocking the pyrophosphate-binding site of DNA polymerase. These agents which block DNA
polymerase directly without processing by viral thymidine kinase. Foscarnet is reported to be less toxic than PAA.

W O 98/04576 PCT~US97/13346 ii) Other Antivirals Although applicants do not intend to be bound by a particular mechanism of antiviral action, the antiherpes-virus agents described above are believed to act through inhibition of viral DNA polymerase~
However, viral replication requires not only the replication of the viral nucleic acid but also the production of viral proteins and other essential components. Accordingly, the present invention contemplates treatment of KS by the inhibition of viral proliferation by targeting viral proteins other than DNA polymerase ( e. g., by inhibition of their synthesis or activity, or destruction of viral proteins after their synthesis). For example, administration of agents that inhibit a viral serine protease, e . g., such as one important in development of the viral capsid will be useful in treatment of viral induced KS.
Other viral enzyme targets include: OMP decarboxylase inhibitors (a target of, e. g., parazofurin), CTP
synthetase inhibitors (targets of, e.g., cyclopentenylcytosine), IMP dehydrogenase, ribonucleotide reductase (a target of, e. g., carboxyl-containing N-alkyldipeptides as described in U.S.
Patent No. 5,110,799 (Tolman et al., Merck)), thymidine kinase (a target of, e.g., 1- [2-(hydroxymethyl)cycloalkylmethyl]-5-substituted -uracils and -guanines as described in, e . g., U. S .
Patent Nos. 4,863,927 and 4,782,062 (Tolman et al., Merck) as well as other enzymes. It will be apparent to one of ordinary skill in the art that there are additional viral proteins, both characterized and as yet to be discovered, that can serve as target for ant iVl ral agents.

W098/04576 PCT~S97/13346 Kutapressin is a liver derivative available from Schwarz Parma of Milwaukee, Wisconsin in an injectable form of 25 mg/ml. The recommended dosage for herpesviruses is from 200 to 25 mg/ml per day for an average adult of 150 pounds.

Poly(I) Poly(C12U), an accepted antiviral drug known as Ampligen from HEM Pharmaceuticals of ~ockville, MD has been shown to inhibit herpesviruses and is another antiviral agent suitable for treating KS. Intravenous injection is the preferred route of administration.
Dosages from about 100 to 600 mg/m2 are administered two to three times weekly to adults averaging 150 pounds. It is best to administer at least 200 mg/m2 per week.

Other antiviral agents reported to show activity against herpes viruses ( e . g., varicella zoster and herpes simplex) and will be useful for the treatment of herpesvirus-induced KS include mappicine ketone (SmithKline Beecham); Compounds A,79296 and A,73209 (Abbott) for varicella zoster, and Compound 882C87 (Burroughs Wellcome) (see, The Pink Sheet 55(20) May 17, 1993).

Interferon is known inhibit replication of herpes viruses. See Oren and Soble, supra. Interferon has known toxicity problems and it is expected that second generation derivatives will soon be available that will retain interferon's antiviral properties but have reduced side affects.

It is also contemplated that herpes virus-induced KS
may be treated by administering a herpesvirus reactivating agent to induce reactivation of the latent virus. Preferably the reactivation is combined W O 98/04576 PCTrUS97/13346 with simultaneous or sequential administration of an anti-herpesvirus agent. Controlled reactivation over a short period of time or reactivation in the presence of an antiviral agent is believed to minimize the adverse effects of certain herpesvirus infections ( e . g., as discussed in PCT Application WO 93/04683).
Reactivating agents include agents such as estrogen, phorbol esters, forskolin and ~-adrenergic blocking agents.
Agents useful for treatment of herpesvirus infections and for treatment of herpesvirus-induced KS are described in numerous U.S~ Patents. For example, ganciclovir is an example of a antiviral guanine acyclic nucleotide of the type described in US Patent Nos. 4,355,032 and 4,603,219.

Acyclovir is an example of a class of antiviral purine d e r i v a t i v e s , i n c l u d i n g 9 - (2 -hydroxyethylmethyl)adenine, of the type described inU.S~ Pat. Nos. 4,287,188, 4,294,831 and 4,199,574.

Brivudin is an example of an antiviral deoxyuridine derivative of the type described in US Patent No.
4,424,211.

Vidarabine is an example of an antiviral purine nucleoside of the type described in British Pat.
1,159,290.
Brovavir is an example of an antiviral deoxyuridine derivative of the type described in US Patent Nos.
4,542 ! 210 and 4,386,076.

BHCG is an example of an antiviral carbocyclic nucleoside analogue of the type descri~ed in US Patent Nos. 5,153,352, 5,034,394 and 5,126,345.

W O 98/04576 PCTrUS97/13346 HPMPC is an example of an antiviral phosphonyl methoxyalkyl derivative with of the type described in US Patent No. 5,142l051.

CDG (Carbocyclic 2'-deoxyguanosine) is an example of an antiviral carbocyc1ic nucleoside analogue of the type described in US Patent Nos. 4,543,255~ 4,855,466, and 4,894,458.

Foscarnet is described in US Patent No. 4,339,445.

Trifluridine and its corresponding ribonucleoside is described in US Patent No. 3,201,387.

UOS~ Patent No. 5,321,030 (Kaddurah-Daouk et al.;
Amira) describes the use of creatine analogs as antiherpes viral agents. U.S. Patent No. 5,306,722 (Kim et al.; Bristol-Meyers Squibb) describes thymidine kinase inhibitors useful for treating HSV
infections and for inhibiting herpes thymidine kinase.
Other antiherpesvirus compositions are described in U.S. Patent Nos. 5,286,649 and 5,098,708 (Konishi et al., Bristol-Meyers Squibb~ and 5,175,165 (Blumenkopf et al.; Burroughs Wellcome). U.S. Patent No.
4,880,820 (Ashton et al ., Merck) describes the antiherpes virus agent (S)-9-(2,3-dihydroxy-1-propoxymethyl)guanine.

U.S. Patent No. 4,708,935 (Suhadolnik et al ., Research Corporation) describes a 3'-deoxyadenosine compound effective in inhibiting HSV and ~BV. U.S. Patent No.
4,386,076 (Machida et al., Yamasa Shoyu Kabushiki K a i s h a ) d e s c r i b e s u s e o f (E)-5-(2-halogenovinyl)-arabinofuranosyluracil as an antiherpesvirus agent. U.S. Patent No. 4,340,599 (Lieb et al., Bayer Aktiengesellschaft) describes phosphonohydroxyacetic acid derivatives useful as antiherpes agents~ U.S. Patent Nos 4,093,715 and 4,093,716 (Lin et al ., Research Corporation) describe 5~-amino-5'-deoxythymidine and 5-iodo-5'-amino-2',5'-dideoxycytidine as potent inhibitors of herpes simplex virus. U.S. Patent No 4,069,382 (Baker et al ., Parke, Davis ~ Company) describes 9-(5-O-Acyl-beta-D-arabinofuranosyl)adenine compounds useful as antiviral agents. U.S. Patent No. 3,927,216 (Witkowski et al . ) describes the use of 1, 2 , 4 - t r i a z o l e -3 - c a r b o x a m i d e a n d 1,2~4-triazole-3-thiocarboxamide for inhibiting herpes virus infections. Patent No. 5,179,093 (Afonso et al., Schering) describes quinoline-2,4-dione derivatives active against herpes simplex virus 1 and 2, cytomegalovlrus and Epstein Barr virus.

iii) Administration The subjects to be treated or whose tissue may be used herein may be a mammal, or more specifically a human, horse, pig, rabbit, dog, monkey, or rodent. In the preferred embodiment the subject is a human.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each subject.
Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals b~ a subsequent - 35 injectlon or other administration.

W O 98/04576 PCTrUS97/13346 As used herein administration means a method of administering to a subject~ Such methods are well known to those skilled in the art and include, but are not limited to, administration topically, parenterally, orally, intravenously, intramuscularly, subcutaneously or by aerosol. Administration of the agent may be effected continuously or intermittently such that the therapeutlc agent in the patient is effective to treat a subject with Kaposi's sarcoma or a subject infected with a DNA virus associated with Kaposi's sarcoma.

The antiviral compositions for treating herpesvirus-induced KS are preferably administered to human patients via oral, lntravenous or parenteral administrations and other systemic forms. Those of skill in the art will understand appropriate administration protocol for the individual compositions to be employed by the physician.
The pharmaceutical formulations or compositions of this lnvention may be in the dosage form of solid, semi-solid, or liquid such as, e.g., suspensions, aerosols or the like. Preferably the compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts. The compositions may also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used ~o formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, W O 98/04576 PCT~US97/13346 ad~uvantsi or nontoxic, nontherapeutic, nonimmunogenic stabilize~s and the like. Effective amounts of such diluent or carrier are those amounts which are effective to obtain a pharmaceutically acceptable formulation in terms of solubilit~ of components, or biological activity, etc.

V. Immunoloqical Approaches to Therapy Having identified a primary causal agent of KS in humans as a novel human herpesvirus, there are immunosuppressive therapies that can modulate the immunologic dysfunction that arises from the presence of viral-infected tissue. In particular, agents that block the immunological attack of the viral-infected cells will ameliorate the symptoms of KS and/or reduce disease progression. Such therapies include antibodies that prevent immune system targeting of viral-infected cells. Such agents include antibodies which bind to cytokines that otherwise upregulate the immune system in response to viral infection.

The antibody may be administered to a patient either singly or in a cocktail containing two or more antibodies, other therapeutic agents, compositions, or the like, including, but not limited to, immuno-suppressive agents, potentiators and side-effect re-lieving agents. Of particular interest are immuno-suppressive agents useful in suppressing allergic re-actions of a host. Immunosuppressive agents of inter-est include prednisone, prednisolone, DECADRON (Merck, Sharp & Dohme, West Point, PA), cyclophosphamide, cyclosporine, 6-mercaptopurine, methotrexate, azathioprine and i.v. gamma globulin or their combination. Potentiators of interest include monensin, ammonium chloride and chloroquine. All of these agents are administered in generally accepted ., ~ ., , . _ W 098/04576 PCT~US97/13346 74 efficac1ous dose ranges such as those disclosed in the Physiciarl Desk Reference, 41st Ed. (1987)~ Publisher Edward R. Barnhartl New Jersey.

Immune globulin from persons previously infected with human herpesvlruses or related viruses can be obtained using standard techniques. Appropriate titers of antibodies are known for this therapy and are readily applied to the treatment of KS. Immune globulin can be administered via parenteral injection or by intrathecal shunt. In brief, immune globulin preparations may be obtained from individual donors who are screened for antibodies to the KS-associated human herpesvirus, and plasmas from high-titered donors are pooled. Alternatively, plasmas from donors are pooled and then tested for antibodies to the human herpesvirus of the invention; high-titered pools are then selected for use in KS patients.

Antibodies may be formulated into an injectable preparation. Parenteral formulations are known and are suitable for use in the invention, preferably for i.m. or i.v. administration. The formulations containing therapeutically effective amounts of antibodies or immunotoxins are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients.
Lyophilized compositions are reconstituted with suitable diluents, e.g., water for injection, saline, 0.3~ glycine and the like, at a level of about from .01 mg/kg of host ~ody weight to 10 mg/kg where appropriate. Typically, the pharmaceutical compositions containing the antibodies or immunotoxins will be administered ln a therapeutically effective dose in a range of from about .01 mg/kg to about 5 mg/kg of the treated mammal. A preferred therapeutically effective dose of the pharmaceutical composition containing antibody or immunotoxin will be in a range of from about 0.01 mg/kg to about 0.5 mg/kg body welght of the treated mammal administered over several days to two weeks by daily intravenous infusion, each given over a one hour period, in a sequential patient dose-escalation regimen.

Antibody may be administered systemically by injection i.m., subcutaneously or intraperitoneally or directly into KS lesions. The dose will be dependent upon the properties of the antibody or immunotoxin employed, e.g., its activity and biological half-life, the concentration of antibody in the formulation, the site and rate of dosage, the clinical tolerance of the patient involved, the disease af~licting the patient and the like as is well within the skill of the physician.

The antibody of the present invention may be administered in solution. The pH of the solution should be in the range of pH 5 to 9.5, preferably pH

6.5 to 7.5. The antibody or derivatives thereof should be in a solution having a suitable pharmaceutically acceptable buffer such as phosphate, tris (hyd~oxymethyl) aminomethane-HCl or citrate and the like. Buffer concentrations should be in the range of 1 to 100 mM. The solution of antibody may also contain a salt, such as sodium chloride or potassium chloride in a concentration of 50 to 150 mM.
An effective amount of a stabilizlng agent such as an albumin, a globulin, a gelatin, a protamine or a salt of protamine may also be included and may be added to a solution containing antibody or immunotoxin or to the composition from which the solution is prepared.
Systemic administration of antibody is made daily, generally by intramuscular injection, although W 098/04576 PCTrUS97/13346 intravascular infusion is acceptable. Administration may also be intranasal or by other nonparenteral routes. Antibody or immunotoxin may also be administered vla microspheres, liposomes or other microparticulate delivery systems placed in certain tissues including blood.

In therapeutic applications, the dosages of compounds used in accordance with the invention vary depending on the class of compound and the condition being treated. The age, weight, and clinical condition of the recipient patient; and the experience and judgment of the clinician or practitioner administering the therapy are among the factors affecting the selected dosage. For example, the dosage of an immunoglobulin can range from about 0.1 milligram per kilogram of body weight per day to about 10 mg/kg per day for polyclonal antibodies and about 5~ to about 20~ of that amount for monoclonal antibodies. In such a case, the immunoglobulin can be administered once daily as an intraveno,us infusion. Preferably, the dosage is repeated daily until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. Generally~ the dose should be sufficient to treat or ameliorate symptoms or signs of KS without producing unacceptable toxicity to the patient.

An effective amount of the compound is that which provides either subjective relief of a symptom(s) or an ob~ectively identifiable improvement as noted by the clinician or other qualified observer. The dosing range varies with the compound used, the route of administration and the potency of the particular compound.

W O 98/04576 PCT~US97/13346 VI Vaccines and Prophylaxis for KS

This invention provides substances suitable for use as vaccines for the prevention of KS and methods for administering them. The vaccines are directed against KSHV and most preferably comprise antigens obtained from KSHV. In one embodiment, the vaccine contains attenuated KSHV. In another embodiment, the vaccine contains killed KSHV. In another embodiment, the lo vaccine contains a nucleic acid vector encoding a KSHV
polypeptide. In another embodiment, the vaccine is a subunit vaccine containing a KSHV polypeptide.

This invention provides a recombinant KSHV virus with a gene encoding a KSHV polypeptide deleted from the genome. The recomblnant virus is useful as an attenuated vaccine to prevent KSHV infection.

This invention provides a method of vaccinating a subject against Kaposi~s sarcoma, comprising administering to the subject an effective amount of the peptide or polypeptide encoded by the isolated DNA
molecule, and a suitable acceptable carrier, thereby vaccinating the subject. In one embodiment naked DNA
is administered to the subject in an effective amount to vaccinate the subject against Kaposi's sarcoma.

This invention provides a method of immunizing a subject against disease caused by KSHV which comprises administering to the subject an effective immunizing dose of an isolated herpesvirus subunit vaccine.

A. Vaccines The vaccine can be made using synthetic peptide or recombinantly-produced polypeptide described above as antigen. Typically, a vaccine will include from about W 098/04576 PCTrUS97113346 1 to 50 micrograms of antigen More preferably, the amount of polypeptide is ~rom about 15 to about 45 mlcrograms~ Typically, the vaccine is formulated so that a dose includes about 0.5 millilitersn The vaccine may be administered by any route known in the art. Preferably, the route is parenteral. More preferably, it is subcutaneous or intramuscular.

There are a number of strategies for amplifylng an antigen's effectiveness, particularly as related to the art of vaccines. For example, cyclization or circularization of a peptide can increase the peptide's antlgenic and immunogenic potency. See U.S.
Pat. No. 5,001~049. More conventionally, an antigen can be conjugated to a suitable carrler, usually a protein molecule. This procedure has several facets.
It can allow multiple copies of an antigen, such as a peptide, to be conjugated to a single larger carrier molecule. Additionally, the carrier may possess properties which facilitate transport, binding, absorption or transfer of the antigen.

For parenteral administration, such as subcutaneous injectlon, examples of suitable carriers are the tetanus toxoid, the diphtheria toxoid, serum albumin and lamprey, or keyhole limpet, hemocyanin because they provide the resultant conjugate with minimum genetic restriction~ Conjugates including these universal carriers can function as T cell clone activators in individuals having very different gene sets.

The conjugation between a peptide and a carrier can be accomplished using one of the methods known in the art. Specifically, the conjugation can use bifunctional cross-linkers as binding agents as detailed, for example, by Means and Feeney, "A recent W O 98/04576 PCT~US97/13346 review of protein modification techniques,"
Bloconjugate Chem 1, 2-12 (1990)~

Vaccines against a number of the Herpesviruses have been successfully developed. Vaccines against Varicella-Zoster Virus using a live attenuated Oka strain lS effectlve in preventing herpes zoster in the elderly, and in preventing chickenpox in both immunocompromised and normal children (Hardy, I., et al., 1990, Inf. Dis. Clin. N. Amer. 4, 159; Hardy, I.
et al., 1991, New Engl. J~ Med. 325, 1545i Levin, M.J.
et al., 1992t J. Inf. Dis~ 166, 253; Gershon, A.A., 1992, J. Inf. Des. 166(Suppl), 563. Vaccines against Herpes simplex Types 1 and 2 are also commercially available with some success in protection against primary disease, but have been less successful in preventing the establishment of latent infection in sensory ganglia (Roizman, B., 1991, RevO Inf. Disease 13(Suppl. 11), S8g2; Skinner, G.R. et al., 1992, Med.
Microbiol. Immunol. 180, 305).

Vaccines against KSHV can be made from the KSXV
envelope glycoproteins. These polypeptides can be purified and used for vaccination (Lasky, L.A., 1990, J. Med. Virol. 31, 59). MHC-binding peptides from cells infected with the human herpesvirus can be identified for vaccine candida~es per the methodology of Marloes, et al., 1991, Eur. ~. Immunol. 21, 2963-2970.
The KSHv antiyen may be combined or mixed with various solutions and other compounds as is known in the art.
For example, it may be administered in water, saline or buffered vehicles with or without various adjuvants or imm~nodiluting agents. Examples of such adjuvants or agents lnclude aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), WO 98/04576 PCTrUS97113346 berylllum sulfate, silica~ kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions r muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionibacterium acnes)/
Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran/ blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example/ Merck Adjuvant 65 (Merck and Company/ Inc., Rahway/ N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan). Other suitable adjuvants are Amphigen (oil-in-water)/ Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel.
Only aluminum is approved for human use.

The proportion of antigen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide can be present in an amount of about 0.5~ of the vaccine mixture (Al2O3 basis)~ On a per-dose basis, the amount of the antigen can range from about 0.1 ~g to about 100 ~g protein per patient. A preferable range is from about 1 ~g to about 50 ~g per dose. A
more preferred range is about 15 ~g to about 45 ~g.
A suitable dose size is about 0.5 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.5 ml containing 45 ~g of antigen in admixture with 0.5~ aluminum hydroxide. After formulation, the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4~C, or it may be freeze-dried. Lyophilization permits long-term storage in a stabilized form.

W 098/04576 PCTrUS97/13346 The vaccines may be administered by any conventional method for the administration of vaccines including oral and parenteral (e.g., subcutaneous or intra-muscular) injection. Intramuscular administration is preferred. The treatment may consist of a single dose of vaccine or a plurality of doses over a period of time. It is preferred that the dose be given to a human patient within the first 8 months of life. The antigen of the invention can be combined with appropriate doses of compounds including influenza antigens, such as influenza type A antigens. Also, the antigen could be a component of a recombinant vaccine which could be adaptable for oral administration.
Vaccines of the invention may be combined with other vaccines for other diseases to produce multivalent vaccines. A pharmaceutically effective amount of the antigen can be employed with a pharmaceutically acceptable carrier such as a protein or diluent useful for the vaccination of mammals, particularly humans.
Other vaccines may be prepared according to methods well-known to those skilled in the art.

Those of skill will readily recognize that it is only necessary to expose a mammal to appropriate epitopes in order to elicit effective immunoprotection. The epitopes are typically segments of amino acids which are a small portion of the whole protein. Using recombinant genetics, it is routine to alter a natural protein's primary structure to create derivatives embracing epitopes that are identical to or substantially the same as (immunologically equivalent to) the naturally occurring epitopes. Such derivatives may include peptide fragments, amino acid substitutions, amino acid deletions and amino acid additions of the amino acid sequence for the viral W 098/04576 PCTrUS97/13346 polypeptides from the human herpesvirus. For example, it is known in the protein art that certain amino acid residues can be substituted with amino acids of similar size and polarity without an undue effect upon the biological activity of the protein. The human herpesvirus polypeptides have significant tertiary structure and the epitopes are usually conformational.
Thus, modifications should generally preserve conformation to produce a protective immune response~

B. Antibody Prophylaxis Therapeutic, intravenous, polyclonal or monoclonal antibodies can been used as a mode of passive immunotherapy of herpesviral diseases including perinatal varicella and CMV. Immune globulin from persons previously infected with the human herpesvirus and bearing a suitably high titer of antibodies against the virus can be given in combination with antiviral agents (e.g. ganciclovir), or in combination with other modes of immunotherapy that are currently being evaluated for the treatment of KS, which are targeted to modulating the immune response (i.e.
treatment with copolymer-1, antiidiotypic monoclonal antibodies, T cell "vaccination~). Antibodies to human herpesvirus can be administered to the patient as described herein. Antibodies specific for an epitope expressed on cells infected with the human herpesvirus are preferred and can be obtained as described above.

A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are W O 98/04576 PCTrUS97/13346 formed with inorganic acids such as; for exampleO
hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic~ tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

C. Monitorinq TheraPeutlc EfficacY

This invention provides a method for monitoring the therapeutic efficacy of treatment for Kaposi's sarcoma which comprises: (a) determining in a first sample from a subject with Kaposi's sarcoma the presence of the isolated nucleic acid molecule; (b) administering to the subject a therapeutic amount of an agent such that the agent is contacted to the cell in a sample;
(c) determining after a suitable period of time the amount of the isolated nucleic acid molecule in the second sample from the treated subject; and (d~
comparing the amount of isolated nucleic acid molecule determined in the first sample with the amount determined in the second sample, a difference indicating the effectiveness of the agent, thereby monitoring the therapeutic efficacy of treatment for Kaposi's sarcoma. As defined herein "amount" is viral load or copy number. Methods of determining viral 3G load or copy number are known to those skilled in the art.

VII. Screeninq Assays For Pharmaceuticals for Alleviatlnq the Symptoms of KS
Since an agent involved in the causation or progression of KS has been identified and described, W O 98/04576 PCT,~S97/13346 assays directed to identifying potential pharmaceutical agents that inhibit the biological activity of the agent are possible. KS drug screening assays which determine whether or not a drug has activity against the vlrus described herein are contemplated in this invention~ Such assays comprise incubatlng a compound to be evaluated for use in KS
treatment with cells which express the KS assoclated human herpesvirus polypeptides or peptides and determining therefrom the effect of the compound on the activity of such agent. In ~i tro assays in which the virus is maintained in suitable cell culture are preferred, though in vivo animal models would also be effective.
Compounds with activity against the agent of interest or peptides from such agent can be screened in in vi tro as well as in vivo assay systems. In vi tro assays include infecting peripheral blood leukocytes or susceptible T cell lines such as MT-4 with the agent of interest in the presence of varying concentrations of compounds targeted against viral replication, including nucleoside analogs, chain terminators, antisense oligonucleotides and random polypeptides (Asada et al., 1989, J. Clin. Microbiol.
27, 2204; Kikuta et al., 1989, Lancet Oct. 7, 861).
Infected cultures and their supernatants can be assayed for the total amount of virus including the presence of the viral genome by quantitative PCR, by dot blot assays or by using immunologic methods. For example, a culture of susceptible cells could be infected with KSHV in the presence of various concentrations of drug, fixed on slides after a period of days, and examined for viral antigen by indirect immunofluorescence with monoclonal antibodies to viral polypeptides ~Kikuta et al., supra). Alternatively, chemically adhered MT-4 cell monolayers can be used for an infectious agent assay using indirect immunofluorescent antibody staining to search for focus reduction (Higashi et al ~, 1989, J. Clin Micro 27, 2204).

As an alternative to whole cell in vitro assays, purified KSHV enzymes isolated from a host cell or produced by recombinant techniques can be used as targets for rational drug design to determine the effect of the potential drug on enzyme activity. KSHV
enzymes am~n~hle to this approach include, but are not limited to, dihydrofolate reductase (DHFR), thymidylate synthase (TS), thymidine kinase or DNA
polymerase. A measure of enzyme activity indicates effect on the agent itself.

Drug screens using herpes viral products are known and have been previously described in EP 0514830 (herpes proteases) and WO 94/04920 (UL13 gene product).
This invention provides an assay for screening anti-KS
chemotherapeutics. Infected cells can be incubated in the presence of a chemical agent that is a potential chemotherapeutic against KS (e.g., acyclo-guanosine).
The level of virus in the cells is then determined after several days by immunofluorescence assay for antigens, Southern blotting for viral genome DNA or Northern blotting for mRNA and compared to control cells. This assay can quickly screen large numbers of chemical compounds that may be useful against KS.

Further, this invention provides an assay system that is employed to identify drugs or other molecules capable of binding to the nucleic acid molecule or proteins, either in the cytoplasm or in the nucleus, thereby inhibiting or potentlating transcriptional activity. Such assay would be useful in the CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 development of drugs that would be specific agalnst particular cellular activity, or that would potentiate such activity, in time or in level of activity.

This invention provides a method of screening for a KSHV-selective antiviral drug in vivo comprising~ (a) expression of KSHV DHFR or KSHV TS in a bacterial auxotroph (nutritional mutant); (b) measuring bacterial growth rate in the absence and presence of the drug; and (c) comparing the rates so measured so as to identify the drug that inhibits KSHV DHFR or KSHV TS in vivo.

Methods well known to those skilled in the art allow selection or production of a suitable bacterial auxotroph and measurement of bacterial growth.

The following reviews of antifolate compounds are provided to more fully describe the state of the art, particularly as it pertains to inhibitors of dihydrofolate reductase and thymidylate synthase: ~a) Unger, 1996, Current concepts of treatment in medical oncology: new anticancer drugs, ~ournal of Cancer Research & Clinical Oncology 122, 189-lg8; (b) Jackson, 1995, Toxicity prediction from metabolic pathway modelling, Toxicology 102, 197-205; (c) Schultz, 1995, Newer antifolates in cancer therapy, Progress in Drug Research 44, 129-1~7; (d) van der Wilt and Peters, 1994, New targets for pyrimidine antimetabolites in the treatment of solid tumours 1:
Thymidylate synthase, Pharm World Sci 16, 167; (e) Fleisher, 1993, Antifolate analogs: mechanism of action, analytical methodology, and clinical efficacy, Therapeutic Drug Monitoring 15, 521-526; ( f) Eggott et al., 1993, Antifolates in rheumatoid arthritis: a hypothetical mechanism of action, Clinical &
Experimental Rheumatology 11 Suppl 8, S101-S105; (g) Huennekens et al., 1992, Membrane transport of folate compounds, Journal of Nutritional Science &
Vitaminology Spec No, 52-57i (h) Fleming and Schilsky 1992, Antifolates: the next generation, SPmin~rs i~
Oncology 19~ 707-719; and (i) Bertino et al., 1992, Enzymes of the thymidylate cycle as targets for chemotherapeutic agents: mechanisms of resistance, Mount Sinai Journal of Medicine 59, 391-395.

This invention provides a method of determining the health of a subject with AIDS comprising: (a) measuring the plasma concentration of vMIP-I, vMIP-II
or vMIP-III; and (b) comparing the measured value to a standard curve relating AIDS clinical course to the measured value so as to determine the health of the subject~

VIII. Treatment of HIV
This invention provides a method of inhibiting HIV
replication, comprising administering to the subject or treating cells of a subject with an effective amount of a polypeptide which is encoded by a nucleic acid molecule, so as to inhibit replication of HIV.
In one embodiment, the polypeptide is one from the list provided in Table 1.

This invention is further illustrated in the Experimental Details Sections which follow. These sections are set forth to aid in understanding the invention but is not intended to, and should not be construed to ! limit in any way the invention as set forth in the claims which follow thereafter.

EXPERIMENTAL DETAILS SECTION I

W 098/04576 PCTrUS97/13346 NUCLEOTIDE SEQUENCE OF THE KAPOSI'S SARCOMA-ASSOCIATED
HERPESVIRUS

The genome of the Kaposi's sarcoma-associated herpesvlrus (KSHV or HHV8) was mapped wlth cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3 kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5 kb long unique coding region (LUR) flanked by multiple G+C rich 801 bp terminal repeat sequences.
A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derlved strain. At least 81 open reading frames (ORFs), including 66 with similarity to herpesviru~ saimiri ORFs, and 5 internal repeat regions are present in the LUR. The virus encodes genes similar to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleu~in-6), dihydrofolate reductase, bc1-2, interferon regulatory factor, IL-8 receptor, NCAM-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor. The complete genome sequence is set forth in Genbank Accession Numbers U75698 (LUR), U75699 (TR) and U75700 (ITR).

Kaposi's sarcoma is a vascular tumor of mixed cellular composition (Tappero et al ., 1993, ~. Am. Acad.
Dermatol. 28, 371-395). The histology and relatively benign course in persons without severe immunosuppression has led to suggestions that KS tumor cell proliferation is cytokine induced (Ensoli et al., 1992, Tmm~nol. Rev. 127, 147-155). Epidemiologic studies indicate the tumor is under strict immunologic control and is likely to be caused by a sexually W 098/04576 PCT~US97/13346 transmitted infectious agent other than HIV (Peterman et a7. 1993~ AIDS 7, 605-611). KS-associated herpesvlrus (KSHV) was discovered in an AIDS-KS lesion by representational difference analysis (RDA! and shown to be present in almost all AIDS-KS lesions (Chang et al., 1994, Science 265, 1865-1869). These findings have been confirmed and extended to nearly all KS lesions e~mlned from the various epidemiologic classes of KS (Boshoff et al., 1995, Lancet 345, 1043-1044; Dupin et al., 1995, Lancet 345, 761-762;
Moore and Chang, 1995, New Eng~ J. Med. 332, 1181-1185~ Schalling et al., 1995, Nature Med. 1, 707-708; Chang et al., 1996, Arch. Int. Med. 156, 202-204). KSHV is the elghth presumed human herpesvirus (HHV8~ identified to date.

The virus was initially identifed from two herpesvirus DNA fragments, KS330Bam and KS631Bam (Chang et al., 1994, Science 265, 1865-1869). Subsequent sequencing of a 21 kb AIDS-KS genomic library fragment (KS5) hybridizing to KS330Bam demonstrated that KSHV is a gammaherpesvirus related to herpesvirus saimiri (HVS) belonging to the genus Rhadinovirus (Moore et al., 1996, J. Virol. 70, 549-558). Colinear similarity (synteny) of genes in this region is maintained between KSHV and HVS, as well as Epstein-Barr virus (EBV) and equine herpesvirus 2 (EHV2)~ A 12 kb region (L54 and SGL-1) containing the KS631Bam sequence includes cyclin D and IL-8Ra genes unique to rhadinoviruses.

KSHV is not readily transmitted to uninfected cell lines (Moore et al., 1996, J. Virol. 70, 549-558~, but it lS present in a rare B cell primary effusion (body cavity-based) lymphoma (PEL) frequently associated with KS (Cesarman et al., 1995, New Eng. J.
~ed. 332, 1186-1191). BC-1 is a PEL cell line W O 98/04576 PCTrUS97/13346 containing a high KSHV genome copy number and lS
coinfected with EBV ~Cesarman et al ., 1995~ Blood 86, 2708-2714) The KSHV genome form in BC-1 and its parental tumor comigrates with 270 kb linear markers on pulsed field gel electrophoresis (PFGE) (Moore et al., 1996, J. Virol. 70~ 549-558). However, the genome size based on encapsidated DNA from an EBV-negative cell line (Renne et al., 1996, Nature Med. 2, 342-346) is estimated to be 165 kb (Moore et al., 1996, J. Virol. 70, 549-558). Estimates from KS
lesions indicate a genome size larger than that of EBV
(172 kb) (Decker et al ., 1996, J. Exp. Med. 184, 283-288~.

To determine the genomic sequence of KSHV and identify novel virus genes, contiguous overlapping virus DNA
inserts from BC-1 genomic libraries were mapped. With the exception of a small, unclonable repeat region at its right end, the genome was sequenced to high redundancy allowing definition of the viral genome structure and identification of genes that may play a role in KSHV-related pathogenesis.

MATERIALS AND METHODS
Library generation and screening. BC-1, HBL-6 and BCP-1 cells were maintained in RPMI 1640 with 20 fetal calf serum (Moore et al., 1996, ~. Virol. 70, 549-558; Cesarman et al., 1995, Blood 86, 2708-2714;
3n Gao et al., 1996, Nature Med. 2, 925-928). DNA from BC-1 cells was commercially cloned (Sambrook et al., 1989, Molecular Cloning: A laboratory manual, Cold Spring Harbor Press, Salem, Mass.) into either Lambda FIX II or S-Cosl vectors (Stratagene, La Jolla, CA).
Phage and cosmid libraries were screened by standard methods ~Benton et al., 1977, Science 196, 180-182;

CA 0226ll64 l999-0l-22 W O 98/04576 PCTrUS97/13346 Hanahan and Meselson, 1983, Methods Enzymol. 100, 333-342).

Initial library screening was performed uslng the KS330Bam and KS631Bam RDA fragments (Chang et al., 1994, Science 265, 1865-1869). Overlapping clones were sequentially identified using probes synthesized from the ends of previously identified clones (Figure 1) (Feinberg and Vogelstein, 1983, Anal. Biochem. 132, 6; Melton et al., 1984, Nucl. Acids Res. 12, 7035-7056). The map was considered circularly permuted by the presence of multiple, identical TR
units in cosmids Z2 and Z6. Each candidate phage or cosmid was confirmed by tertiary screening.
Shotqun se~uencin~ and sequence verification Lambda and cosmid DNA was purified by standard methods (Sambrook et al., 1989, Molecular Clonlng: A
laboratory manual, Cold Spring Harbor Press, Salem, Mass.). Shotgun sequencing (Deininger, 1983, Anal.
Biochem. 129, 216-223; Bankier et al.~ 1987, Meth.
Enzymol. 155, 51-93) was performed on sonicated DNA
A 1-4 kb fraction was subcloned into M13mpl9 (New England Biolabs, Inc., Beverly, MA) and propagated in XL1-Blue cells (Stratagene, La Jolla, CA) ~Sambrook et al., 1989, Molecular Cloning: A laboratory manual, Cold Spring Harbor Press, Salem, Mass.) M13 phages were positively screened using insert DNA from the phage or cosmid, and negatively screened with vector arm DNA or ad~acent genome inserts.

Automated dideoxy cycle sequencing was performed with M13 (-21) CS+ or FS dye primer klts (Perkin-Elmer, Branchburg NJ) on ABI 373A or 377 sequenators (ABI, Foster City, CA). Approximately 300 Ml3 sequences were typically required to achieve initial coverage ., ~ ... .... . . .. . . . ..

for each 10 kb of insert sequence~ Minlmum sequence fidelity standards were defined as complete bidirectional coverage with at ieast 4 overlapping sequences at any given site. For regions with sequence gaps, ambiguities or frameshifts that did not meet these criteria, primer walking was done with custom primers (Perkin-Elmer) and dye terminator chemistry (FS or Ready Reaction kits, Perkin-Elmer).
An unsequenced 3 kb region adjacent to the right end TR sequence in the Z2 cosmid insert could not be cloned into M13 or Bluescript despite repeated efforts.

Sequence assembly and oPen readinq frame analysis Sequence data were edited using Factura (ABI, Foster City, CA) and assembled into contiguous sequences using electropherograms with AutoAssembler (ABI, Foster City, CA) and into larger assemblies with AssemblyLIGN (IBI-Kodak, Rochester NY). Base positions not clearly resolved by multiple sequencing attempts (less than 10 bases in total) were assigned the majority base pair designation. The entire sequence (in 1-5 kb fragments) and all predicted open reading frames (ORFs) were analyzed using BLASTX, BLASTP and BLASTN (Altschul et al., 1990, J. Mol.
Bi ol . 215, 403-410). The sequence was further analyzed using MOTIFS (Moore et al., 1996, J. Virol.
70, 549-558), REPEAT and BESTFIT (GCG), and MacVector (IBI, New Haven, CT).

ORF assiqnment and nomenclature All ORFs with similarities to HVS were identified.
These and other potential ORFs having >100 amino acids were found using MacVector. ORFs not similar to HVS
ORFs were included in the map (Fig. 1) based on WO 98/04576 PCTrUS97/13346 similarity to other known genes, optimum initiation codon context (Kozak, 1987, Nucl. Acids Res. 15, 8125-8148), size and position. Conservative selections were made to minimize spurious assignments;
this underestimates the number of true reading frames.
KSHV ORF nomenclature is based on HVS similarities, KSHV ORFs not similar to HVS genes are numbered in consecutive order with a K prefix. ORFs with sequence but not positional similarity to HVS ORFs were assigned the HVS ORF number (e.g., ORF 2). As new ORFs are identified, it is suggested that they be designated by decimal notation. The standard map orientation (Fig. 1) of the KSHV genome is the same as for HVS (Albrecht et al., 1992, J. Viro7. 66, 5047-5058) and EHV2 (Telford et al., 1995, J. Mol.
Biol . 249, 520-528), and reversed relative to the EBV
standard map (Baer et al., 1984, Nature 310, 207-211).

RESULTS
Genomic mappinq and sequence characteristics Complete genome mapping was achieved with 7 lambda and 3 cosmid clones (Fig. 1). The structure of the BC-l KSHV genome is similar to HVS in having a long unique region (LUR) flanked by TR units. The ~140.5 kb LUR
sequence has 53.5~ G+C content and includes all identified KSHV ORFs. TR regions consist of multiple 801 bp direct repeat units having 84.5~ G+C content (Fig. 2A) with potential packaging and cleavage sites.
Minor sequence variations are present among repeat units. The first TR unit at the left (Z6) TR junction (205bp) is deleted and truncated in BC-l compared to the prototypical TR unit.
The genome sequence abutting the right terminal repeat region is incomplete due to a 3 kb region in the Z2 W 098/04576 PCTrUS97/13346 cosmid insert that could not be c~oned into sequencing vectors. Partial sequence information from primer walkins indicates that this region contains stretches of 16 bp A+G rich imperfect direct repeats interspersed with at least one stretch of 16 bp C+T
rich imperfect direct repeats. These may form a larger inverted repeat that could have contributed to our difficulty in subcloning this region. Greater than 12-fold average sequence redundancy was achieved for the entire LUR with complete bidirectional coverage by at least 4 overlapping reads except in the unclonable region.

The BC-1 TR region was examined by Southern blotting since sequencing of the entire region is not possible due to its repeat structure. BC-l, BCP-1 (an EBV-negative, KSHV infected cell line) and KS lesion DNAs have an intense ~800 bp signal consistent with the unit length repeat sequence when digested with enzymes that cut once in the TR and hybridized to a TR
probe (Figs. 2B and 2C). Digestion with enzymes that do not cut in the TR indicates that the BC-1 strain contains a unique region buried in the TR, flanked by ~7 kb and ~35 kb TR sequences (Figs. 2C and 2D). An identical pattern occurs in HBL-6, a cell line independently derived from the same tumor as BC-1, suggesting that this duplication was present in the parental tumor (Figs. 2C and 2D). The restriction pattern with Not I, which also cuts only once within the TR but rarely within the LUR t suggests that the buried region is at least 33 kb. Partial sequencing of this region demonstrates that it is a precise genomic duplication of the region beginning at ORF K8.
The LUR is 140 kb including the right end unsequenced gap (~3kb). The estimated KSHV genomic size in BC-l and HBL-6 (including the duplicated region) is approximately 210 kb.

W 098/04576 PCTrUS97/13346 Based on the EBV replication model used in clonality studies (Raab-Traub and Flynn, 198 6 ~ Cel 1 4 7, 883-889), the polymorphic BCP-1 laddering pattern may reflect lytic virus replication and superinfection (Fig. 2C). The EBV laddering pattern occurs when TR
units are deleted or duplicated during lytic replication and is a stochastic process for each infected cell (Raab-Traub and Flynn, 1986, Cell 47, 883-889). No laddering is present for BC-l which is under tight latent KSHV replication control (Moore et al., 1996, J. Virol. 70, 549-558). KS lesion DNA
also shows a single hybridizing band suggesting that virus in KS tumor cells may be of monoclonal origin.

Features and codinq reqions of the KSHV LUR

The KSHV genome shares the 7 block (B) organization (B1-B7l Fig. 1) of other herpesviruses (Chee et al ., 199O, Curr . Topi cs Mi crobi ol . Irrlmunol . 154, 125-169) with sub-family specific or unique ORFs present between blocks (interblock regions (IB) a-h, Fig. 1).
ORF analysis indicates that only 79% of the sequenced 137.5 kb LUR encodes 81 identifiable ORFs which is likely to be due to a conservative assignment of ORF
positions. The overall LUR CpG dinucleotide observed/expected (O/E) ratio is 0.75 consistent with a moderate loss of methylated cytosines, but there is marked regional variation. The lowest CpG O/E ratios (c0.67) occur in IBa (bp 1-3200), in B5 (68,602-69,405) and IBh (117,352-137,507). The highest O/E ratios (>0.88) extend from B2 to B3 (30,701-47,849), in IBe (67,301-68,600), and in B6 (77,251-83,600). Comparison to the KS5 sequence (Moore et al., 1996, J. Virol. 70, 549-558) shows a high sequence conservation between these two strains with only 21 point mutations over the comparable 20.7 kb region (0.1%). A frameshift within BC-1 ORF 28 CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 (positlon 49,004) compared to KS5 ORF 28 was not resolvable despite repeated sequencing of KS5 and PCR
products amplified from BC-l Two additional frameshifts in noncoding regions (bp 47,862 and 49,338) are also present compared to the KS5 sequence.

Several repeat regions are present in the LUR (Fig.
1). A 143 bp sequence is repeated within ORF K11 at positions 92,678-92,820 and 92,852-92,994 (waka/jwka).
Complex repeats are present in other regions of the genome: 2 0 and 30 bp repeats in the region from 24,285-24,902 (frnk), a 13 bp repeat between bases 29,775 and 29,942 (vnct), two separate 23 bp repeat stretches between bases 118,123 and lI8, 697 (zppa), and 15 different 11-16 bp repeats throughout the region from 124,527 to 126,276 (moi). A complex A-G
rich repeat region (mdsk) begins at 137,099 and extends into the unsequenced gap.

Conserved ORFs with similar genes found in other herpesviruses are listed in Table 1, along with their polarity, map positions, sizes, relatedness to HVS and EBV ORFs, and putative functions. Conserved ORFs coding for viral structural proteins and enzymes include genes involved in viral DNA replication (e.g., DNA polymerase (ORF 9)), nucleotide synthesis (e.g., dihydrofolate reductase (DHFR, ORF 2), thymidylate synthase (TS, ORF 70)), regulators of gene expression (R transactivator (LCTP, ORF50)) and 5 conserved herpesvirus structural capsid and 5 glycoprotein genes.

Several genes that are similar to HVS ORFs also have unique features. ORF 45 has sequence similarity to nuclear and transcription factors (chick nucleolin and yeast SIR3) and has an extended acidic domain typical for transactivator proteins between amino acids 90 and W O 98/04576 PCTrUS97/13346 115. ORF73 also has an extended acidic domain separated into two regions by a glutamine-rich sequence encoded by the moi repeat. The first region consists almost exclusively of aspartic and glutamic acid residue repeats while the second glutamic acid rich region has a repeated leucine heptad motif suggestive of a leucine zipper structure. ORF 75, a putative tegument protein, has a high level of similarity to the purine biosynthetic enzyme of E.
coli and D. melanogaster N-formylglycinamide ribotide amidotransferase (FGARAT).

ORFs K3 and K5 are not similar to HVS genes but are similar to the major immediate early bovine herpesvirus type 4 (BHV4) gene IE1 (12 and 13 identity respectively) (van Santen, 1991, J. Virol.
65, 5211-5224). These genes have no significant similarity to the herpes simplex virus I (HSV1~ aO
(which is simi~ar to BHV4 IE1), but encode proteins sharing with the HSV1 ICP0 protein a cysteine-rich region which may form a zinc finger motif (van Santen, 1991, J. Virol. 65, 5211-5224). The protein encoded by ORF K5 has a region similar to the nuclear localization site present in the late form of the BHV4 protein. ORF K8 has a purine binding motif ~GLLVTGKS) in the C-terminus of the protein which is similar to a motif present in the KSHV TK (ORF21)(Moore et al ., 1996, J. Virol. 70, 549-558).

No KSHV genes with similarity to HVS ORFs 1 t 3, 5, 12, 13, 14, 15, 51 and 71 were identified in the KSHV LUR
sequence. HVS ORF 1 codes for a transforming protein, - responsible for HVS-induced in vitro lymphocyte transformation (Akari et al., 1996, Virology 218, 382-388) and has poor sequence conservation among HVS
strains (Jung and Desrosiers, 1991, ~. Virol. 65, 6953-6960; Jung and Desrosiers, 1995, Molec. Cell~lar W O 98/04576 PCTrUS97/13346 Biol. 15, 6506-6512~. Functlonal KSHV genes similar to this gene may be present but were not identifiable by sequence comparison. Likewise, no KSHV genes similar to EBV latency and transformation-associated proteins (EBNA-1, EBNA-2~ EBNA-LP, LMP-1~ LMP-2 or gp350/220) were found despite some similarity to repeat sequences present in these genes~ KSHV also does not have a gene similar to the BZLF1 EBV
transactivator gene.
Several sequences were not given ORF assignments although they have characteristics of expressed genes.
The sequence between bp 90,173 and 90,643 is similar to the precursor of secreted glycoprotein X (gX), encoded by a number of alphaherpesviruses (pseudorabies~ EHV1), and which does not form part of the virion structure. Like the cognate gene in EHVl, the KSHV form lacks the highly-acidic carboxy terminus of the pseudorabies gene.
Two polyadenylated transcripts expressed at high copy number in BCBL-1 are present at positions 28,661-29,741 (T1.1) in IBb and 118,130-117,436 (T0.7) in IBh. T0.7 encodes a 60 residue polypeptide (ORF
K12, also called Kaposin) and T1.1 (also referred to as nut-1) has been speculated to be a U RNA-like transcript.

Cell cYcle requlation and cell siqnalinq Proteins A number of ORFs which are either unique to KSHV or shared only with other gammaherpesviruses encode genes similar to oncoproteins and cell signaling proteins.
ORF 16, similar to EBV BHRF1 and HVS ORF16, encodes a functional Bcl-2-like protein which can inhibit Bax-mediated apoptosis. ORF 72 encodes a functional cyclin D gene, also found in HVS (Nicholas et al ., W O 98/04576 PCTrUS97/13346 1992, Nature 355~ 362-365)1 that can substitute for human cyclin D in phosphorylating the retinoblastoma tumor suppressor protein.

KSHV encodes a functionally-active IL-6 (ORF K2) and two macrophage inflammatory proteins (MIPs) (ORFs K4 and K6) which are not found in other human herpesviruses. The vIL-6 has 62~ amino acid similarity to the human IL-6 and can substitute for human IL-6 in preventing mouse myeloma cell apoptosis.
Both MIP-like proteins have conserved C-C dimer signatures characteristic of ~-chemokines and near sequence identity to human MIP-1~ in their N-terminus regions. vMIP-I (ORF K6) can inhibit CCR-5 dependent HIV-1 replication. An open reading frame spanning nucleotide numbers (bp) 22,529-22,185 (vMIP-III) has low conservation with MIP 1~ (BLASTX poisson p=0.0015) but retains the C-C dimer motif. ORF K9 (vIRF1) encodes a 449 residue protein with similarity to the family of interferon regulatory factors (IRF) (David, 1995, Pharmac. Ther. 65, 149-161). It has 13.4~ amino acid identity to human interferon consensus sequence binding protein and partial conservation of the IRF
DNA-binding domain. Three additional open reading frames at bp 88,910-88,410 (vIRF2), bp 90,541-89,600 (vIRF3) and bp 94,127-93,636 (vIRF4) also have low similarity to IRF-like proteins ~p > 0.35). No conserved interferon consensus sequences were found in this region of the genome.
Other genes encoding signal transduction polypeptides, which are also found in other herpesviruses, include a complement-binding protein (v-CBP, ORF 4), a neural cell adhesion molecule (NCAM)-like protein (v-adh, ORF
K14) and an IL8 receptor (ORF 74). Genes similar to ORFs 4 and 74 are present in other rhadinoviruses and ORF 4 is similar to variola B19L and D12L proteins.

W O 98/04576 PCTrUS97/13346 O~F K14 (v-adh) is similar to the rat and human OX-2 membrane antigens, various NCAMs and the poliovirus receptor-related protein PRR1. OX-2 is in turn similar to ORF U85 of human herpesviruses 6 and 7 but there is no significant similarity between the KSH~
and betaherpesvirus OX-2/NCAM ORFs. Like other immunoglobulin family adhesion proteins, v-adh has V-like, C-like, transmembrane and cytoplasmic domains, and an RGD binding site for fibronectin at residues lC 268-270. The vIL-8R has a seven transmembrane spanning domain structure characteristic of G-protein coupled chemoattractant receptors which includes the EBV-induced EBI1 protein (Birkenbach et al., 1993, Virol. 67, 2209-2220).
DISCUSSION

The full-length sequence of the KSHV genome in BC-1 cells provides the opportunity to investigate molecular mechanisms of KSHV-associated pathogenesis.
The KSHV genome has standard features of rhadinovirus genomes including a single unique coding region flanked by high G+C terminal repeat regions which are the presumed sites for genome circularization. In addition to having 66 conserved herpesvirus genes involved in herpesvirus replication and structure, KSHV is unique in encoding a number of proteins mimicking cell cycle regulatory and signaling proteins.
Our estimated size of the BC-1 derived genome (210 kb including the duplicated portion) is consistent with that found using encapsidated virion DNA ~Zhong et al., 1996, Proc. Natl. Acad. Sci. USA 93, 6641-6646).
Genomic rearrangements are common in cultured herpesviruses (Baer et al., 1984, Nature 310, 207-211;
Cha et al ., 1996, J. Virol . 70 , 78-83). However, the W O 98/04576 PCT~US97/13346 genomic duplication present in the BC-1 KSHV probably did not arise during tissue culture passage. TR
hybridization studies indicate that this insertion of a duplicated LUR fragment into the BC-1 TR is also present in KSHV from the independently derived HBL-6 cell line (Gaidano e t al., 1996, Leukemi a 10, 1237-40).

Despite this genomic rearrangement, the KSHV genome is well conserved within coding regions. There is less than 0.1~ base pair variation between the BC-1 and the 21 kb KS5 fragment isolated from a KS lesion~ Higher levels of variation may be present in strains from other geograpnic regions or other disease conditions.
Within the LUR, synteny to HVS is lost at ORFs 2 and 70 but there is concordance in all other regions conserved with HVS. Several conserved genes, such as thymidine kinase (TK) (Cesarman et al., 1995, Blood 86, 2708-2714), TS and DHFR (which is present in HVS, see Albrecht et al ., 1992; J. Virol . 66 , 5047-5058, but not human herpesviruses), encode proteins that are appropriate targets for existing drugs.

Molecular mimicry by KSHV of cell cycle regulatory and signaling proteins is a prominent feature of the virus. The KSHV genome has genes similar to cellular complement-binding proteins ~ORF 4), cytokines (ORFs K2, K4 and K6), a bc1-2 protein (ORF 16), a cytokine transduction pathway protein (K9), an IL-8R-like protein (ORF74) and a D-type cyclin (ORF72).
Additional regions coding for proteins with some similarity to MIP and IRF-like proteins are also present in the KSHV genome. There is a striking parallel between the KSHV genes that are similar to cellular genes and the cellular genes known to be induced by EBV infection. Cellular cyclin D, CD21/CR2, bc1-2, an IL-8R-like protein (EBI1), IL-6 CA 0226ll64 l999-0l-22 W O 98/04576 PCTrUS97/13346 and adhesion molecules are upregulated by EBV
infection (Birkenbach et al.~ 1993~ J~ Virol. 67, 2209-2220i Palmero et al., 1993, Oncogene 8, 1049-1054; Finke et al., 1992i B700d 80r 459-469;
Finke et al., 1994, Leukemia & Lymphoma 12, 413-419;
Jones et al., 1995, J. Exper. Med. 182, 1213-1221).
This suggests that KSHV modifies the same signaling and regulation pathways that EBV modifies after infection, but does so by introducing exogenous genes from its own genome.

Cellular defense against virus infection commonly involves cell cycle shutdown, apoptosis (for review, see Shen and Shenk, 1995, Curr. Opin. Genet. Devel. 5~
105-111) and elaboration of cell-mediated immunity (CMI). The KSHV-encoded v-bc1-2, v-cyclin and v-IL-6 are active in preventing either apoptosis or cell cycle shutdown (Chang et al., 1996, Nature 382, 410).
At least one of the ~-chemokine KSHV gene products, v-MIP-I, prevents CCR5-mediated HIV infection of transfected cells. ~-chemokines are not known to be required for successful EBV infection of cells although EBV-infected B cells express higher levels of MIP-1~ than normal tonsillar lymphocytes (Harris et al., 1993, 151, 5975-5983). The autocrine dependence of EBV-infected B cells on small and uncharacterized protein factors in addition to IL-6 (Tosato et al., 1990, J. Virol. 64, 3033-3041) leads to speculation that ~-chemokines may also play a role in the EBV life cycle.

KSHV has not formally been shown to be a transforming virus and genes similar to the major transforming genes of HVS and EBV are not present in the BC-1 strain KSHV. Nonetheless, dysregulation of cell proliferation control caused by the identified KSHV-encoded proto-oncogenes and cytokines may W 098/04576 PCT~US97/13346 contribute to neopiastic expansion of virus-infected cells. Preliminary studies suggest that subgenomic KSHV fragments can transform NIH 3T3 cells. If KSHV
replication, like that of EBV, lnvolves recombination of TR units (Raab-Traub and Flynn, 1986, Cell 47, 883-889), a monomorphic TR hybridization pattern present in a KS lesion would indicate a clonal virus population in the tumor. This is consistent with KS
being a true neoplastic proliferation arising from single transformed, KS-infected cell rather than KSHV
being a "passenger virus". Identification of KSHV
genes similar to known oncoproteins and cell proliferation factors in the current study provides evidence that KSHV is likely to be a transforming virus.

W 098/0~576 PCTAUS97/13346 EXPERIMENTAL DETAILS SECTION II:

MOLECULAR MIMICRY OF HUMAN CYTOKINE AND CYTOKIN~
RESPONSE PATHWAY GENES BY KSHV

Four virus genes encoding proteins similar to two human macrophage inflammatory protein (MIP) chemokines, arl IL-6 and an interferon regulatory factor (IRF or ICSBP) polypeptide are present in the genome of Kaposi's sarcoma-associated herpesvirus (KSHV~. Expression of these genes is inducible in nfected cell lines by phorbol esters. vIL-6 is functionally active in B9 cell proliferation assays.
It is primarily expressed in KSHV-infected hematopoietic cells rather than KS lesions. vMIP-I
inhibits replication of CCR5-dependent HIV-1 strains in vitro indicating that it is functional and could contribute to interactions between these two viruses.
Mimicry of cell signaling proteins by KSHV may abrogate host cell defenses and contribute to KSHV-associated neoplasia.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus related to Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS). It is present in nearly all KS lesions including the various types of HIV-related and HIV-unrelated KS (Chang et al., 1994, Science 265, 1865-1869; Boshoff et al., 1995, Lancet 345, 1043-1044; Dupin et al., 1995, Lancet 345, 761-762; Schalling et al., 1995, Nature Med. 1, 707-708). Viral DNA preferentially localizes to KS
tumors (Boshoff et al., 1995, Nature Med. 1, 1274-1278) and serologic studies show that KSHV is specifically associated with KS. Related iymphoproliferative disorders frequently occurring in patients with KS, such as primary effusion lymphomas WO 98/04576 PCTrUS97113346 (PEL), a rare B cell lymphoma, and some forms of Castleman's disease are also associated with KSHV
infection (Cesarman et al., 1995, New Eng. J. Med.
332, 1186-1191; Soulier et al.i 1995, Blood 86, 1276-1280). Three KSHV-encoded cytokine-like polypeptides and a polypeptide similar to interferon regulatory factor genes have now been identified.
Paradoxically, while cytokine dysregulation has been proposed to cause Kaposi's sarcoma (Ensoli et al.~
1994, Nature 371, 674-680j Miles, 1992, Cancer Treatment & Research 63, 129-140), in vitro spindle cell lines used for these studies over the past decade are uniformly uninfected with KSHV (Ambroziak et al., Science 268, 582-583; Lebbé et al., 1995, Lancet 345, 1180).

To identify unique genes in the KSHV genome, genomic sequencing ( see METHODS) was performed using Supercos-l and Lambda FIX II genomic libraries from BC-l, a nonHodgkln's lymphoma cell line stably infected with both KSHV and EBV (Cesarman et al., 1995, Blood 86, 2708-2714). The KSHV DNA fragments KS330Bam and KS631Bam (Chang et al., 1994, Science 265, 1865-1869) were used as hybridization starting points for mapping and bi-directional sequencing.
Open reading frame (ORF) analysis (see METHODS) of the Z6 cosmid sequence identified two separate coding regions (ORFs K4 and K6) with sequence similarity to ~-chemokines and a third coding region (ORF K2) similar to human interleukin-6 (huIL-6); a fourth coding region (ORF K9) is present in the Z8 cosmid insert sequence with sequence similarity to interferon regulatory factor (IRF) polypeptides (Figures 3A-3C).
None of these KSHV genes are similar to other known viral genes. Parenthetically, a protein with conserved cysteine motifs similar to ~-chemokine motif signatures has recently been reported in the molluscum W O 98/04576 PCT~US97/13346 contagiosum virus ~MCV) genome Neither vMIP-I nor vMIP-II has significant similarity to the MCV protein The cellular counterparts to these four viral genes encode polypeptides involved in cell responses to infection. For example, the MIP/RANTES (macrophage inflammatory protein/regulated on actlvation, normal T cell expressed and secreted) family of 8-10 kDa ~-chemoattractant cytokines (chemokines) play an important role in virus infection-mediated inflammation (Cook et al., 1995, Science 269, 1583-1585). ~-chemokines are the natural ligand for CCR5 and can block entry of non-syncytium inducing (NSI~, primary lymphocyte and macrophage-tropic HIV-1 strains in vitro by binding to this HIV co-receptor (Cocchi et al., 1995, Science 270, 1811-1815). IL-6, initially described by its effect on B cell differentiation (Hirano et al., 1985, Proc Natl Acad Sci, USA 85, 5490; Kishimoto et al., 1995, Blood 86, 1243-1254), has pleiotropic effects on a wide variety of cells and may play a pathogenic role in multlple myeloma, multicentric Castleman's disease (a KSHV-related disorder), AIDS-KS and EBV-related postransplant lymphoproliferative disease (Klein et al., 1995, Blood 85, 863-872; Hilbert et al., 1995, J
Exp Med 182, 243-248; Brandt et al., 1990, Curr Topic Mi crobi ol Immunol 166, 37-41; Leger et al., 1991, Blood 78, 2923-2930; Burger et al., 1994, Annal Hematol 69, 25-31; Tosato et al., 1993, J Clin Invest 91, 2806-2814) IL-6 production is induced by either EBV or CMV infection and is an autocrine factor for EBV-infected lymphoblastoid cells that enhances their tumorigenicity in nude mice (Tosato et al., 1990, Virol 64, 3033-3041; Scala et al., 1990, J Exp Med 172, 61-68; Almeida et al., 1994, Blood 83, 370-376).
Cell lines derived from KS lesions, although not infected with KSHV, also produce and respond to IL-6 W O 98/04576 PCT~US97113346 (Miles et al., 1990 ! Proc Natl Acad Sci US~ 87, 4068-4072i Yang et al., 1994, ~ Immunol 152, 943-955).
While MIP and IL-6 are secreted cytokines, the IRF
family of polypeptides regulate interferon-inducible genes in response to ~- or ~ -interferon cytokines by binding to specific interferon consensus sequences (ICS) within interferon-inducible promoter regions.
A broad array of cellular responses to interferons is modulated by the repressor or transactivator functions of IRF polypeptides and several members (IRF-1 and IRF-2) have opposing anti-oncogenic and oncogenic activities (Sharf et al., 1995, J Biol Chem 270, 13063-13069; Harada et al., 1993, Science 259, 971-974; Weisz et al., 1994, Internat Immunol 6, 1125-1131; Weisz et al., 1992, ~ Biol Chem 267, 25589-25596) The 289 bp ORF K6 (ORF MIP1) gene encodes a 10.5 kDa polypeptide (vMIP-I; MIP1) having 37.9~ amino acid identity (71~ similarity) to huMIP-l~ and slightly lower similarity to other ~-chemokines (Figure 3A).
ORF K4 also encodes a predicted 10.5 kDa polypeptide (vMIP-II; vMIP1~-II) with close similarity and amino acid hydrophobicity profile to vMIP-I. The two KSHV-encoded MIP ~-chemokines are separated from each other on the KSHV genome by 5.5 kb of intervening sequence containing at least 4 ORFs ( see METHODS).
Both polypeptides have conserved ~-chemokine motifs (Figure 3A, residues 17-55) which include a characteristic C-C dicysteine dimer (Figure 3A, residues 36-37), and have near sequence identity to human MIP-l~ at residues 56-84. However, the two polypeptides show only 49.0~ amino acid identity to each other and are markedly divergent at the nucleotide level indicating that this duplication is not a cloning artifact. The two viral polypeptides are more closely related to each other phylogenetically than to huMIP-l~, huMI~ or huRANTES suggesting that they arose by gene duplication rather than independent acquisition from the host genome (see Sequence alignment in METHODS).
The reason for this double gene dosage in the viral genome is unknown.

The KSHV ORF K2 (Figure 3B) encodes a hypothetical 204 residue, 23.4 kDa IL-6-like polypeptide with a hydrophobic 19 amino acid secretory signaling peptide having 24.8~ amino acid identity and 62.2~ similarity to the human polypeptide vIL-6 also has a conserved sequence characteristic for IL-6-like interleukins (amino acids 101-125 of the gapped polypeptide) as well as conserved four cysteines which are present in IL-6 polypeptides (gapped alignment residue positions 72, 78, 101 and 111 in Figure 3B). IL-6 is a glycosylated cytokine and potential N-linked glycosylation sites in the vIL-6 sequence are present at gapped positions 96 and 107 in Figure 3C. The 449 residue KSHV vIRF polypeptide encoded by ORF K9 has lower overall amino acid identity (approximately 13~) to its human cellular counterparts than either of the vMIPs or the vIL-6, but has a conserved region derived from the IRF family of polypeptides (Figure 3C, gapped residues 88-121). This region includes the tryptophan-rich IRF ICS DNA binding domain although only two of four tryptophans thought to be involved in DNA binding are positionally conserved. It is preceded by an 87-residue hydrophilic N-terminus with little apparent IRF similarity. A low degree of amino acid similarity is present at the C-terminus corresponding to the IRF family transactivator/repressor region.
The four KSHV cell signaling pathway genes show similar patterns of expression in virus-infected lymphocyte cell lines by Northern blotting (see METHODS). Whole RNA was extracted from BCP-1 (a cell - line infec~ed with KSHV alone~ and BC-1 (EBV and KSHV
coinfected, see Cesarman et al ., 1995 ~ Blood 86 , 2708-2714) with or without pretreatment with 20 ng/ml 12-o-tetradecanoylphorbol-l3-acetate (TPA, Sigma, St.
Louis MO) for 48 hours. While constitutive expression of these genes was variable between the two cell lines, expression of all four gene transcripts increased in BCP-1 and BC-1 cells after TPA induction (Figures 4A-4D). This pattern is consistent w1th expression occurring primarily during lytic phase virus replication. Examination of viral terminal repeat sequences of BCP-1 and BC-1 demonstrates that low level of virus lytic replication occurs in BCP-1 but not BC-1 without TPA induction ( see METHODS), and both cell lines can be induced to express lytic phase genes by TPA treatment despite repression of DNA
replication in BC-1. Lower level latent expression is also likely, particularly for vIL-6 (Figure 4C) and vIRF (Figure 4D), since these transcripts are detectable without TPA induction in BC-1 cells which are under tight latency control. To determine if in vitro KS spindle cell cultures retain defective or partial virus sequences that include these genes, DNA
was extracted from four KS spindle cell lines (KS-2, KS-10, KS-13 and KS-22) and PCR amplified for vMIP-I, vMIP-II, vIL-6 and vIRF sequences (see METHODS). None of the spindle cell DNA samples were positive for any of the four genes.

vIL-6 was examined in more detail using bioassays and antibody localization studies to determine whether it is functionally conserved. Recombinant vIL-6 (rvIL-6) 3~ is specifically recognized by antipeptide antibodies which do not cross-react with huIL-6 (Figures 5A-5B) (see METHODS). vIL-6 is produced constitutively in W O 98/04576 PCTrUS97/13346 BCP-1 cells and increases markedly after 48 hour TPA
induction, consistent with Northern hybridization experiments. The BC-l cell line coinfected with both KSHV and EBV only shows vIL-6 polypeptide expression after TPA induction (Figure 5A, lanes 3-4~ and control EBV-infected P3HR1 cells are negative for vIL-6 expression ~Figure 5A, lanes 5-6). Multiple high molecular weight bands present after TPA induction (21-25 kDa) may represent precursor forms of the polypeptide. Despite regions of se~uence dissimilarity between huIL-6 and vIL-6l the virus interleukin 6 has biologic activity in functional bioassays uslng the IL-6-dependent mouse plasmacytoma cell line B9 (see METHODS)~ COS7 supernatants from the forward construct (rvIL-6) support B9 cell proliferation measured by 3H-thymidine uptake indicating that vIL-6 can substitute for cellular IL-6 in preventing B9 apoptosis (Figure 6). vIL-6 supported B9 proliferation is dose dependent with the unconcentrated supernatant from the experiment shown in Figure 6 having biologic activity equivalent to approximately 20 pg per ml huIL-6.

Forty-three percent of noninduced BCP-l cells (Figure 7A) have intracellular cytoplasmic vIL-6 immunostaining (see METHODS) suggestive of constitutive virus polypeptide expression in cultured infected cells, whereas no specific immunoreactive staining is present in uninfected control P3HRl cells (Figure 7B). vIL-6 production was rarely detected in KS tissues and only one of eight KS lesions examined showed clear, specific vIL-6 immunostaining in less than 2~ of cells (Figure 7C). The specificity of this low positivity rate was confirmed using preimmune sera and neutralization with excess vIL-6 peptides. Rare vIL-6-producing cells in the KS lesion are positive for either CD34, an endothelial cell marker (Figure W098/04576 PCT~S97/13346 8A), or CD45, a pan-hematopoietic cell marker (Figure 8B)/ demonstrating that both endothelial and hematopoietic cells in KS lesions produce vIL6 It is possible that these rare vIL-6 positive cells are entering lytic phase replication which has been shown to occur using the KSHV T1.1 lytic phase RNA probe.
In contrast, well over half (65~) of ascitic lymphoma cells pelleted from an HIV-negative PEL are strongly positive for vIL-6 (Figure 7E) and express the plasma cell marker EMA (Cesarman et al., 1995, Blood 86, 2708-2714) indicating that either most PEL cells in vivo are replicating a lytic form of KSHV or that latently infected PEL cells can express hlgh levels of vIL-6. No specific staining occurred with any control tissues examined including normal skin, tonsillar tissue, multiple myeloma or angiosarcoma using either preimmune or post-immune rabbit anti-vIL-6 antibody (Figure 7E and 7F).

Virus dissemlnation to nonKS tissues was found by ex~m' ni ng a lymph node from a patient with AIDS-KS who did not develop PEL. Numerous vIL-6-staining hematopoietic cells were present in this lymph node (Figure 8C) which was free of KS microscopically.
vIL-6 positive lymph node cells were present in relatively B-cell rich areas and some express CD20 B
cell surface antigen (Figure 8D), but not EMA surface antigen (unlike PEL cells) (Cesarman et al., 1995, Blood 86, 2708-2714)~ No colocalization of vIL-6 positivity with the T cell surface antigen CD3 or the macrophage antigen CD68 was detected, although phagocytosis of vIL-6 immunopositlve cells by macrophages was frequently observed.

To investigate whether the vMIP-I can inhibit NSI
HIV-1 virus entry, human CD4+ cat kidney cells (CCC/CD4) were transiently transfected with plasmids 11~
expressing human CCR5 and vMIP-I or its reverse construct I-PIMv ( see CCR5 and vMIP-I cloning in METHODS). These cells were infected with either M23 or SF162 primary NSI HIV-1 isolates which are known to use CCR5 as a co-receptor (Clapham et al~, 1992, J
Viro~ 66, 3531-3537) or with the HI~-2 variant ROD/B
which can infect CD4+ CCC cells without human CCR5.
Virus entry and replication was assayed by immunostaining for retroviral antigen production (Figure 9). vMIP-I cotransfection reduced NSI HIV-1 foci generation to less than half that of the reverse-construct negative control but had no effect on ROD/B HIV-2 replication Molecular piracy of host cell genes is a newly recognized feature of some DNA viruses, particularly herpesviruses and poxviruses (Murphy, 1994, Infect Agents Dis 3, 137-154; Albrecht et al ., 1992, J Virol 66, 5047-5058i Gao and Murphy, 1994, J Biol Chem 269, 28539-28542i Chee et al ., 1990, Curr Top Microbiol ~mmunol 154, 125-169; Massung et al ., 1994~ Virol 201, 215-240). The degree to which KSHV has incorporated cellular genes into its genome is exceptional. In addition to vMIP-I and vMIP-II, vIL-6 and vIRF, KSHV
also encodes polypeptides similar to bc1-2 (ORF 16), cyclin D (ORF 72), complement-binding proteins similiar to CD21/CR2 (ORF 4), an NCAM-like adhesion proteln (ORF K14), and an IL-8 receptor (ORF 74). EBV
also either encodes (BHRFl/bc1-2) or induces (CR-2;
cyclin D; IL-6; bc1-2; adhesion molecules and an IL-8R-like EBI1 protein) these same cellular polypeptides (Cleary et al ., 1986, Cell 47, 19-28;
Tosato et al., 1990, J Virol 64, 3033-3041; Palmero et al., 1993/ Oncogene 8! 1049; Larcher et al ., 1995, Eur J Immunol 25, 1713-1719; Birkenbach et al., 1993, Virol 67~ 2209-2220). Thus, both viruses may modify similar host cell signaling and regulatory pathways.

CA 0226ll64 l999-0l-22 W 098/04576 PCTrUS97/13346 EBV appears to effect these changes through induction of cellular gene expression whereas KSHV introduces the polypeptides exogenously from its own genome.

Identification of these virus-encoded cellular-like polypeptides leads to speculation about their potential roles in protecting against cellular antiviral responsesO huIL-6 inhibits ~-interferon-induced, Bax-mediated apoptosis in myeloma cell lines (Lichtenstein et al ., 1995, Cellular Immunology 162, 248-255) and vIL-6 may play a similar role in infected B cells. KSHV-encoded vIRF, vbcl-2 and v-cyclin may also interfere with host-cell mediated apoptosis induced by virus infection and v-cyclin may prevent G1 cell cycle arrest of infected cells. Interference with interferon-induced MHC antigen presentation and cell-mediated immune reponse (Holzinger et al., 1993, Immunol Let 35, 109-117) by vIRF is also possible.
The ~-chemokine polypeptides vMIP-I and vMIP-II may have agonist or antagonist signal transduction roles.
Their sequence conservation and duplicate gene dosage are indicative of a key role in KSHV replication and survival.
Uncontrolled cell growth from cell-signaling pathway dysregulation is an obvious potential by-product of this virus strategy. Given the paucity of vIL-6 expressing cells in KS lesions, it is unlikely that vIL-6 significantly contributes to KS cell neoplasia.
KSHV induction of hu-IL6, however, with subsequent induction of vascular endothelial growth factor-mediated angiogenesls (Holzinger et al., 1993, Immunol Let 35, 109-117), is a possibility. vIL-6 could also potentially contribute to the pathogenesis of KSHV-related lymphoproliferative disorders such as PEL or the plasma cell variant of Castleman's disease.

CA 0226ll64 l999-0l-22 W O 98/04576 PCTrUS97/13346 The oncogenic potential of cellular cyclin and bc1-2 overexpression is well-established and these virus-encoded polypeptides may also contribute to KSHV-related neoplasia.

KSHV vMIP-I inhibits NSI HIV-1 replication in ~itro (Figure 9). Studies from early in the AIDS epidemic indicate that survival ls longer for AIDS-KS patients than for other AIDS patients, and that 93~ of US AIDS
patients surviving >3 years had KS compared to only 28~ of rem~in~ng AIDS patients dying within 3 years of diagnosis (Hardy, 1991, ~ AIDS 4, 386-391; Lemp et al., 1990, J Am Med Assoc 263, 402-406; Rothenberg et al.~ 1987, New Eng ~ Med 317, 1297-1302; Jacobson et al., 1993, Am ~ Epidemiol 138, 953-964; Lundgren et al., l99S, Am J Epidemiol 141, 652-658). This may be due to KS occuring at relatively high CD4+ counts and high mortality for other AIDS-defining conditions.
Recent surveillance data also indicates that the epidemiology of AIDS-KS is changing as the AIDS
epidemic progresses ~ ibid).

METHODS

Genomic Sequencing. Genomic inserts were randomly sheared, cloned into M13mpl8, and sequenced to an average of 12-fold redundancy with complete bidirectional sequencing. The descriptive nomenclature of KSHV polypeptides is based on the naming system derived for herpesvirus saimiri (Albrecht et al., 1992, J Virol 66, 5047-5058).

Open reading frame (ORF) analysis. Assembled sequence contigs were analyzed using MacVector (IBI-Kodak, Rochester NY) for potential open reading frames greater than 25 amino acid residues and analyzed using BLASTX and BEAUTY-BLASTX (Altschul et al., 1990, J Mol CA 0226ll64 l999-0l-22 W O 98/04576 PCTrUS97/13346 Biol 215, 403-410; Worley et alO, 1995~ Genome Res 5, 173-184i http://dot.imgen.bcm.tmc.edu:9331/seq-search/nucleic_acid-search.html) Similar proteins aligned to the four KSHV polypeptides (in italics:) included (name (species, sequence bank accession number, smallest sum Poisson distribution probability score)): (1) vMIP-I: LD78 (MIP-l~j (human, gi 127077, p=9.8xe-22), MIP-1~ (Rattus, gi 790633, p=3.3xe-20)~
MIP-l~ (Must gl 127079, p=1.7xe-19)/ MIP-l~ (Mus, gi 1346534, p=7.8xe-18), (2) vMIP-II~ LD78 (MIP-la) (human, gi 127077, p=7.1xe-23), MIP-1~ (Must gi 127079, p=8.9xe-21)~ MIP-1~ (~attus, gi 790633, p=1.2xe-20), MIP-1~ (Mus, gi 1346534, p=3.8xe-20); (3) vIL-6: 26 kDa polypeptide (IL-6) (human, gi 23835, p=7.2xe-17), IL-6 (Macaca, gi 514386, p=1.6xe-16); and (4) vIRF: ICS3P (Gallus~ gi662355, p=l.lxe-ll), ICSBP
(Mus, sp p23611~ p=l.Oxe-10)~ lymphoid specific interferon regulatory factor (Mus, gi 972949, p=2.0xe-10), ISGF3 (Mus, gi 1263310, p=8.1xe-10), IRF4 (human, gi 1272477, p=l.Oxe-9), ISGF3 (human, sp Q00978, 3.9xe-9), ICSBP (human, sp Q02556, p=2.3xe-8).

Sequence alignment. Amino acid sequences were aligned using CL~STAL W (Thompson et al., 1994, Nuc Acids Res 22, 4673-4680) and compared using PAUP 3.1.1. Both rooted and unrooted bootstrap comparisons produced phylogenetic trees having all 100 bootstrap replicates with viral polypeptides being less divergent from each other than from the human polypepides.
Northern blotting. Northern blotting was performed using standard conditions with random-labeled probes tChang et al., 1994, Science 265, 1865-1869) derived from PCR products for the following primer sets:
vMIP-I: 5'-AGC ATA TAA GGA ACT CGG CGT TAC-3' (SEQ ID
NO:4), 5'-GGT AGA TAA ATC CCC CCC CTT TG-3' (SEQ ID
NO:5); vMIP-II: 5'-TGC ATC AGC TTC TTC ACC CAG-3' (SEQ

CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 ID NO:6), 5'-TGC TGT CTC GGT TAC CAG AAA AG-3r (SEQ ID
NO:7); vIL-6: 5'-TCA CGT CGC TCT TTA CTT ATC GTG-3~
(SEQ ID NO:8), 5'-CGC CCT TCA GTG AGA CTT CGT AAC-3' (SEQ ID N0:9); vIRF~ 5'CTT GCG ATG AAC CAT CCA GG-3' (SEQ ID N0:10), 5'-ACA ACA CCC AAT TCC CCG TC-3' (SEQ
ID NO:11) on total cell RNA extracted with RNAzol according to manufacturer's instructions (TelTest Inc, Friendswood TX) and 10 ~g of total RNA was loaded in each lane. BCP-1, BC-1 and P3HRl were maintained in culture conditions and induced wlth TPA as previously described (Gao et al., 1996, New Eng ~ Med 335, 233-241). PCR amplification for these viral genes was performed using the vMIP-I, vMIP-II, vIL-6, and vIRF
primer sets with 35 amplification cycles and compared to dilutions of whole BC-1 DNA as a positive control using PCR conditions previously described (Moore and Chang, 1995, New Eng J Med 332, 1181-1185). KS
spindle cell line DNA used for these experiments was described in Dictor et al., 1996, Am J Pathol 148, 2009-2016. Amplifiability of DNA samples was confirmed using human HLA-DQ alpha and pyruvate dehydrogenase primers.

vIL-6 cloning. vIL-6 was cloned from a 695 bp polymerase chain reaction (PCR) product using the following primer set: 5'-TCA CGT CGC TCT TTA CTT ATC
GTG-3' (SEQ ID NO:12) and 5'-CGC CCT TCA GTG AGA CTT
CGT AAC-3' (SEQ ID NO:13), amplified for 35 cycles using the 0.1 ~g of BC-1 DNA as a template. PCR
product was intially cloned into pCR 2.1 (Invitrogen, San Diego CA) and an EcoRV insert was then cloned into the pMET7 expression vector (Takebe et al., 1988, Mol Cell Biol 8, 466-472) and transfected using DEAE-dextran with chloroquine into COS7 cells (CRL-1651, American Type Culture Collection, Rockville MD). The sequence was also cloned into the pM~T7 vector in the reverse orientation (~-LIv) relative to CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 the SRa promoter as a negative control, with orientation and sequence fidelity of both constructs confirmed by bidirectional sequencing us1ng dye-primer chemistry on an ABI 377 sequenator (Applied Biosystems Inc, Foster City CA).

- 15 ml of serum-free COS7 supernatants were concentrated to 1.5 ml by ultrafiltration with a Centriplus 10 filter (Amicon, Beverly MA) and 100 ~l of supernatant concentrate or l ~g of rhuIL-6 (R&D
Systems, Minneapolis MN) was loaded per each lane in Laemmli buffer. For cell ~ysate immunoblotting, exponential phase cells with and without 20 ng/ml TPA
induction for 48 hours were pelleted and 100 ~g of whole cell protein solubilized in Laemmli buffer was loaded per lane, electrophoresed on a 15~
SDS-polyacrylamide gel and im~llnohlotted and developed using standard conditions (Gao et al., 1996, New Eng ~ Med 335, 233-241) with either rabbit antipeptide antibody (1:100-1:1000 dilution) or anti-huIL-6 (1 ~g per ml, R&D Systems, Minneapolis MN).

Cell line B9. B9 mouse plasmacytoma cell line were maintained in Iscove's Modified Dulbecco's Medium (IMDM) (Gibco, Gaithersburg, MD), 10% fetal calf serum, 1~ penicillin/streptomycin, 1~ glutamine, 50 ~M
~-mercaptoethanol, and 10 ng per ml rhuIL-6 (R~D
Systems, Minneapolis, MN). 3H-thymidine uptake was used to measure B9 proliferation in response to huIL-6 or recombinant supernatants according to standard protocols (R&D Systems, Minneapolis, MN). Briefly, serial 1:3 dilutions of huIL-6 or Centriplus 10 concentrated recombinant supernatants were incubated with 2x104 cells per well in a 96 well plate for 24 hours at 37~C with 10 ~1 of thymidine stock solution (50 ~l of lmCi/ml 3H-thymidine in 1 ml IMDM) added to each well during the final four hours of incubation.

CA 0226ll64 l999-0l-22 W O9X/04576 PCTnJS97/13346 Cells were harvested and incorporated 3H-thymidlne determined using a liquid scintillation counter~ Each data point is the average of six determinations with standard deviations shown.

vIL-6 immunostaining. Immunostaining was performed using avidin-biotin complex (ABC) method after deparaffinization of tissues and quenching for 30 minutes with 0.03~ H2O2 in PBS. The primary antibody was applied at a dilution of 1:1250 after blocking with 10~ normal goat serum, 1% BSA, 0.5~ Tween 20.
The secondary biotinylated goat anti-rabbit antibody (1:200 in PBS~ was applied for 30 minutes at room temperature followed by three 5 minute washes in PBS.
Peroxidase-linked ABC (1:100 in PBS) wa~ applied for 30 minutes followed by th~ee 5 minute wa~hes in PBS.
A diamino-benzldine (DAB) chromogen detection solution (0.25~ DA~3, 0.01~ H2O2 in PBS) was applied for 5 minutes. Slides are then washed, counterstained with hematoxylin and coverslipped. Amino ethyl carbazole (AEC) or Vector Red staining was also used allowing better discrimination of double-labeled cells with Fast Blue counterstaining for some surface antigens.
For CD68, in which staining might be obscured by vIL-6 cytoplasmic staining, double label immunofluorescence was used. Microwaved tissue sections were blocked with 2~ human serum, 1~ bovine serum albumin (BSA) in PBS for 30 minutes, incubated overnight with primary antibodies and developed with fluorescein-conjugated goat anti-rabbit IgG (1:100, Sigma) for vIL-6 localization and rhodamine-conjugated horse anti-mouse IgG (1:100, Sigma) for CD68 iocalization for 30 minutes. After washing, secondary antibody incubation was repeated twice with washing for 15 minutes each to amplify staining. For the remaining membrane antigens, slides were developed first for vIL-6 and then then secondly with the cellular antigen, as well W098t04576 PCT~S97/13346 as the reverse localization (cellular antigen antibody first, anti-vIL-6 second) to achieve optlmal visualization and discrimination of both antigens. In each case, the first antibody was developed using A~C
(Sigma) with blocking solution preincubation (1~ BSA, 10~ normal horse serum, 0.5~ Tween 20 for 30 minutes) - and development per manufacturer's instructions. The second antibody was developed using the ABC-alkaline phosphatase technique with Fast Blue chromagen. Both microwaving and trypsinization resulted in poorer localization and specificity of vIL-6 immunolocalization. In cases where this was required for optimal localization of membrane antigen, these techniques were applied after vIL-6 AEC localization.
Vector-Red (Vector, Burlingame, CA) staining was used as an alternative stain to AEC to achieve optimal discrimination and was performed per manufacturer's protocol using the ABC-alkaline phosphatase technique.
Cell antigen antibodies examined included CD68 ~1:800, from clone Kim 6), epithelial membrane antigen (EMA, 1:500, Dako, Carpinteria, CA), CD3 (1:200, Dako), CD20, (1:200, Dako), OPD4 (1:100, Dako), CD34 (1:15, Dako)/ CD45 (1:400, from clone 9.4), L26 (1:100, Immunotech, Westbrook, ME) and Leu22 (1:100, Becton-Dickinson, San Jose, CA) on tissues prepared according to manufacturer~s instructions. Specific vIL-6 colocalization was only found with CD34 and CD45 in KS lesions, EMA in PEL, and CD20 and CD45 in lymph node tissues.
Immunohistochemical vIL-6 localization was performed on exponential phase BCP-1 cells with or without 48 hour TPA incu~ation after embedding in 1~ agar in saline. The percentages of positive cells were determined from cell counts of three random high power microscopic fields per slide. Lower percentages of BCP-1 cells stain positively for vIL-6 after TPA

treatment possibly reflecting cell lysis and death from lytic virus replication induction by TPA.
Immunostainlng of cells and tissues was demonstrated to be specific by neutralization using overnight incubation of antisera with 0.1 ~g/ml vIL-6 synthetic peptides at 4~C and by use of preimmune rabbit antisera run in parallel with the postimmune sera for the tissues or cell preparations. No specific staining was seen after either peptide neutralization or use of preimmune sera.

CCR5 and vMIP-I cloning. CCR5 was cloned into pRcCMV
vector (Invltrogen) and both forward and reverse orientations of the vMIP-I gene were cloned into pMET7 after PCR amplification using the following primer pairs: 5'-AGC ATA TAA GGA ACT CGG CGT TAC-3' (SEQ ID
NO:14), 5'-GGT AGA TAA ACT CCC CCC CTT TG-3' (SEQ ID
NO:15). CCR5 alone and with the forward construct (vMIP-I), the reverse construct (I-PIMv) and empty pMET7 vector were transfected into CCC/CD4 cells (CCC
cat cells stably expressing human CD4, see McKnight et al., l9g4, Virol 201, 8-18) using Lipofectamine (Gibco). After 48 hours, media was removed from the transfected cells and 1000 TCIDso of SF162, M23 or ROD/B virus culture stock was added. Cells were washed four times after 4 hours of virus incubation and grown in DMEM with 5~ FCS for 72 hours before immunostaining for HIV-1 p24 or HIV-2 gplO5 as previously described. Each condition was replicated 3-4 times (Figure 9) with medians and error bars representing the standard deviations e~pressed as percentages of the CCR5 alone foci.

CA 0226ll64 l999-0l-22 EXPERIMENTAL DETAILS SECTION III:

The following patents are hereby incorporated by reference to more fully describe the invention described herein:

1. Fowlkes, CARBOXY TERMINAL IL-6 MUTEINS ! PATENT NO.
5,565,336, ISSUED October 15, 1996;

2. Skelly et al. ~ METHOD OF MAKING CYSTEINE DEPLETED
IL-6 MUTEINS, PATENT NO. 5,545,537, ISSUED August 13, 1996;

3. Ulrich, COMPOSITION AND METHOD FOR TREATING
INFLAMMATION, PATENT NO. 5,376,368, ISSUED December 27, 1994;

4. Skelly et al ., CYSTEINE DEPLETED IL-6 MUTEINS, PATENT NO. 5,359,034, ISSUED October 25, 1994, 5. Williams, ULTRAPURE HUMAN INTERLEUKIN 6, PATENT
NO. 5,338,834, ISSUED August 16, 1994, 6. Fowlkes, CARBOXY TERMINAL IL-6 MUTEINS, PATENT NO.
5,338,833, ISSUED August 16, 1994;

7. Ulrich, COMPOSITION AND METHOD FOR TREATING
INFLAMMATION, PATENT NO. 5,300,292, ISSUED April 05, 1994;

8. Mikayama et al., MODIFIED HIL-6, PATENT NO.
5,264,209! ISSUED November 23, 1993;

9. Park, HYPERGLYCOSYLATED CYTOKINE CONJUGATFS, PATENT NO. 5,217,881, ISSUED June 08, 1993;

W 098/04576 PCT~US97/13346 10. Goldberg and Faquin, INTERLEUKIN 6 TO STIMULATE
ERYTHROPOIETIN PRODUCTION, PATENT NO 5,188, 828 ISSUED February 23t 1993;

ll. Miles et al. METHOD TO TREAT KAPOSI'S SARCOMA, PATENT NO. 5,470,824, ISSUED November 28, 1995;

12. Li and Ruben, MACROPHAGE INFLAMMATORY PROTEIN
- 3 AND -4 LIsolate d polynucleotide encoding said polypeptide~, PATENT NO. 5,504,003, ISSUED April 02, 1996;

13 ~ Gewirtz, SUPPRESSION OF MEGAKARYOCYTOPOIESIS BY
MACROPHAGE INFLAMMATORY PROTEINS [Reducing number of circulating platelets in bloodstream], PATENT NO.
5,306,709, ISSUED April 26, 1994 ;

14. Fahey et al.~ METHOD AND AGENTS FOR PROMOTING
WOUND HEALING, PATENT NO. 5,145,676, ISSUED September 8, 1992;

15. Rosen et al., POLYNUCLEOTIDE ENCODING MACROPHAGE
INFLAMMATORY PROTEIN GAMMA, PATENT NO. 5,556,767, ISSUED September 17, 1996;

16. Chuntharapai et al., ANTIBODIES TO HUMAN IL-8 TYPE A RECEPTOR, PATENT NO. 5~ 543,503, ISSUED August 06, 1996;
17. Chuntharapai et al., ANTIBODIES TO HUMAN IL-8 TYPE B RECEPTOR [A monoclonal antibody as antiin~1ammatory agent treating an inflammatory disorder], PATENT NO. 5,440,021, ISSUED August 08, 1995 ;

CA 0226ll64 l999-0l-22 W 098/04576 PCTrUS97/13346 18~ Kunkel e t al ., LABELLED MONOCYTE CHEMOATTRACTANT
PROTEIN MATERIAL AND MEDICAL USES THEREOF, PATENT NO.
5,413,778, ISSUED May 9, 1995;

19. Lyle and Kunkel~ LABELLED INTERLEUKIN-8 AND
MEDICAL USES THEREOF [Radionuclide labeled chemokines, imaging agents], PATENT NO. 5,346,686/ ISSUED
September 13, 1994;

20. Jones et al., ANTI-CANCER QUINAZOLINE
DERIVATIVES, PATENT NO. 4,564,616, ISSUED January 14, 1986i 21. DeGraw e t al ., ANTIINFLAMMATORY AND

5,10-DIDEAZA~MINOPTERINS, PATENT NO. 5,536,724, ISSUED July 16l 1996;

22. Mahan et al., IN VIVO SELECTION OF MICROBIAL
VIRULENCE GENES [Genetic engineering and expression using auxotrophic or antibiotic sensitive microorganism's chromosome], PATENT NO. 5,434,065, ISSUED July 18, 1995;

23. DeGraw et al ., 8,10-DIDEAZATETRAHYDROFOLIC ACID
DERIVATIVES [Antitumor agents], PATENT NO. 5,167,963, ISSUED December 1, 1992; and 24. Watanabe, 6, 7-DIHYDROPYRROL[3, 4-C] PYRIDO [2, 3-D]
PYRIMIDINE DERIVATIVES [STRUCTURALLY SIMILAR TO
THYMIDYLIC ACID], PATENT NO. 4,925, 939, ISSUED May 15, 1990 .

CA 0226ll64 l999-0l-22 W 098/04576 PCTrUS97113346 REFERENCES

1~ Chang, Yuan, E Cesarman, MS Pessin, F Lee, J Culpepper, DM Knowles and Patrick S Moore (1994) Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science 265, 1865-1869.

2. Moore, Patrick S and Yuan Chang (1995) Detection of herpesvirus-like DNA sequences in Kaposi's sarcoma in patients with and those without HIV infection. New Eng J Med 332, 1181-1185.

3. Cesarman, E, Yuan Chang, Patrick S Moore, JW
Said and DM Knowles (1995) Kaposils sarcoma-associated herpesvirus-like DNA sequences are present in AIDS-related body cavity based lymphomas. New Eng J Med 332, 1186-1191.

4. Cesarman, E, Patrick S Moore, PH Rao, G
Inghirami, DM Knowles and Yuan Chang ( 1995) In vitro establishment and character1zation of two AIDS-related lymphoma cell lines containing Kaposi's-sarcoma associated herpesvirus-like (KSHV) DNA sequences.
Blood 86, 2708-2714.

W098/04576 PCT~S97/13346 Table 1. KSHV Genome ORFs and their similarity to genes in other herpesviruses.

Name Pol Start Stop Size HVS HVS EBV Name EBV EBV
~Sim ~Id ~Sim ~Id K1 + 105 974 289 ORF4~ + 1142 2794 550 45.3 31.2 ** 46.4 34.0 ORF6 + 3210 6611 1133 74.1 55.2 BALF2 65.6 42.1 ORF7 + 6628 8715 695 65.0 44.7 BALF3 59.9 41.3 ORF8 + 8699 11,236 845 72.5 54.9 BALF4 62.1 42.6 ORF9 + 11,363 14,401 1012 77.6 62.1 BALF5 70.9 55.6 ORF10 + 14,519 15,775 418 50.4 26.2 ORF11 + 15,790 17,013 407 49.4 28.9 Raji LF2 44.4 27.9 K2 - 17,875 17,261 204 ORF02 - 18,553 17,921 210 65.8 48.4 K3 - 19,609 18,608 333 ORF70 - 21,104 20,091 337 79.5 66.4 K4 - 21,832 21,548 94 K5 - 26,483 25,713 257 K6 - 27,424 27,137 95 K7 + 28,622 29,002 126 ORF16 + 30,145 30,672 175 50.0 26.7 BHRF1 46.3 22.8 ORF17 - 32,482 30,821 553 60.3 42.9 BVRF2 58.8 34.3 ORF18 + 32,424 33,197 257 70.6 48.4 ORFl9 - 34,843 33,194 549 62.8 43.8 BVRFl 62.5 42.0 ORF20 - 35,573 34,611 320 59.6 42.7 BXRFl 54.7 34.6 ORF21 + 35,383 37,125 580 50.9 32.5 BXLFl 50.7 28.2 ORF22 + 37,113 39,305 730 53.9 35.1 BXLF2 48.3 26.5 ORF23 - 40,516 39,302 404 57.4 33.7 BTRF1 51.0 31.0 ORF24 - 42,778 40,520 752 65.8 45.6 BcRF1 56.4 37.7 ORF25 + 42,777 46,907 1376 80.9 65.8 BcLF1 74.8 56.8 ORF26 + 46,933 47,850 305 76.8 58.3 BDLF1 73.4 48.8 ORF27 + 47,873 48,745 290 49.6 29.6 BDLF2 43.3 19.6 ORF28 + 48,991 49,299 102 42.2 21.7 BDLF3 ORF29b - 50,417 49,362 351 41.8 17.0 BDRF1 43.3 16.3 ORF30 + 50,623 50,856 77 52.1 31.0 BDLF3.5 ORF31 + 50,763 51,437 224 63.0 43.5 BDLF4 58.9 36.4 ORF32 + 51,404 52,768 454 51.7 30.1 BGLF1 47.0 26.6 ORF33 + 52,761 53,699 312 58.6 36.4 BGLF2 52.8 32.2 ORF29a - 54,676 53,738 312 41.9 15.8 BGRF1 57.1 40.6 ORF34 + 54,675 55,658 327 58.9 42.7 BGLF3 54.8 33.0 ORF35 + 55,639 56,091 151 60.0 31.7 BGLF3.5 ORF36 + 55,976 57,310 444 49.4 31.1 BGLF4 50.0 30.2 ORF37 + 57,273 58,733 486 65.9 50.4 BGLF5 60.1 42.7 ORF38 + 58,688 58,873 61 58.6 39.7 BBLFl 52.5 23.0 ORF39 - 60,175 58,976 399 73.2 52.1 BBRF3 65.2 43.6 ORF40 + 60,308 61,681 457 51.9 28.1 BBLF2 47.1 23.3 ORF41 + 61,827 62,444 205 53.4 29.2 BBLF3 ORF42 - 63,272 62,436 278 55.8 38.9 BBRF2 52.9 33.0 ORF43 - 64,953 63,136 605 74.9 60.5 BBRFl 67.6 50.1 ORF44 + 64,892 67,258 788 75.5 61.4 BBLF4 67.8 51.1 ORF45 - 68,576 67,353 407 50.2 30.7 BKRF4 48.9 26.2 ORF46 - 69,404 68,637 255 73.0 59.5 BKRF3 69.2 54.8 ORF47 - 69,915 69,412 167 53.0 29.9 BKRF4 53.8 24.2 ORF48 - 71,381 70,173 402 47.3 24.4 BRRF2 46.1 18.8 ORF49 - 72,538 71,630 302 45.4 21.2 BRRF1 49.8 28.0 ORF50 + 72,734 74,629 631 46.5 24.9 BRLF1 41.4 19.0 K8 + 74,850 75,569 239 ORF52 - 77,197 76,802 131 50.0 33.3 BLRF2 54.6 36.9 ORF53 - 77,665 77,333 110 59.6 36.0 BLRF1 58.1 40.9 ORF54 + 77,667 78,623 318 55.0 35.5 BLLF3 53.7 32.4 ORF55 - 79,448 78,765 227 64.4 46.4 BSRFl 61.6 44.0 ORF56 + 79,436 81,967 843 62.5 44.3 BSLF1 56.6 35.4 CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 ORF57 + 82,717 83,544 275 56~9 31.5 BMLF1 45.1 22.0 K9 - 85,209 83,860 449 K10 - 88,164 86,074 696 K11 - 93,367 91,964 467 ORF58 - 95,544 94,471 357 55.9 28.7 BMRF2 50.6 25.3 ORF59 - 96,739 95,549 396 54.1 32.3 BMRF1 50.7 28.3 ORF60 - 97,787 96,870 305 79.3 64.6 BaRF1 74.8 57.3 ORF61 - 100,194 97,816 792 69.4 52.4 BORF2 64.1 43.6 ORF62 - 101,194 100,199 331 64.6 40.2 BORFl 57.7 34.7 ORF63 + 101,208 103,994 927 53.1 32.1 BOLF1 47.0 24.5 ORF64 + 104,000 111,907 2635 50.1 29.7 BPLF1 46.6 26.1 ORF65 - 112,443 111,931 170 60.4 40.3 BFRF3 49.4 27.8 ORF66 - 113,759 112,470 429 58.7 34. 7 BFRF2 50.0 28.0 ORF67 - 114,508 113,693 271 71.8 53.0 BFRFl 62.8 39.5 ORF68 + 114,768 116,405 545 64.7 45.4 BFLF1 58.3 36.2 ORF69 + 116,669 117,346 225 71.1 53.6 BFLF2 60.7 41.7 K12 - 118,101 117,919 60 K13 - 122,710 122,291 139 ORF72 - 123,566 122,793 257 53.0 32.5 ORF73 - 127,296 123,808 1162 51.2 31.8 K14 + 127,883 128,929 348 ORF74 + 129,371 130,399 342 57.8 34.1 ORF75 - 134,440 130,550 1296 54.8 36.3 BNRF1 K15 - 136,279 135,977 100 Name Function ORF4* Complement binding protein (v-CBP) **
ORF6 ssDNA binding protein (SSBP) ORF7 Transport protein ORF8 Glycoprotein B (gB) ORF9 DNA polymerase (pol) K2 vIL-6 ORF70 Thymidylate synthase (TS) K4 vMIP-II

K6 vMIP-I

ORF16 Bc1-2 ORF17 Capsid protein I

ORF19 Tegument protein I

ORF21 Thymidine kinase (TK) ORF22 Glycoprotein H (gH) ORF25 Major capsid protein (MCP) ORF26 Capsid protein II

ORF29b Packaging protein II

CA 0226ll64 l999-0l-22 W 098/04576 PCTrUS97/13346 ORF29a Packaging protein I

ORF36 Viral proteln kinase ORF37 Alkaline exonuclease (AE) ORF39 Glycoprotein M (gM) ORF40 Helicase-primase, subunit l ORF41 Helicase-primase, subunit 2 ORF43 Capsid protein III
ORF44 Helicase-primase, subunit 3 ORF45 Virion assembly protein ORF46 Uracil DNA glycosylase (UDG) ORF47 Glycoprotein ~ (gL) ORF50 Transactivator (LCTP) ORF54 dUTPase ORF5s ORF56 DNA replication protein I
ORF57 Immediate-early protein II (IEP-II) K9 vIRFl(ICSBP) Kll ORF58 Phosphoprotein ORF59 DNA replication protein II
ORF60 Ribonucleotide reductase, small ORF61 Ribonucleotide reductase, large ORF62 Assembly/DNA maturation ORF63 Tegument protein II
ORF64 Tegument protein III
ORF65 Capsid protein IV

ORF67 Tegument protein IV
ORF68 Glycoprotein Kl2 Kaposin Kl3 ORF72 Cyclin D
ORF73 Immediate-early protein (IEP) Kl4 OX-2 (v-adh) ORF74 G-protein coupled receptor ORF75 Tegument protein/FGARAT
Kl5 Legend to Table 1. Name (e.g. K1 or ORF4) refers to the KSHV ORF designationi Pol signifies polarity of the ORF within the KSHV genome; Start refers to the position of the first LUR nucleotide in the start codon; Stop refers to the position of the last LUR
nucleotide in the stop codon; Size indicates the number of amino acid residues encoded by the KSHV ORF;
HVS%Sim indicates the percent similarity of the indicated KSHV ORF to the corresponding ORF of CA 0226ll64 l999-0l-22 herpesvirus salmiri; HVS~Id indicates the percent identity of the indicated KSHV ORF to the corresponding ORF of herpesvirus saimiri~ EBV Name indicates the EBV ORF designation; EBV~Sim indicates the percent similarity of the indicated KS~V ORF to the named Epstein-Barr virus ORF; EBV~Id indicates the percent identity of the indicated KSHV ORF to the named Epstein-Barr virus ORF~ The asterisks in the KSHV Name column indicate comparison of KSHV ORF4 to HVS ORF4a (*) and HVS ORF4b (**). The entire unannotated genomic sequence is deposited in GenBank~
under the accession numbers: U75698 ~LUR), U756g9 (terminal repeat), and U75700 (incomplete terminal repeat). The sequence of the LUR (U75698~ is also set forth in its entirety in the Sequence Listing below.
Specifically, the sequence of the LUR is set forth in 5' to 3' order in SEQ ID Nos:17-20. More specifically, nucleotides 1-35,100 of the I.UR are set forth in SEQ ID NO:17 numbered nucleotides 1-35,100, respectivelyi nucleotides 35,101-70,200 of the LUR
are set forth in SEQ ID NO:18 numbered nucleotides 1-35,100, respectively; nucleotides 70,201-105,300 of the LUR are set forth in SEQ ID NO:19 numbered nucleotides 1-35,100, respectively; and nucleotides 105,301-137,507 of the LUR are set forth in SEQ ID
NO: 20 numbered nucleotides 1-32,207, respectively.

CA 0226ll64 l999-0l-22 W O g8/04576 PCTrUS97/13346 SEQUENCE LISTING
(1) ~.ENERAL INFORMATION:
(1) APPLICANT: The Trustees of Columbia Unlversity in the City of New York (ii) TITLE OF INVENTION: UNIQUE ASSOCIATED KAPOSI'S SARCOMA VIRUS
SEQUENCES AND USES THEREOF
(iii) NUMBER OF SEQUENCES: 20 (iv) CORRESPO~N~ ADDRESS:
~A~ ADDRESSEE: Cooper & Dunham LLP
(B) STREET: 1185 Avenue of the Americas (C) CITY: New York (D) STATE: New York ~E) COUNTRY: U.S.A.
~F) ZIP: 10036 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: White, John P.
(B) REGISTRATION NUMBER: 28,678 (C) REFERENCE/DOCKET NUMBER: 45185-G-PCT/JPW
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 278-0400 ~B) TELEFAX: (212) 391-0525 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 338 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Met Phe Pro Phe Val Pro Leu Ser Leu Tyr Val Ala Lys Lys Leu Phe ~ry Ala Arg Gly Phe Arg Phe Cys Gln Lys Pro Gly Val Leu Ala Leu Ala Pro Glu Val Asp Pro Cys Ser Ile Gln His Glu Val Thr Gly Ala Glu Thr Pro His Glu Glu Leu Gln Tyr Leu Arg Gln Leu Arg Glu Ile CA 0226ll64 l999-0l-22 W O 98/04576 PCTrUS~7/13346 Leu Cys Arg Gly Ser Asp Arg Leu Asp Arg Thr Gly Ile Gly Thr Leu ~er Leu Phe Gly Met Gln Ala Arg Tyr Ser Leu Arg Asp His Phe Pro ~eu Leu Thr Thr Lys Arg Val Phe Trp Arg Gly Val Val Gln Glu Leu Leu Trp Phe Leu Lys Gly Ser Thr Asp Ser Arg Glu Leu Ser Arg Thr Gly Val Lys Ile Trp Asp Lys Asn Gly Ser Arg Glu Phe Leu Ala Gly Arg Gly Leu Ala Hls Arg Arg Glu Gly Asp Leu Gly Pro Val Tyr Gly ~he Gln Trp Arg His Phe Gly Ala Ala Tyr Val Asp Ala Asp Ala Asp ~yr Thr Gly Gln Gly Phe Asp Gln Leu Ser Tyr Ile Val Asp Leu Ile Lys Asn Asn Pro His Asp Arg Arg Ile Ile Met Cys Ala Trp Asn Pro Ala Asp Leu Ser Leu Met Ala Leu Pro Pro Cys His Leu Leu Cys Gln Phe Tyr Val Ala Asp Gly Glu Leu Ser Cys Gln Leu Tyr Gln Arg Ser ~ly Asp Met Gly Leu Gly Val Pro Phe Asn Ile Ala Ser Tyr Ser Leu ~eu Thr Tyr Met Leu Ala His Val Thr Gly Leu Arg Pro Gly Glu Phe Ile His Thr Leu Gly Asp Ala His Ile Tyr Lys Thr His Ile Glu Pro Leu Arg Leu Gln Leu Thr Arg Thr Pro Arg Pro Phe Pro Arg Leu Glu Ile Leu Arg Ser Val Ser Ser Met Glu Glu Phe Thr Pro Asp Asp Phe Arg Leu Val Asp Tyr Cys Pro His Pro Thr Ile Arg Met Glu Met Ala Val (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Thr His Tyr Ser Pro Pro Lys Phe Asp Arg W 098/04~76 PCT~US97/13346 (2l INFORMATION FOR SEQ lD NO:3:
) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 10 amino acids (B~ TYPE: amino acid (D) TOPOLOGY: linear (il) MOLECULE TYPE: peptide (xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:3:
Pro Asp Val Thr Pro Asp Val His Asp Arg (2) INFORMATION FOR SEQ ID NO:4:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
lxi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: slngle (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

~2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) W O 98/04576 PCT~US97/13346 (iiii HYPOTHETICAL N
~iv) ANTI-SENSE N
(xi) SEQUENCE DESCRIPTION SEQ ID NO 6 TGCATCAGCT TCTTCACCCA G 2l ~2) INFORMATION FOR SEQ ID NO 7 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 23 base pairs (B~ TYPE nucleic acid (C) STRANDENESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N
(lV) ANTI-SENSE N
(xi) SEQUENCE DESCRIPTION SEQ ID NO 7 TG~ CG GTTACCAGAA AAG 23 (2) INFORMATION FOR SEQ ID NO 8 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 24 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY iinear (ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N
(iv) ANTI-SENSE N
(xi) SEQUENCE DESCRIPTION SEQ ID NO B

t2) INFORMATION FOR SEQ ID NO g (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 24 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single ~D) TOPOLOGY linear (ii) MOLECULE TYPE DNA (genomic) (iii) HYPOTHETICAL N

(iv) ANTI-SENSE N
(xi) SEQUENCE DESCRIPTION SEQ ID NO 9 (2) INFORMATION FOR SEQ ID NO l0 CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 (1! SEQUENCE CHARACTERISTICS~
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~S: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

(2) INFORMATION FOR SEQ ID NO:12:
(i) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 24 base pairs (Bj TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N

W 098/04576 PCT~US97/13346 (iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

(2) INFORMATION FOR SEQ ID NO:14:
(i) ~QU~ CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECVLE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

(2) INFORMATION FOR SEQ ID NO:15:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOT~ETICAL: N
(iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 801 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N

(iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 GTGCGAGGAG TCTGGGCTGC l~l~l~lGAG C-"l~l"l"lGGG GGAGCCTCCT CAGTGCTTGC 780 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35100 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

TCTGCAGTCT GGCGGTTTGC TTTCGAGGAC TATTAAGCCT TTCTCTGCTA TC~lLlC~AA 180 ATTTGTGCCC TGGAGTGATT TCAACGCCTT ACACGTTGAC ~~ ~LC~l~ AATGCATCCT 240 CAGTCACTTG TGGTCAGCAT GTTACTTTGT All~ll~lAC CTCTGGA~AT AATGTTACCG 480 TTTGGCATCT ACCA~ACGGA CGAAATGAAA CCGTGTCACA AACTAAATAC TATAATTTTA 540 CGCTGATGAG CCAAACTGAG GGGTGTTATA ~~ AA CGGGCTGTCG TCTCGCCTGT 600 CA~ATCGTAT A~l~lllllGG GCGCGTTGTG CCAATATAAC TCCAGAAACT CATACTGTAT 660 ATGCAACCAC ACGTGATGTA GTTGTAGTGA AAGAAGCA~A ATCTACACAT TTTCATATTG 780 CA 02261164 l999-0l-22 W O 98/04576 PCT~US97/13346 ~l~llllATG TTTATAGCTA CAAATGTTTT ATGCAAAATA CATTTTATGA GGTCGGATAC 1080 TTATTAAAAG CAll~l-ll'A AGTACATTAA AAGGACATTG TATAACCGTG CTACTTACAG 1140 TGGTGGACAA GATGCCTTAA AATATGGGGC AAACATTTCA TAl~lll~lA ATGAAGGATA 1500 'llllllGGTT GGTCGAGAAT ACGTGCGATA TTGTATGATT GGAGCATCTG GCCAAATGGC 1560 ACGTTTGCAT CCAACACCCA ATGAAAAACC AAATGGTAAT ~~ AAC GCTCAAACTA 2040 TGAAGAAGGC CCATCCAATT CTACTACTTC AGAAAAGGCC ACTTCCTCTA ~l~l~l~ACA 2460 GACACACAAT AAAACAACCA GTAATCCTGC CAlll~lllA ACAGATTCTG CAGATGTGCC 2640 GATTACCTTA TTTCACTATC l~ll~lllCG TTAGCCTAGA ACTTGCTCCA GTGTTAGACA 2820 CA 0226ll64 l999-0l-22 W O 98/04576 PCTrUS97/13346 l"l'll"l'~'l''l~L CTTGGCCAAT CGTGTCTCCA TGGCGCTAAA GGGACCACAA ACCCTCGAGG 3240AAAATATTGG GTCTGCGGCC CCCACTGGTC CCTGCGGGTA CCTCTATGCC TATCTGACAC 3300 CGGTGCACAA GAAAATCGAT GCAACCACAG ~"l"l.l~lGAA ATTAACTTCA TACCACAGGG 3480 AAAAGTTATG TCGAGAGAGC CGAGAGCTGT TTGGATTTTC AAC~llL~ll GAGCAACAAC 3600 TGTATGACGA GGA'L-l'~ll"l' GGTCCAAGTC GCGCCCAAGA ACTATGTAGG TTTTACAACC 3840 AGGCCTTGCA TATTGGCGCC CAG~L~ll"lG CGGCCAACTC TGTGCTCTAC CTGACCAGAG 4260 ACGCGTCCTC TTTCTCCCCA CATCTCCTGG CAAGGATGTG TTACTATCTG CA~l"l~"l"lGC 4500 CACCTAGTCA AATGTGTGAC ~L~l~lCAGG GGCAATGTCC AGCTGTATGC ATCAACACGC 4620 'L~Lll'ACAG GATGAAGGAC AGGTTCCCAC ~l'~'l'l~'l'~l'C AAACGTTAAG AGAGACCCAT 4680ATGTGATCAC GGGCACAGCG GGAACGTACA ATGACCTAGA GATTCTCGGA AACTTTGCCA 4740 CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 CAAAAAATCC CATACTACCA G~l~l~lCGG GGGAACACCT AACGGAGTTA TGTAATTATG 5640 TCGCCGGCAG ~l~l~lAACC ATTGTACAGT CAACACTGAA GCAAGCTGTT TCCACCAACG 5940 GGAACACAAA C~lClllCAC TGTGCAAACC TGGGATACTT CTCGGGGAGA GGGGTGGACA 6060 TTGGGGACGA GGTC~ A TTACTGAGCA CAGTGGGCCA GGCGGGGGTG CCATGGACGG 6420 CCGAGGGTGT GGCCTCGGTC ATCCAGGACA TAATAGATGA TTGCGAGTTA CA~lll~lGG 6480 CGGGCGTCCC AAGCCTGACA GTGGGTAAAA AACGAAAAAT CGCATCCCTG ~lCl~lGACC 6600 TGGATTTGTA ~ll~l~l'ACC CGTAACGATG GCAAAGGAAC TGGCGGCGGT CTATGCCGAT 6660 GTGTCAGCCC TAGCCATGGA CW ~l~l~ll CTTAGTTACG CAGACCCGGC AACACTGGAC 6720 CAGA,~GAGCA lllllCCCTC CTGCATTTGG ACACAACATG CAACAAGCAA CCGGAGCGTT 6960 CA 0226ll64 l999-0l-22 TGTACCCCCG CATGCGGTGA GCCTGTCCGG GGC~l~lllG AGAACGAGCT AAAACAGCTC 7320 llllllGACT GCTTTCGCCC AGACTCCCTA GAAACCCTTT TCTGTGGTGG l~lllllAGC 7680 CTGGCAGCTG GTCAGCTAAA TTTGGGCA~A l~llC~ACTG AAAGTTGCCA ATCCGAGGCC 7860 AGACACCAGG GCCACATCCT GTCTCAGACC CTGGGTCTAA GACTGTGGGG ~l~l~l~ATC 8040 AALl~l~l~l ACTGCCAGCG TCTGGGGCGG GAACACGTAG AGATCCTGAC ACTGGAGTTC 8220 AGAGAGCTGG TATTATCGGT ll~ AC AACAGAACTT GGGAGAGGGA GCTAAAAATA 8520 GGGCTGTACC TAACATTTGA GACATCTGCG CC~ll~l~l TGGTGGATAA AAAATATGGC 8640 Al~llC~l~l AAGGCCCCCA CCAAACCTGG TGAGGAAGCA TCTGGTCCTA AGAGTGTGGA 8880 CTTTTACCAG TTCAGAGTGT GTAGTGCATC GATCACCGGG GAGLlllllC GGTTCAACCT 8940 CA 0226ll64 l999-0l-22 ~ GACC TCGGATATCA ACACCACGCT AAACGCCAGC AAGGCCAAAC TGGCGAGCAC 9840 AGCCAGTACC GCTGCTGCCG GCGGCGGGGG GTCCACGGAC AAC~ l ACACGCAGCT 10080 CCCC~CCAGC GTTATGACAG CCATCTACGG TCGACCTGTA TCCGCCAAGT TCGTAGGAGA 10260 CCTCAGAACC AATAGTAAGG AC~l ~'l'~'l"l'A CGCGCGCCCC CTGGTGACGT TTAAGTTTTT 10380 CATAGATATG AACAAGGAGC G~llC~lAAG GGACTTGTCG GAGATAGTGG CGGACCTGGG 10800 CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 GAGGCAGAAG GCGGATGATC TGAAAAAAAG TACACCCTCG ~~ AGC GTACCGCAAA 11160 GGAAACGGGG GAGTGACAGT GGATTCGAGG TTAll~lllG ATGTAAATTT AGGAAACACG 11280 GCGCGCGTAC GACACACAAC AATATGCTGT GCAAAAAATA ACC~l~lCAT CCAGTCCGAT 12000 GATGCGAACG CTTAGCGACC GCCTAACAAC CTGTGGGTGC GAG~l~lllG AGTCCAATGT 12060 GGACGCCATT AGGCGCTTCG TGCTGGACCA CGG~ll~lCG ACATTCGGGT GGTACGAGTG 12120 CATGATTATA CAAATCTCGT ~~ ACA CACAGTCGGC AACGATAAAC CGTACACCCG 12360 AATATCGGGG ATCGTCCCCA TAGACATGTA CCAGGTTTGC AGGGAAAAGC TGA~l~l~lC 12720 CTACTGTGTT ATTGACTCGG TCCTGGTTAT GGAI~ll~lG CTACGGTTTC AGACCCATGT 12900 W 098/04576 PCTrUS97/13346 GAGA~AAGAA ATAAGAAAGA CCCTGGCATC ATGCACGGAC CCCGCACTGA AAACTATTCT 13380 TGCCTCTGGC ATACTGCCTT GCCTA~ACAT AGCGGAGACC GTGACACTAC AAGGGCGAAA 13500 GATGCTGGAG AGATCTCAGG C~~ AGA GGCCATCTCG CCGGAACGCC TAGCGGGTCT 13560 CGACAAGGTT CTGATGAAGG GCGTAGACCT CATTAGGAAA ACAGCCTGTC ~llll~lCCA 13860 GATACACGAC AGAATCCCCT AC~l~llCGT CGACGCCCCA GGTAGCCTGC GCTCCGAGCT 14220 GCAGACAGAG GCAACGTTCA TCCTAGGTGA CTGGGAGATA ACG~l~l~l~ ACTGCCGGTT 14580 AAATATTGAG GAl~l~llAA CCCATGGGTC ATGCGTCGCC GTAGTGGCCG ACGCAAACGC 14760 CACAGGCGGC AACGCGCGAC GCAlC~.CGC GCCTGGCGTG ATAAACAATT TTTCAGAACC 14820 ATACTTTCAC A~lC~l~lCC CG~l~l~lll TTTGGACCTC CTGACATTCG AGTCCATTGG 15120 CA 0226ll64 l999-0l-22 W O 98/04576 PCTrUS97/13346 GCATGGTGCC AACGCCGGAG ACTGCGCCTT TGTCATCATG GGGCTCGCCC GTGAAACA~A 15480 GTCATCTCTA ATCGAGTACC CCTCTTACTA AGAGAACAGC ACATATGTCT CC~-llC~lGC 15960 GTTAGGATCT TGCCCACTGT TGTGGA~AGC TCCTCGAGCG TGCTGATTTT TAGACTGCGT 16140 CTGGTGCATG CCCAAGGAGT GTACCTGCGT TGCGGTAAGG AC~111~1AC ACCACACTGC 16260 ACTGGCATTC CAGTGACATC CTCGAACTTA AACCAATGCT A~lll~-lGGT AAGAAAGCCA 16380 GC~lllGATC GTAACCCGTA CAATCACGAG ACATTTGCCT GTAATGCCAA GCACTACATC 16620 CACAACAACA GCTACACGTC ~-lCC~GCCA TGCAAAGTCA CAGCCATCGT GTCAAACCAC 16740 CTTTATTGCT TTCAAATAAA ACG~l~ll~l GTCAACCTCC TCCGGGCTCA CTAGTATTGT 17100 CA 0226ll64 l999-0l-22 W O 98/~4576 PCTrUS97/13346 CGGCGAGCTT TTTAAGGCAG CTAGTCTCAT TA~ATCCTAT TAACCCGCAG TGATCAGTAT 17640 ~l~l~lGAAT GAGAAGATCC TTTTCAAACT CGGGGGCGTC CGGCAACTTG CCCCGCGTTC 17820 CGCCAGGCTT GGGGTTCACG CGGGCATACG CAGCCAAGCT ATCATGCGAG AGA~ACACGT 18120 CAGGGGGCGG G~lll~lAAC GTGCGACTTA GAACCACATT GATTCTACCC GCCAATGGTC 18360 GACAGCCCGC GGGAATCGAA AGCCATGTGC GCCGCCCCAT AACAACCATG llll~llllC 18420 ACGGGAGACA l~l~llllll CCGATCCCGA ~lllG~lATC AACCGCAACT ACACAGTA~A 18540 TGGAACCATA GCCACCCCCA GGCA~ACCCT GTGGAAGGAT ATCAACTAGA GAGGAGGGTC 18720 CCGCGGGCCC ~lC~lC~lCC TGGTTATCCC CACGGGGAAG AAlllC-lGA AGCTCGATCT 19080 CCTCTACTGC ACA~l-lG~l GATGTCGGCC GAGGTCTATA TGGAAACACT TCAACCCGCG 19140 W 098/04576 PCT~US97/13346 AAGTCAGCCG AGGCAATAGC GTCATTTCGC GCAAGGGTCG CCAGACCACG CGC~~ ~L 19440 AACTTCTATG GAC~ll-lCG AGCTCTCCTG TGCATCCACA GGCTCTAAAT CTCTCATTTC 19560 CGCGTCAGCT GCAGCCGTAG TGGCTCTATA TGC~llLl~l AGATGTGGGC ATCTCCCAAC 20280 GCCATCAACG ACAAGTCCGC CGGGTTCCAC GCACACATAA TGAlL~ l ATCGTGCGGA 20520 TTALlllllA TTAAATCCAC AATGTACGAC AATTGGTCAA ACCCCTGGCC TGTATAGTCA 20580 AAATCCCCCT CC~Ll~l~lG CGCCAGGCCG CGCCCGGCCA GGAACTCCCT GGAGCCATTT 20700 TTGTCCCATA TCTTGACTCC L~~ lGAA AGCTCCCTGG AGTCAGTACT CCCCTTCAGA 20760 AACCAAAGCA GCTCTTGCAC TACGCCTCGC CAAAACACCC G~lLlGl'GGT TAGTAAGGGA 20820 A~llC~lCGT GGGGCGTCTC AGCCCCAGTT ACCTCATGCT GAATCGAACA AGGGTCAACC 21000 AA~llllLGG CGACATACAA GCTTAAAGGT ACAAACGGAA ACATGATAGA TCCTGGAAGT 21120 TATACTCAGG ~l~Ll~lATA AACCCTCCCC AAAAGTTTAT AAAACACCGT ACGTAATACA 21240 CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 GAAAACATCA CAAGAACAAA AA~l~l~l~l CTGACATTCA CATTTATTTT TACAAGACAA 21360 GGGAGATCCC ACTCCTTGGC AGGCACGTTT CACGAAACGC l~ll~l~lCG CTGGCCTTAG 21480 GCATTCTTCA GCGAGCAGTG ACTGGTAATT GCTGCATCAG ~ll~llCACC CA~l~lllCG 21600 AACCGAGACA GCA~ll~lCC GGTCTATGCC AGGACGCTCC CAGCGTGTCC CCAGATTGCA 21780 lC~l~lGlCC TCGTGTAAAT GCGAAACGGC GATGTTAGGT CAGGCGCGGT AAACAGCTCA 21900 CAAAACAACT TGACACAGGG GAAACACCAG GGGCGGCGGA G~ll~lCAAT AGTGTCCAGT 22860 AlllC~llAG ACGCGGGTTC TTGGACCCGA TGTCCCAGGT CATTAAAGTC TCAAATGGGA 22920 CA 0226ll64 l999-0l-22 W O 98/04576 PCTrUS97/13346 TCTGCCCCGC TCCCATTGGT CCGCCGGCCC GTCAATCA~A GTTTTCCGAG CCGCCATTGG 23400 TTGGCCCAGA GCGGGAACCA ATCAGCGATT AGA~llll~l TTTGATTTTT CCTATATATA 23580 TCAACCCTCC 'l''l"l'~'l"l"l"l'CC GGAAGTATAT CCATTTATGG AAATCAGCTG GGTCACTCTA 23880 ACCCCTTCGC CGGGAACGCT ATA~AAACGA GGGACAGCAG CCCCCCCTCG CGCACTGCGC 24240 GCGCGGCGGC ACGTGGGACG GAl~l~llGG ATTTACCCGT AACGAGGAGC CCCGGCAGCA 24300 AGTATTAATG ~lGlllAA~A CGTTCTACAC GTACGGCGGA CCGCATCCGT CGCAAGCACG 25020 AAACATCGTT ATCCAATATC ATTAAAAACC ACACCGA~AT TTACACAGGT AGCACGTCAC 25140 C~L~llAGTG TCACCCACTG TACACAAGGC GTGTCGTATA TGTAGTATAG GTATTTGATG 25200 AGGCGGAAGC ATATCCCGCT TCCAGCGAAC GGA~ATAAGA ATCATCCGTT CCAGCATTTA 25260 TTCA~AGAGG GCACAGAGGA TTCACATTGT TTAGAGAGAG 'l"l"l"l''l~''l''l'AG TCACCATTCC 25320 CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 GAAAACTAGA GGACACGGAT GGAAAACATA TCGCACGCGG ~~ GAA AGTCAACAGC 25560 TA~ll~llll TAATGAGGAC AGATTTGGGC ACAGGCCAGA GGGTAAAGCC CTACGTGTGC 25620 CGTCACATAT ~l-l~l'GCAC CCAAGTGGTT GTTCAACCGT l~llllllGG ATGATTTTTC 25740 CGCACCGGCT ~ GLGGG CGCGCATAGG TCGGTACGCG CTGTCCCCCT AAGTCCCGCA 25800 CG~lC~llCG GGCCCCCGTC CGGCTCGTCT CCGGATGAAC CGTCACGTTC TTTGTCTCCA 25860 ACGCGGCATA TGCCGCCAAA TGCAAACACA ATAAATATTT GGTA~AACCC AAAGAAG QG 26100 AACAGTTCAA AAAlll~llG GCGCTCCATC TCCGGCCACA GGTTAAGGCG ACTACGCCAC 26280 TGCGTGCGCG TGCGGTATAT AACGCGACAC ATTTGACAGG CCGl~lllCG AGACACTGTT 26340 TGTATGCCCT CGTTCCCCAC ~lCll-CCCTA CATATCCAGC AGATGGGTCC CTCTACACCC 26460 CGCCTTGACA GATGTCTAAC GTATTCACGG ACGCCACATG l~l~l~lATT TTCCTACATC 26640 GTTTACGCAC AGGATCAACG ~llC~lGCCC GTCCACCCCC GCG~l~l-'CG C~l~l~lllG 26760 CCCCACCCCC CGCGTACGAC AGGCGTTTCT GTGGTGCGCT TCTGGGA~AA AC~lllllCC 27000 CCCATTTCTT CCTCGACAGG '~ lAAGG TAGATAAATC CCCCCCCTTT GCGCGTCTCC 27060 TTTTGGAAGG GTCTGCG Q G ATCTGACGCC CTCGCTTGGT CAGCA~AATA ACTCCGGGTT 27240 GCTGGAACCC GTAGCAGCAG CTATTAGGCG TGTACGACAC GAGTGACCCC GCG~ lG 27360 CA 0226ll64 l999-0l-22 W O 98/04576 PCTtUS97tl3346 T~l~ll~l~l CAGATGTAAC GCCGAGTTCC TTATATGCTT ACCTGATTCT GGTCTCACCT 27540 CGCATAGTCT '~ ATTT GTCAACCAAC CAGTCAATCA CCTGTCATCG CCGCTCAGAA 28140 GCACACGTCT TCGGCCAATG CC~l~ll~GC GGGTTTGACC ACGGTTACTG ATAGGTAGAC 28200 GGTTGGTAGA AGCAAATTAT CCAATGGTCG TGTTTGGGTT l~llllGGGG TTATCTACAT 28320 CTGGATGAAC ATTAATGAAA GTTTATTAAT GTTCATCCGT All~l~lATA TGTAATTTGG 28500 TCTGGTTTTC ATTGGTGCCG CCGATTGTGG GTTGATTGCG TCG~LlllGG CAATATACCC 28800 TCAATCTCCA GGCCAGTTGT AGCCCCCTTT TATGATATGC GAGGATACTT AAC~l~l~lG 29040 CTACCTATCT GCTCATTCGT l~lllCGGTT ~''l'~l~'l~"l'~'l' CTGATTCTTA GATAGTGTTG 29160 CTGGATGTGT ATCTTATTGG TGC~ll~lGA AGCATTTTAA AATGCGTTTT AGATTGTATC 29280 TAGGCGACAA AGTGAGGTGG CATTTGTCAG AAGTTTCA~A ~lC~'l~l'AAG AACATTGGAC 29400 CA 02261l64 l999-0l-22 W 098/04576 PCT~US97/13346 TAAAGTGGTG TGCGGCAGCT GGGAGCGCTC TTTCAATGTT AAl~llllAA TGTGTATGTT 29460 GTGTTGGAAG TTCCAGGCTA ATATTTGATG TTTTGCTAGG TTGACTAACG AT~llll~ll 29520 GTAGGTGAAA GC~ll~l~lA ACAATGATAA CGGl~llllG GCTGGGTTTT TCCTTGTTCG 29580 CACCGGACAC CTCCAGTGAC CAGACGGCAA G~lllllATC CCAGTGTATA TTGGAAAAAC 29640 AATGCAACTT ACAACATAAA TAAAGGTCAA TGTTTAATCC ATATTTCCTG A~ll~l~l~l 29760 GCC~llllAC CTGTATATGC GTATGAAGCA ATCGGACCCC AGTGGTTTCG CGCTCGCGGA 30540 ACAACATAAA CATTAAATGA AcAll~ll~A AAACGTATGT TTAlllllll TCAAACAGGG 30780 GAGTAGGGTA GGAAGGGTAC GTCTAATACG TAA~l~llCG CTACTGCTTG TTCAGGAGCT 30840 GGTTCGATGT AGGGTTCGGC GTAGGAGCGT ~lll~lCCAC CGCCGCGCAT GGTGTATGCG 30960 TGGTCTCCGG TGC~l~l~l TGGATGCTCT GC'~lG~lGGA GGCGGGGGTG GGTTCAGCGG 31020 GGTGTACGTA GGTTCCTCCG TG~lC~ A TTGTCGGGAA TTGACACGGG ACCGCTGAAT 31200 TCTTTGACAT GGACAAGATA Tc~ll~l~AT ACGCCGGCTC CTCTCCTGGA AAGAGGTGTC 31380 CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 AGTAATACCA TGGATCGTAT GG~l"l~ll~l AAGCGTAGCC GTATGGTGGC GCTGGGTTTG 31500 TTAAATACGC CGGGCTCAAT ATGCTGGCCA CACCTCTGTC A~llllCAAT AGGTCGAGGC 31800 CGGCCACTGT GCCGCGTCGG CGCCCCAGGG CGCATAGTGA TAC~l~LlGA AACACGGGAC 32040 CGGTGCAGAA AATACCGTGG GACACTTGAA ATAGACCCAG TGTCCAGCCC A~ll~lVl~l 32280 CTGGTAGGTG TTCGATTGTT ATTGGAAGGG ~'ll'~'l'~'l'~AC TGGGAGATAA TCCGTCACCT 32340 ACGCCCAAGA GCTGCGTTTT ATTCATTTGG ll~l~lGCAG GATGTACAAT TTCGGTCTAA 32580 GACGCAAGGT TCCTGCGGAG GTGTGGAAGC lC~l~lACGA TGGGCTCGAG GAGATGGGCG 32700 ACGACGTTCG CATCGCCCCG GAAAACCTGG TGGACGGAAA ~''l"l"l''l''l'~l''l'l' AATCTGGGAA 32880 ATGAACACGA ACGCTGGGTG CG~ll~llCG CCCAGAAGCT TTTCATTTGC TACCTGATAG 33000 ACAACAACGC GGTTTA~ACG ACCGCGAGGA CCACCGGCAG GCAGCCAAGA ACCATAAAGT 33240 'l'~lCGC CTGGAGCAGC GCCAGTGGAT CTCGGAATGT AAGCTGCTGG TTCAGGATTT 33420 CGAATATCTC ATTAAACCTA CTGC~ A GATTTACAAA TGGTCCGGGT 'l'~lll~lGGG 33480 CA 0226ll64 l999-0l-22 W O 98l04576 PCTAUS97/13346 GTCCCAGCAG GGATACCAGG TTCAAGCGGC GGTTTGGGTG CCCTCGCGCG ACTTGCCCA~ 33720 ATACCAAGTC CATGGCGGGC GCTGTCCCTG GCGCGCCCGT ACC~ lG TGGGGAAATA 33900 TGAAGTCCCC AGGACCGCTT TC~ A GCTGAGTGAT TAGCAGGTCT AGCTTTTGAG 34080 GAACAAGCCG GTCCAGCTCT AGGGAAAGCA GGTGTGCCTT l~l~lllCGT TCTCGATTTC 34680 GCACGAGTTG GCTGCGCAGT CCAAGGGCGA CC~ ll~l TTCTTCCATG GTGGGCTTGT 34740 GAATAAACAG CAC~lll~l~CC GGGTGTGGGG CCCAGAATCT TCCCGCCTCT GTCCATCTTC 34800 G~llllllGG GTACCTTAGA TAGGACCTTT CTGATGTCAG CATTTTCTCT AGCAGTGAGA 34860 AGGCCTTGAG CTCGCTGTGA C~l L ~ ACG GTGTTGGTTG GGATCAGCTG GTGACTCAGA 35100 (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35100 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 (xij SEQUENCE DESCRIPTION: SEQ ID NO:18:

CAGTCGGCAA ~ll~l'~lGCCC TAGAGTCACC TCAAAGAATA ATCTGTGGTG TCCAAGGGGA 120 CTGCTCAGGG A'lll~'llAAC CTC'GGCCTCG GTTGGACGTA CCATGGCAGA AGGCGGTTTT 300 CTCCCTGAGG AGAGGAAACC ACTAACGGGA AA~lCl~'l'AA AAACCTCGTA CATATACGAC 480 AACGTTGGGG GTGGGTCTGG GAGGGGCACT CACTGGTGCG 'l'~'ll"l'~ATAG GCATCTCCTC 1380 ll'~'l"l''l'~AAT TA-lllC~AT CTTTAGAGCC ACAGAAGGCG ACGTGGTCGC CATTCTCACC 1500CTCTCCAGCG CCGAGTCGTT GCGGCGGGTC AGGGCGAGGG GAAGAAAGAA CGACGGGACG 1560 CCCGTCGTGA TCGAGCTTTG ~''l"l'l''l'~l''llC TTCACAGAGC TGAGAAAATT ACAATTTATC 1860 W O 98/04576 PCTrUS97/13346 AACTGGAATT CAACGGCACT A~lllll"l"lC TAAATTGGCA A~Al-'L~llG AATGTGATCA 2220 CTCTGA~AAA GAGGCAAAGT ~''Lllll'l"l'CC CCAACAAGAC A~'lL~l'GATC TCTGGAGACG 2340GCCATCGCTA TACGTGCGAG GTGCCGACGT CGTCGCAAAC TTATAACATC ACCAAGGGCT 2400 TGTTTTACCC AATGATTGTC ATGGCCGTCA AGTTTTCCAT ATCCATTGGC AACAACGAGT 2~80 GGTCCAAAGA GGCTAACGAG ACGGCGTCCC Al~ L~ll CGGTCTCCCG GATTCACTGC 2700 TCTTGGTGTT GTTTCAGATG TTGGTGGCAC A~ll'l~'l'l~'l TGCGCGGGGC ATTACCGAGC 3000 ACCGATTTGT GGAGGTGGAC TGC~L~l~lC GGCAGTATGC GGAACTGTAT 'lLl-lCCGCC 3060 GCATCTCGCG TCTGTGCATG CCCACGTTCA CCA~l~LCGG GTATAACCAC ACCACCCTTG 3120 TGTTCACATC AATGTGTACC AACATAGAGC TGGGCGAAAT GATCGCCCGC llllc~AAAc 3480 CCTTGCCTCA CGTCACGTAT ATCATCAGTT CCGAAGCACT CTCGAACGCT ~l-l~l~-lAcG 3780 CCGGCTTTAA ~llll~l~AG ATTGATAGGC ACATTCCCAT AGTCTACAAC ATCAGCACAC 3900 TGCAGTCTCT CATGTATGTC ACTAATGAAA GGGTGCAGAC CAACCl~lLl TTAGATAAGT 4020 CA 0226ll64 l999-0l-22 'l'~l~'l"l"l''l'AT TGG~'ll~'lCG GGGGTTATCT A~lll~lllA CAGACTGTTT TCCATCCTTT 4200 ATTAGACGGT CAATAAAGCG TAGATTTTTA AAAGGTTTCC TGTGCATTCT 'l''ll"l~'l'ATGG 4260 TGAGTCTTTC TAGCAGAGCG CCGAAGAACT CCCGCTCGTG T~llllCGCA GGGGCAAGTT 4560 CTGCGCCGTA CAGCGATGAG AAACACGACA CGAl~llllC CAGCCCCATG CTGCGCAGCA 4620 ACACGTGCTT CAGGAACAGG 'l'~'l"l'~'l'AGCC GGTTCAGTTT TAGCTTGGGT AGAAAAGTTA 4680TCGAGTTGTT AGCACGCTCC ATGATGGTAA CGGTGTTGAA GTCACAGACC GGG~lll~lC 4740 CGAGTCTCGG CCGCCTGAGT CCAATCATGT AGAACATAGA CGCGGCCTCG 'l"l'~'l'~''l'~'l'~l' 4800 GCTTGGAGTG CAGGTAAACG CCAAGAGATG CG~l~l~llC GCCTACGCAC AAGTGGCTTC 5160 CCGCGTAGAG TGGCAGGGTA GACGAGTCCG GAGTCCCAAA ~llllCGAAC AACAGTGGCA 5280 GCG~ lG ~lC~llGGAC GCGGCCGTTC GGTGGCGCCA GTGCAGGCCT AGTTTGCGAA 5520 ACGAAATGAA GTTTGCATTG CGGCCCAACT CGTCTAGCCT G~l~ llG TTTCGGGCAT 5640 AGATTTTCGG GATTAGGTTA CA~lllllAT ATCCCAGTAC TGCGCACTCG TGTTTGCTTT 5700 ACACGTTTAG CCCATCCTTG CTGGAGACCA CAGATGGAAA ~lll~lGGTC CAAAATACGT 6060 CA 02261164 l999-0l-22 W O 98/04576 PCTrUS97/13346 TGAAGTTTTC CAAACTGACG 'l~llll~lGG GTTCCAGCAT GTCTGACACT GTAGAGCTGC 6480 CGATGCAGTC TGCCACTGCC ATACACATGA CGA~l~l~lA GATGGCCGGT GTGCCCGGAT 6720 ACGCATGAAA ACA-l-~llCG AACTCCCAGA ACTCCAGGTA CCTGCACACT ATCCTGAACA 7200 TGG~lll~lA ACATATGGTG CACGTTAGTA GCGCGGGAAG ATACAGCGAG CGTAGCTCCC 7260 CGCCGCTGTA CCGTCGACCC A~llllCCCA A~AGAGTCCC ll~llGATGT ATAAAAGGGT 7440 GCTGCATCAT CTTATCAAGA C~ll~lAAGG TCAGCTCTGC CTGCAGGTGC GA~l~ ~G 7560 AGGCGACCTT GGAGCAACGA C-lllCCCGT ACCTCGCCAC GGAGGCCAAC CTCCTAACGC 7740 ACGCCAGAGA AGGCAGTGTC C~lllCGAAG CGCTACTGGG CGTATATACC AATGTGGTGG 7860 A~lll~llAA GTTTCTGGAG ACCGCCCTCG CCGCCGCTTG CGTCAATACC GAGTTCAAGG 7920 AGCACCACAT CGGTGCGGAG ATTGAGCTTG CGGCCGCAGA CATCGAGCTT ~l~llCGCCG 8100 CA 0226ll64 l999-0l-22 W 098/04576 PCTrUS97/13346 AACTTCGGCA CGCTCCACCC ~l~''l"l"l'ATTT TAAAGACGCT GGGCGATCCC GTCTACTCTG 8280 TAGAACATTC Allllll~lA GATAAGGCCG AGCTCATGAC AAGGGGGAAG CAGTATGTCC 8400 CCTACGCCAG CTAC~ll~lC AGGGGTGCCA ACCTCGTCAC CGCCGTTAGC TACGGAAGGG 8640 TCGCCGCCTC l~lC~l~AAG ATAGGGGATA A~lll~l~GC CATTGAAAGT TTGCAGCGCA 8820 TCC~l~llGG CCTTCACCTT CCCGTGCCCC GCTACTCGAC ATCC~l~l~'A GTCAGGGGCG 8940 ATCTACTGCA CCCAACCTCT CACCGTCTCC TCAGATTGGA GGTCCACCCC l"l~l"l"lGATT 9300 ~ll'lll~lGCA CCCCTGTCCT GGAGCGAGAG GATCGTACCG CGCCACCCAC AGAACAATGG 9360 GGGTCAACTC GGGAAAACTG GC~lll~lGA ACAGTTATCA CATGGTTAGA TTCATCTGTA 9660 CGCATATGGG GAATGGAAGC ATCCCTAAGG AGGCGCACGG CCACTACCGG A~AATCTTAG 9720 GCATGGAGGA CCTAGTCAAT AAC~ll~llA ACATTTACCA GACAAGGGTC AATGAGGACC 10020 . , CA 0226ll64 l999-0l-22 WO 98104~76 PCTAUS97/13346 TTCTGACGTG CC~lllC~lC ACCCAGGCCG CTCGCGTGAT CACAAAGCGG GACCCGGCCC 10380 CAAACTATGT CACCAGGCTC CCCAACCAGA GAAACGCGGT G~l~lllAAC GTGCCATCCA 10620 CGTGCGTGGT GTCGTGTGAT GCTTACAGTA ACGA~AGCGC AGAGCGTTTG CTCTACGACC 11340 CGCCGATGAA GAGACTATTA AAGCTCGGAA ACAAGGTGGT GTATTAGCTA ACC-ll~lAG 11820 GCTGATGAAC TGGCCGCCCT TC~GTCAAAA ATAGGGAGCG TACTGCCGCT CGGAGATTGC 11940 AACGTATATG CCCC~lllll TCAGTGGGAC AGCAACACCC AGCTAGCAGT GCTACCCCCA 12180 CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 1~9 ~ lllAGcc GAAAGGATTC CACCATTGTG CTCGAATCCA ACGGATTTGA CCTCGTGTTC 12240 TCGTGGTGCC CTGCCCTCAA ATTCTCACAA CGGCTTGAGG ATGGTGCTTT ~ ATTG 13020 AACTCCCTCA AGCATTGAGT TTGCCGAGCC C~ll~lGGCA CCTGAGGTGC TCTTCCCACA 13140 CCCGGCTGAG Al~l~lCGCG GTTGCGATGA CGCGATTTTC TGTAAACTGC CCTATACCGT 13200 GTGGGAAAAT CTGCTGGCTA l~llll~l~l GATTATCTAT GCCTTAGATC ACAACTGTCA 13440 CTCTCAGCCC CGGAGCGTGC CCTCGCCTAC CCCTTGCGAC ~l~lC~l~GG AAGATATCTA 13560 CTCACTGAGA l~l~lllllA ACCGCTAAGG GATTATACCG GGATTTA~AA CCGCCCACTG 13740 CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 ~ AG TATAGTGTAG AAAATGTATG GGGAGCGGGC ATAlllC~ll AAGGACGGTT 14640 GGAATGTGCA ~l~l~lAACA AGGACAGGAC ACTAGTGCGT CTTGCAGGTG GAAATCTTCG 15180 CGGTGGTCCG CACACACGTA ACTGACCACA TTCAGCATCT lllC~lGGGC ~llCClGAGG 15240 GC~l~lll~l GAAGCATGAA ACCCAGAATA GCCGGCAGTG CATCCTTTTT AATAAAATTC 15360 ~l~ll~ll~'l TGGGAATAAA AGGGGGCGTG TGTGCCGATC GTATGGGTGA GCCAGTGGAT 15540 AGTTCCACGG C~l~llllGC CTGTACCAGT GTCGCCAGTG CCTGGCATAC CAC~l~l~lG 15840 CA 0226ll64 l999-0l-22 W 098/04576 PCTrUS97/13346 ~ lGCTG CATCACGCCA TACCCCTGGA GCCCGAGATC ATCTTTTCCA CCTACACCCG 16440 AGGGGAGGAA AACCAACTTG C~l~llCACC TTCTGGCTTG GCGCTTAGCC TGCCTCTGTT 16560 TGCTCCCTAC TAl~l~l~lG TTTACGAACG CGGTGGCCGT CAGGAAGACG ACTGGCTGCC 17100 CGACTTGTTC ATTAACACGA AGCAGTGCGA ~lll~l-GGAC ACGCTAGAGG CCGCCTGTCG 17220 CGCAGACGCA GTTAAATCGC A~lllllAGA GGCGTGCCTA GTGTTACGGG GGCTGGCTTC 17340 G~ll~l~llA CCTGGGGGTT TTGCTATTAA AGGCCGCTAT AGGGCGTCGA AGGAGGATCT 17640 CTGCTTACAA l~-l~l~l~l'G GCTATCTTGC TGCATAAAGT CATGGGACCG TGTGTGGCTG 17880 TGGGAATTAA CGGAGAAATG ATCATGTACG TCGTAAGCCA ~l~l~lll~"l' GTGCGGCCCG 17940 TCCCGGGGCG CGATGGTATG GCGCTCATCT ACTTTGGACA ~lll~lGGAG GAAGCATCCG 18000 TGGGTCTCGA ATTCAGGAAT GTGAACCCTT ll~lllGGCT CGGGGGCGGA TCGGTGTGGC 18180 TGCTGTTCTT GGGCGTGGAC TACATGGCGT l~l~lCCGGG TGTCGACGGA ATGCCGTCGT 18240 TGGCAAGAGT GGCCGCCCTG CTTACCAGGT GCGACCACCC AGA~l~l~lC CACTGCCATG 18300 CA 0226ll64 l999-0l-22 W O 98/04576 PCTrUS97/13346 ACGACGGCAC CTTGGTGCCG TCCGTCCAAG GCACCCTGGG lC~l~llACG AAl~l~lGAC 18600 TAATGTTCTC TACGGATGCC AGTAGCATGC TGATGATCGC CACCACTATC CAl~l~lllC 18900 TTGGAGTTTC AAATAAACCG AAGTACTGCT TAAACAATCC AAACAACTGG TGC~l~llll 19020 GTGGGGCCTT GATTGAAACC AAAAAGAAAA AAGTGTGCAT TACTAGCTGC l~llGGAAGG 19080 GAAAAGCCTG AAGTTCGCGG TAGACAGAGC AGGCGTGCAG GGA~lC~l~l ~lllll~lGG 19200 GCATCTGCAA GTATGTTGAT AGGGACTCCA ATAGGCGCGG CTTTGCGGGG AC~ll~lCCT 19320 AAGGGTGCGC CATCCGTGCC ~llllGGGAC AGTGTCGCGT GAATGTCGGG GCACTCAGTT 19500 ACTATGCTAC CGAGGAGTGG ACGTGGGCTT TGACTCTGAA TAAGGATGCG CTC~llCGGG 19980 AGGCTGTAGA TGGCCTGTGT GACCCCGGAA CTTGGAAGGG 'l'~-L'l~l lCCT GACGACCCCC 20040 TTCCGTTGCT ATGGCTGCTG TTCAACGGAC CCGC~l~lll TTGTCGGGCC GACTGTTGCC 20100 CCAAACGGGA l~llll~-l-CG llc~-llAATc ATGCCCTGAA GTACACCAAG TTTCTATACG 20220 CA 0226ll64 l999-0l-22 W 098/04576 PCTrUS97/13346 GC~l~ll~lG TCACATATAT CAGCAAAATA GCATAATTGC GGGTCAGGGG ACCCACGTGG 20400 CGAGGATCTG TGTGTGAAGC AGTTTGATAG CCGCCGGGAG ~ 'lACG AGGCAATTGC 21240 CGGCCTAATG GCGGCGGTGT C~"~ AAA CAGATACTGT GGCATGGTGC ACTGCGACGT 21480 TTGTAAGCAT CTCTATAAGC C~l~llGCGT CCTCTTCCAG TGTTACCTAT CCAGTCTCGG 21720 CATCGACATG lC~lC~llGG GCTACACTCT GCTGACATGC CTGGAACTCT ATCTCGATCT 21840 GCCGCTAAAC AACCCTCTGA A~ll~llGGG TTCAGCCACC AGAGACGGAC GCCCCGAACC 21900 GTGGACCATG ACGCTTGACC TGGGACTAGA TTGCACCGGC A~AGCCCAGG CGATTCCCAT 22020 GGCAGAGTCG TTAGCGAACT GCTCCGATAA GCTA~ACTGC CCCATGTTAA A~l~l~lC~l 22140 TAGA~AGCTA CTAGAGCGAG A~lllll~AA CCATGGAGGC CACCCCCACA CCCGCGGACT 22200 l~ll"ll~lGA AGACTATCTG GTTGACACCC TGGATGGGTT AACAGTGGAT GACCAACAGG 22260 CA 0226ll64 l999-0l-22 ~l~l~lllGG TGGTGTCAGG TTACTGGACG TGGCCAGCGT GTACGCCGCC TGTTCGCAAA 22500 TGCTGGCCAC C~llllGCAC CCGGACGAGA CAAATTGTCT CGATTATGGG TTTATGCAGA 22800 GTCCGCAAAA TGGAATATTT GGCL~ CGC TGGATTTCGC GGCGAACGTC AAAACTGACA 22860 AGGCACCCGG AAAGCTGGCA CTGAAGGACT lLllLlATAG CATTTCCAAG CCTGCGGTTG 23040 AGTACGTGGG ACTTGGAAAA CTGCCCAGTG AATCTGATTA ~llGLlGGCT TATGATCAGG 23100 AATGGGAGGC ~lGlC~lCGC AAAAAGAGGA AATTAACGCC CCTTCACAAT CTTATTAGGG 23160 CGGGGAACCG CTCGACGTAG TCGTGGACTA TGACCCCATT CGC~lllLAG AAAAGGGCAT 23700 CTGACACGAG CACGTAAAAG CTGTTGCCAA CGGCCATCAT GGTGCTCAAT GA~AACAGCA 24420 CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 CCAGCATCTC TG~lll~l~l AAAAACAGGG TCGGGGTGAG GTGCTTCGCT GAGTTGCGCA 24900 TCG~l~llll TAACTTTCGC CAGGCGGCGC TGCGGCGGGA GAGCCAATCT GATGCCACTG 25140 CAGCGTTCAC GTCCACTGCC GCATTATTGT CTGGCAGGTT AAllll~lAC CCCTGGACCC 25320 AGAl~ L~'l''l"l' ~'1 ~lGl~ A CGTATGATAT CCCTATCGCA GGGCGGGTCC 'lll~l~lGCT 25860 ACCCC~l~lG TGGTTGTACG TGTTCCCGGG AGCTGTGGAA GAGGGCACAG CCTTTGCGCC 26040 GCGAACTTCT TTGGCAGATA CA~l~lllGG GTACGACTCC CTGGCCATTT CAAGGGAATG 26220 CCTGTTGCGC ACCGACTCCG TATTATCGCG GGTCTCGTCC All~l-l~AC TCGATACGCT 26400 W 098/04576 PCT~US97/13346 GTCGACTGTA TTATCATGGT CCACCTCTAG GGGTCACAAA TGGGCCGCAA TCGTGAAGTG 2652 n CCACGGCACA GGACCCATTA ACTTTCCTAT ~ -ATTT TTAGCAATGG TCTCCAGAAT 26700 GGGATCTTGT ATTCTCATCG CATGCGAACT TG~l~llllC AACCTCGATG GGATATTTCC 26820 ACCGGGCAGG ~llllCGCTT CCTGTGGATG TTAATTACCT GTTCTATTTA GAGCAGACTC 27240 TTGGTAAAGA TGGTATCACA ATAAAAAATG TTTACTGGGT CCGCGCAGGT ll~lll~lCA 27480 GTCTCTATAC CGTGCATGAC GAAGGCCGCG TCCATCCCCG GCGTCCTCTC Al~l~l~lll 27720 CTGGCGCGAC AAATAATAGA TCTCAAAAAC GTTGGTGACA l~l~lCGACA ~ll~Lc~AGc 27780 CGCTCTTGGA ACACGTGAGG GTGTAGGTCT ATGTGGGTCA CCAl~l~llC GTGCTCCACC 27900 AGGCACACCA CCGTA~ATCC CACAAAGTTG GGCGAGGACA GGCGAGATTT CACGTGCTCC 27960 ACGGGACAGG C-l~ll-lGA CTCGCGAGTC TTCGGGGCAT GAGTACTCAT TGGCACTCCA 28140 lll~"l~lCCC ACGTCGGTTA TATACACAAA GAGTCTGCTA GTCTGATATA AATAGGCCGC 28260 CGCGATCACA TACGTGAATG GACCAAGCAG GATGGATATG ~lGlC~lGAG AATAGGTGAC 28380 GCTTTCCCAC AG~-lC~lGA GAl~lllCAT G~lll~l~lC ACTGGGGGTA TGTAAGAAGA 28500 CA 0226ll64 l999-0l-22 W 098/04576 PCTrUS97/13346 GGGGTCCTGT AAACGTCCCA GAGATTGAAA L~l~llGGCG GTCAGCAGAT TCACACTCCC 28620 CTTCATGCAG AGCTCCCTCT C~Llll~'AAG TTGAGTTATT GTGTCAAATT GTTCGTTTAT 28740 C~L~l~llGG CCTGTGCCGA ATAGTTTATT ~ lACT Al~llllGGG ACACGTCGGT 28860 GACAAAGTCC TCCACGACGT CGGTGACACC GCTCACTGTC Ll~llllCTG CCAGTTTCAT 28920 GAGCAGGTTG AGGAGCTCTC GCTTGGGGTC l~ lGA GAGGCCTGCT CCAGGTGGGT 28980 GG~l~l~laC TTGACGACTG GTACCATTCC TACCGTGACC ACCCAGTCTA CGTATCTCTC 29460 ATACGAGAGC l~L~l~llGG CGTAGAGGAC CCGGTTGATG GCATTGAGAA GCAGGTGGTC 29520 TAATGTCATG CGCATAGTCT GGGCCCAGGA GTCGAAGGTT GAC~ll~l~l AAGACCCCCA 29580 CTGTGCTTCC LLllClGGCC ACCTGGTTTT TGCTGAGGAC TCGTATGTCC TCCAGTCGGA 29640 TGACCTAGAG TTCGATCCAC TGGAAGACGA AGGCCCCTTT CTGCC~llLl CGGCATACGT 30000 CAACTGCCTA ATTACGGGGG CTACAGTGGT AGCGGCACAG AAl~LllC~A GGGCTTTAAA 30120 TTTGACGGCC GTG~l~ll~l TCTATTGGTT TTACAACAGT TGGCTGGACA CCCCGCTATA 30480 CA 0226ll64 l999-0l-22 TGTGTCCACA AAGCTTTTCA CCTGCCCAGT G~l~l~lGAG ~~ ACAG AGCCATTTGA 30960 l~llll~AGG CTAAGTAACT ACTCGCAGTT TGCTGATCAG GACATGGCTG TGGTTGGGAC 31080 CGGGGTGAAG CAGGGGCATG AAGAATTCCT CAGGGACCTC AGGGAACTGC CG~l~l~lCA 31380 AGAGCTGATC TCTGAGATGA GCTCCGAGGA C~ll~lGGGG CAGGAGGGGG ACACAGATGC 31440 AGATGGCGTG GA~lll~l~l CCACTTCCCC CGGGCTCCAC GGTCTAGTGG CATACGCATC 31680 AGGTGGCGAC ~l~lllAACA TAl~ll~lGC AGGGGACTAC GGTATCAGTT CTAATCTGGC 31920 CC~lLlCCAC ACAGGGGTGG CGTGGAGGCC AGGATGCGGG TTGGGTCGCT GCACCTGGAC 32340 GGGAGGTGAC C~lll~lGCT CGACGGAGAC ACGATCACGC TCACGGCGGA CGAGGGCTCC 32460 TC~1~1~1~L CACTCCCCGA GGATATAATT ATCACGGACG CCACTGCTTT GCGGCTTAAG 32520 ~llG~ll~lC TCTGGCAGCG CACCACATCC TCGCTACCAG AGGAGGCGGT AGACTGCCTT 32580 CA 0226ll64 l999-0l-22 W O 98t04576 PCTtUS97/13346 CTGTATGGTG AA~ TCCAGGTACA CTATTTCTGG AAGCAGGTGA AAGTCCGTAT 32880 GCAGGGTACC CCTCTGGCTC GTCTTCCTCC TGAACATCGT CAllll~-llC TTCATCTTCA 33180 TCTTCCTCAT C~lC~l-ATA TTCAGATTCG CCGCTCGACT GATCCGGGGA TATCTGTAGA 33240 GGAAGCATTC TCTCTTCATC ~l~l~lGCTA GACGAGGTCC TCACAAACAT CGCCATGGCC 33480 ~ ~AA TGGCCTTGCG CCCCCACAGG AGAAAAACGC AAl~Ll~lAA CTTTGAGGAT 33780 CTCCC~ L CCACCGTCAA AA~L~l~lll AGTAGCAACA CACCCTGGCG AGCCCAGCTG 33900 TCGAGGCACC CGTGGGAAGG AGTACTGAAA TTGGGGACGG AAGCCTCTAG ~l~L~lAAAG 33960 ATG~ll.l~A AACTGGGTGG AACCTGACAT TGCGGATCCA CACTAAACGC CAGGCCAGTA 34020 AGTTCCCGAA TCTGTCTGAG CAGCGAGAGC A~lll~l~ll TCAGAAATGA TGAGAGGCTC 34200 GATAGGGTGC CCCTAAAGAC C~l~l~llGC AACCATGCGT CCATGTTGAA CTTATTTTCC 34320 AGAGCTATCT GCAGTGGTCG CGTTAAAACC TACAGTATAG GCCGTCAAAC llC~ll~LAA 34500 W O 98/04576 PCT~US97/13346 ATGGGGGAAA TGTGGCATTA CCTGACACGG TTTCAATCAT ACTCATCGTC GGAGCTGTCA 35l00 (2) INF0R~ATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35100 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xij S~:UU~N~ DESCRIPTION: SEQ ID NO:l9:

GGAGACACAG ATGATGGGTT TGAAATGTCC ATACGGGCCG TGTGCACAAG GGTCACGTCC l80 TTTATTATTT CCACAGGAAC CG~111~1~1~ AATTGCATCA CCAGGGTATC CAAAAGCCGG 360 GCTTCCACGT TGATCCGGCT TACCGACAGT TCTTTCCAGG ~l11C~1G~1 GGGGCGCGGC 420 TCTTCCAGGA CACTGGCCAT GCATGACTCC AACCG1~-~A CGTCCGAGGT AATGTGCTCT 540 ATGAAGATGT GGTAGAGCCA GCAGACGTTC AAACACGATG A~ATCAAGCT AAGCTCCCGC 600 AACTCAGAAA AAGACACTGA CCCACCAAGG AGAACCTGGC GTCTTGCA~A GTTGATGAGC 720 CCCGCAGAAA GAA1~1~1~1 CCCGTGGGAC A~AGAGCTTG GGGGGGCAGA GATGGCGCTA 780 CAGTGGGTGA 111~11CTAC CACGGTCATA CATTGGTGGC ACCCACAGGC CTGTTCCAGT 840 TGGCATTTTG CCCGCATGTA CAl11C~1~1 CCCACATATT TTAACATCTG TAATACTGGA 960 W 098/04576 PCTrUS97/13346 CGTCCCCTTT AAAAAGTCAA CCTTACTCCG CAAGGGGTAG l~l~ll~lGA GAATACTGTC 1380 TGAACAATGC ~'l~'lll'ACAA TGGTGTAGGT GGGAGCAGAG TTCGCCAAGC TCTACGTCCG 1500 GAAAGTCCTC GTAAATGACA ~~ laCCA AGAAAAACTT TTTTACCACG CTGGCCATCC 1800 ACTGAAAGGA GGGAGCACAC GTCCCGTTGT GC~ll~llAG GATATCCCTA ACTTCGGAGC 1860 GGAGACGGCC GGACGCTCCC ACAAAATGGG AGAGGCACCA ~l.l~lGCAG TCCGCGGTCT 1920 GGGGTTCTGA TTCCAGGGGC GCC~l~lGGG GGTATTGGAG AGTCAAAACT CTGGGCAGTC 1980 CCTTAATGAG ~L~l~l-lCA A~ACCTATGC AGCCAGCGTC CACTAGTGGC AGCATGCCGT 2040 TAATAACACC CCTTATCTTG TCGTTGCCAA ~'l'll~l'ACAA CTGCTGCAGG GAATAAGCCA 2100 GAATGGTCTT TGCAAGGTAT AGG~l~ll~l CAACGTTTAG AGCGGGTACG TGGCAGTCTG 2220 TAAAAACTAT GACACGCCAC TCTCTCCTTA GGGTAAGAAG CTTCGGCGGT C~l~l~lGGA 2400 AAG~llC~lC GGCCTCTCGG ACGAACTGAA GGCCCAACTC TACCAGTGTG TGCTCCTTAT 2460 .. .. . ... ..

CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 Al~llll~lC GCAGAGAACA CCGGAGATTC TCCCGACAAC CCGAGCTCTT TCCTGACGTC 3600 AGCGGGCCCT GAAACATGGG ATGTCGGGTC GC~l~lClCC CCCACTGACG ACGCGCTGTT 4200 CCCCGCTTCC TTCGGCCCGG A~l~-lCCGGC GGATATACCG TCACCTTCTG GTGGAGAGTA 4320 CGGCACACTG TACCAGCTGC ACCAATGGCG TAATTACTTC CGAGACTGAA ~l~llCGCAA 4440 TCAACGGGCA ACCATTTTTT ACGGACAATA CTGACGGTGG GGAAAACGAA ~l~-l~llGGA 4800 CAAGCTCGCT GTTGTCAACC TACGTAGGTT GCCAGCCCCC GGCCATACCG ~l~lGlGAAA 4860 CCGTGCCTGC ~lcl~lAGTT AAGGTAGGGA ACATCACCCC CCATTATGGG GAAGAACTGA 5160 W O 98/04576 PCTrUS97/13346 CAGCACAGCC CACACATGTC ~l~llll~lC ~l~lllllGT ~LClllAAAG GCCGAAGTAT 5280 CATACACAAG ACAGCTGCAG CAGGTATAGA CGGGAAACAG ~l~l~lATCT TGGCCGGCTG 5400 GTTACTCAAA TGGGAACAAT GGCGCCACCT TG~lGl~lll GTAGGCATTA GAAGAAAAGG 5460 CACCATGTTG AAGCTTGGTT GTGCCGTCGT CCGGGAGAAC CATGCCAGAC lll~l~lGGT 5640 ACCGTCGGTG TGTAGGGATA AAGCGTAACC TTAC~ll~lG TCTCATCTAC AGGATCATAT 6240 AGAAATAGTG GTGCTAGTAA CCGTGTGCCA llll~lGCCA CCACTACAAC GACTAGAGGA 6360 CCCTACATAG TGTAACACAA AACCATAAAA GTAAATAAAC ~l~lllATTG TTCACATGAT 6540 AAAGAGTGGT A~l~lll'ACT GGTTTGGGGG TTGG~ll~lG GCGTGGTGGC TGGTCCGCGG 6600 TTCAGTCATC AACCCCCGCC C~'lG'll~lCG AGGCTCCTCT TCGTCGCCTG TTATTGGCAC 6660 CAGG~GGCGG TTTAGCGGTG CCCCCGTCTG ACATGCAGAC GTCGATTCTA AGCGAAAGTC 6720 CCTTCAGGGC ATCGTCCACT TG-llll~lG TTACAACCTT GCTGAATATT GTCCTGACCC 6780 TGGCTTCGAT lll~llAGcG GCCGCCGCAC TCAGTGCACC CACAGTAGCG GTAAGCTGCG 6840 ~ TCCGAAGCTC CCGATTCTCC ACAGTCAATT GGCTTATCTT TGCGGTTAGG TCTTCCATCG 6960TAAGGTCCTT TTTGGGTCTG CCCCTGGGCG CGGCCATGTC AGGTACGCGT AGATGTACGT 7020 GGACCACCTC GTCCACAAAC TTGAAAAAAC AAAGATATAC CAGATAGA~A AATGTGGCCA 7200 , _ CA 0226ll64 l999-0l-22 W O 98/04576 PCTrUS97113346 CCAGGGTGGA ATAl~L~llC GAAACAAGCA AATTTAGAAT GACGTCGAGA GCAAATGAAG 7620 CGTGGCTGGC GCAGCCCGGT AGCCGCATAT GCCAGATTGT ~ Ll~lGGAA CGCAGACACA 8220 GCG~lll~lG 'l'C~l~l~llC CAGGGTTGAC GAAGGCCGGG GAGGGATTGA CGAATGCATC 8640 GCGGAAACGG ACGG~l~lLC GGTGGGTGGC TTGGGTAAAG TTGCCTCCGG CTGGCGCGTA 8700 GCCCATATTA CCGAGTCGTC TGACGCCATA GCAGTCGCCA ~lLlllCCAT CTCCATGAGC 8820 GAAACGCATT CCCCGGCCCT ~l''l"l'~'L'l lAAG AGGGACTGGA GCGCACTGTC GTCCACGGTA 8880 ATCTCGCCGA CCGCCAAGGC CAGCATTGTG TTCCACACGA C~ll~lGAAT AGACTGCAGT 8940 'L'l l''l''l'~ACcT GG~llll-'AC G~L~lC~lGG CAGCCCGCCG GAATTTTAGC CACGTCAAAA 9000 CGCTTCAGGT A~L~l~lGAT ~ll~'l'llGAC TGTACAGCCA GAAGGTAGGT CTGGTGCAGC 9060 GCC~lC~lGC CAAGGTTCGA CTGGACAACG TCACCCAGAC ACACTCCGGG GGGGAGGCCC 9120 AAATCTATCT CTTGCCGCCA GCG~'L-LGGA CAGCCTTCCA GAGGGTCACC GAGGCGCTTG 9180 CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 GACGACATAC CGCCGCGAGG CGCTGACAGT AAGGGTTATT ~ ~lACGA GTGGCGACAG 9300 CGCCGAGACG ATCGCCGACG TCCTTACGGG GGCCCCAACG TCAGCGTCCT T~llll~l~l 9360 ACTCCACGAC ~llllllATT CCCAGATACT CGCCCCCAGG GTAACCCTAA AATTGTGCCT 9420 G~l~l~lGCA TTTGGCGCTC AGGAAGCCCA ~llC~lCCTG ACCAGCTCAT TCTATTTTTT 9780 TGAACACACT GTG~ l'~''l'~'l'A CCACAGAGAC A~lll~lCAC ~l~l~lAGAC l~llllCGCC 9840TCAACAGGGA CAGACGCTGG TTTCCGTTAC CAGCCACGAG GAGCTGGGGC AGCTATACGG 9900 CAAACGGGCG TTCCTGCGCT ACTCTAGACA GAC~lC~l~l TCAAGTGCTC TAAGGGAGCT 10140 CCTGCCCGTC TGCCGCGACA TTGACAGGAC ATTCGAGGAG GTGCACTCTC l~l-~ll~llC 10800 CAGCACCAGC ll~l~lCGGT GTCATGACAA ACTGGGTATG CGTATTATCG TCCCGTTCCC 10980 AGAAGGAGTA TGC~lC~llG GGTCGGAGCC CATGGTGGCA CTCACTGGCA TTCTAAACAG 11040 CC~lllCGAC TGTGGCATAT ACGGCCGAGG ACGAAGCGTC CGGCTTCCCC ACTGTTACAA 11160 W 098/04576 PCTrUS97/13346 TAAAGCATAC GACACTATAT GTAAATTTTT CCCAGATGAA AAAGCACAAC A~llll~lCA 11520 CAAGTGTAAC AATAATGTTC CCACGGCCCA lllllC~lll GTGGTACCAG TGGGACTGGC 11760 CCA~ lC GACCCAGGTC CCCCTCACCA GTAAACAGGT ACGGTAAAAA AATCAAGTTT 12300 GGAACCGCCG GTCAAAACAC ACGTCCTCCC CCTGAAAAGC ~lC~lCGGCG CAGACCACGC 12360 CTCCACAGAC AGATGCTGTC llll~lGACC AGCCGCCATA ATCAAGCGTA CTGGGTGAGT 12780 TATTTGGTAG CGATGGGAAA CCGCTTAGTA GAGGCATGTA AC~ llGG CGAGGTCAAG 13080 CGCAGAAGTA TTTCTGCGCG CGGACAGGAG ~~ AGAA CACTTCTGGA ATACTACAGG 13200 CA 0226ll64 l999-0l-22 W 098/04576 PC~US97/13346 TCCATGTAAC TCACGTAGCC 1-1--1~1~1AAT AAACAAGCTA CCTGCAAACT ATACACAAAT 13440 GAAATGAGTC AGGCGTGGTC T~ lAC CGTGAATCGC ACCTTAAACA CAACACCAGA 13500 TGGAGCCAAC AAACAAGACA CACCCGCCAA 1~1111GGTC TCTTTATTGA TATGATATAC 13620 TACGCAAAAT ATGCCATTAC AAGAGCTAGT CAGCAGAATG C~1111GCAC ATGCGTCCAG 14040 TGAGATTCGA CAGCCCGACT GG~11~1C~1 CAGTAACTCA TGAACCTGTT CGCCATTATA 14220 TTCCACGCAT l~ll~LlCCT CCGGTTTACG TACTCTAAAG ACCAGAAAAT GGTGTCCATC 14460 CTGAGAAATG CCTTTGCCAA T.1.11~AA ACCCCGCGTC CTGCGTAGCG CGGCAAGCAT 14520 CGTAACTGGT ATACGGAAGC GGGTGCGCTC 11C~1.1 lCC CACTCTACTC CGGGAAATTT 14700 TGACAAACAA G~-L-1--1111GG GTATCGCCCC AGGCGCCCCA AAAGGGTTCG ~1-~1--11-GGCC 15000 GC~111~1~A AATTAACCGA AACTACATTT TTCTATTTTA AGTACGGGAT ACAAAGCAGG 15240 AATACTTTAA TTTGTGCAGG TTTCTTCACC CCACACCTGC 11111~1CTG GTACAAAAAA 15420 CTAGCCATCC TATCACCGCG GATCCGCTGG ACCAATATAC CACGCCCACT l l lc~LAATc 15660 ATCCACAATA AATGGTGCAA TAATTGGCGA AA1~1C~1~1 CTGGTTTATT TGGACTACAA 15840 GGACCTGGAC CTCGGGCGCC GCCTATCCGT GGCCTGTCTG GTTGAGGAGC TCG~11C~1C 16560 TTCGCCACTA CCCGGGCACA CACACTCCCG CCGCTCCAGC 1.1~1CGGTA AATGCGAAAC 16800 CGCCATGTTG TCCCCCTGGC AGACGTACGG TATTCCAGAC GAGGATGGCT C~1~1CGCTC 17040 CGTAGCTGGC G~'L'1'~'1GCCA ATGGACTCCA ATTGTAACAT GATGGTTTCG CATACCCGGG 17280 AGGAGCCCGG C~'1''~1'~1'AG ATGCCCCGCA AGCGCCTTCG GCACCGGTTT CCCGGCGGGG 17400 CA 0226ll64 l999-0l-22 CTGTGCGACG CCGGAGACCC CTGACACAGT ACCGGCAAGC C~l~lllCGT GCTGCGGCTT 17820 TGCCTCCTCG TAG~lll~ll CTCTAAGTAA AAGGCACGAG AGTTAACGTG GTTAGGGTAC 18180 ATAAATCGCT GCA~'lllllC AGGACCATAC GCGGCCCCAT AGCAATACGT ACA~LllLlA 18360 ACC~l~l~lC CCACGACAGG CGATGGTCCG TAGACTCTAT CACACTCCTC ATCAAATGCA 18540 GCAAAAAGAA G~L1~11C-1 ATCAAACCAG GAAAAATAGG GAAACTTATT ~Llll-CAAGG 18660 TAll~l~lGG CACAAAAAAA GCAACTTTTC ACGCACGCAG CATAAGACCC GAGCCAGTCG 19020 CGCCCTCCAT CGCGCCTGCG AATTTTCCCA CCACCCAATA TTGTGGCAGA l.lll~llAT 19080 GTATATGTGG TTACAAACAC CACGCCCCTT AAG~l~lC~l CTCTCCCAAG GGGACTAGAT 19140 GCTTGAGGTT TC~l-l~'llA TCAAACAAAC CTGACCACAA CTGTACAGAG AAAAGTGGGT 19320 W 098/04576 PCTrUS97/13346 CGAGGGGGAC CAT~TCGAAC TGTTCTGCCG ACGTTGGGTC ACCTCCGATG AACACAGTTG 19500 ~ llAAT GTGCTCATGT CCCTGTATGC GATATTGTGC CACATTA~AA ACATCCAGAA 19560 GCCGGCGTCT GGACGCCGGC GGCAGTCCAG CACCATCATC GA~ llCG TCACTTATCT 20100 TCAATGCACA TGCAGATTCT TTATCAAGCA GTGTGAGGTC ATCTTCAACG llGl~l~l~l 20400 ATGGCCTACA GA~IU~AAA CAAATATGTC AACCGGACTA GGGTGGCCAA ACCATTTGCC 20700 CCACCCCTCC CCA-l-lllC CCCAGGGGAC ACATCTTACC TTG~l~ll~l CCGATGCTTC 20760 TCGAGCCGTA CA~l~l~llG ATACAAAATT TCCCATAGTG ATGACCCACT GTGTAGGTGA 20820 ATTACGTCAT TACGGGCAGC CGGAGCA~GA AATGTTCCAG TAGATCTATC TAGCCACTTG 21120 GGTCA~AAAG CATTGCCTGT ATTAGACACA l~l~lll-lC ACTATGAATC GTGCTCTCCA 21360 ATGCATAATG AAGCCCACAT ~'l"l''l'~l'~''ll'A CTTTACTAAC CTCATTACCT TGCATTGCAG 21480 W 098/04576 PCTrUS97/13346 GCCGGCGGCC CAGAAACAGG GACGCGTACC GGGACCCTTC AG~Ll~lCGA TTATGTCGCT 21900 CCACGTCAAA AG~-Ll~llaG AL~lC~lGGC GGTGGGACAG GGGCCTACAT TTGCCTATTC 21960 AAGTTCGGTT TGCGTCCACA CTACAGTGGG CC~ll~lGGG AAGTAAGCAT TTATACGGGG 22140 CCCCCGTTAA ATCAAAGTGG ~l~ll~ACCT TTTCTCCGAA ATAATACACT TCCACCACTA 22260 GGGGCACAAG ~'L'l~ l~ACCC A~lll~lAAA TAGCCTGTTT CTTACTCAGG TATGCTGCCA 22320 AACTCTCAAG GGGATGCGTG GCGGGAAGTA CTGAGACACT ~LCC~.GGAC CCCTCCTCAC 22740 CTGGGGAAAA TGGGGTTTGC GACTGTCCAG CAGGCGGCGC TAATAAGCCT l~l~L~AT 22980 TGTGCATATC CCTCCCTACA AAACCCTGAG CCCCCACCCA AA~lLC~lll TCGCTGTCAC 23220 TCGATTCCGT ATCTTCGCTC TGTGACCGTG ATGAAACTTC AGCTGCGGAG GAL~ll~lGG 23280 GCCCACACAA CCCACCGCCC AGTACATCAA CCATCCTACC TCTGGGCTTT LLll-LAAGG 23460 CTCCTTCTAA GTGCCTTTTC 'l~l~L~lllG TCATCATGGG GATAGATCCC AAACAATGCT 23520 . .

CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 AAGGGCCTCT C~ LGGC TCGATTGGCG GTCCTTAATA GCCGTCCAAA GCAGCCCAGG 23700 AATATTCTCT l"Ll~ll-'ATC CAACCACCCT ACCCCCCAGA AGCGTCCACT GTCTAAAGCA 23880 AGCCACGACA CAAAACACCT TTTCCGTGGG CGA~lllClC GCCACAACTA GCTGGACCCC 24000 CA~ACCAAGA AAAACACATG TATTATTCAA TTAGCCAACA ACTTTATTTA TTACCGACAG 24300 GGCCCTGATG TACACATGCT GTTCCAGGTG CCTA~ATGCC AAAAGTCCCC CGACCAAGAA 24840 GACAATGAAG GGCAGCCAGA AAACGCCGGA CACAAAGACC TTCTTA~ACA ACAGAAGGTA 24900 GTACACCATA AATGCTCCGC AGAAGCCCAG CTCATAGTAC ~l~l~lACTA TTGGCGGCGC 24960 TACAGCAGCA AACACTGCTG ACGCGCAGAT CCATTCCAGC CTCCGGTCCA G~'L~lllllG 25140 GCGCTCACTG TCCAGGCGGC ACATGGTGTC AAATCAGGGG GTTAAATGTG ~llllGGGCA 25380 CCTTCCCACG ATCCCTGGAC TGGCTCGAGT CTGAGCGCCT CTTGTGAGGC ~'L~''l''l ~lGC 25440 l~lC~llAGT TGGCGCCGCT GGGGGGCAGC TGGTGACAGA GGCAGCGTCC TCAGAGGCGT 25500 W O 98/04576 PCTrUS97/13346 CATCCCAGCC AGAAACGGGT TTG~lll~lG GCTTGAAATC AATGATCTTG CTCACGCCAC 25920 ~1111~1111 TTGCTGCCTT AGCCACTTAA GTAGGAATGC ACCCGTTTTG CCACAGAGGA 26040 GAAGCCTGGT GGTCCTACCA CCGGCTTCCA TCCGATCGTG GA~AGGTAGG ATACCCTTTT 26100 GGTCCACCAC G~llll~lGC ACGGTGGAGG TGAGGTTGTC CCCGTAGGAA ATGGTGGTCC 26160 ACGTATTCCT GAAGCTGAAG CTAACGTCTC CACTGCCTTC C~l~l~lCCC ACCAGGGGCG 26340 TAAl~l~llC ATTGACCCTC CTGATTTTAG ACAGGAGGGT CACGTCCACC CTGACCCCAT 26520 AGTGAAAATC CACAGGCATG ATTGCGGCCG TAGACGCACA GAGAAATCAC AGGA~AGCTG 26580 CACGCTTCTG GCGGAGGCGT GCCAAATATG GGAGGAACGA A~ATATCACG CAGAATCCTG 26820 TCTGCGTGAC TGG~lllllC CTGTATCTCC ACCATAGTGT TGTACAACAT ACTGGCGGCC 27000 llG~l~lGCA GCAGCTCGTC CCTGGAAATG TAATCGTTGG CAAGGCACAC CCCGGGCATG 27060 AGCCACTCCA G~ll~lCGCA AAGGGCGGGG TCCAAAATGA TCTTGGCAGC ATATGCTAGA 27240 AGTTCGCCTC GA~l~ll~ll GAAAAATATC TTCAAGATAT TGGCATACAC GACACCGTGG 27300 GTGACAAGGT CCTCAATGTT AAAGTTAACT AGGCGTTCGG CCATTCCCAA A~ACGTAAAC 27420 , . ~ ........... . . . ..

CA 0226ll64 l999-0l-22 W 098/04S76 PCT~US97/13346 GTAGTACATG AGG~lllllA GACCAAGCCT GTATCCATGT AGCAGCAGGT CCCTAAGATA 27840 ~ lAACC ACCCGAAGGT CGTCGGGGAG AAc~ll~llA AAAAAAGTCA CATTGGGCCT 28080 CAACACCTCT TCTTTATTGG TGACCTTGGA AGATATATTA GCA~AAAAGG GGTACACAGA 28140 CTCGGCATAG CCAGTTACTT GCGAGGTCCC AGCCGTCGGC ATCACCGCCA GA~ACTGAGA 28200 CACCGCGGTG TAGTACATAG ACTGGAATAT ATT~ll~l~l AACTCAGCGC TCTCAGCATC 28440 GAGGTACCCG TACCCCAATT CCGCA~ACAC ATCCGCCAAC CCCTGAACAC CAATCCCCAT 28500 CGGC~l~llG GCGTCCGTGG TGCCAACCCT CGCGCTTTCA ACAGTTCTCA GACACTTTGG 28680 AAGGCAGATA TTTGCCAGGT TGCACACCGA A~l~lll~ll CCTGGCAGTT GGACTATCTC 28740 TGCACACAAG TTTGAGCAGT TAATGGCCAT GCCCTGAGTG TCGGTCCAGT G~l~ll~ATT 28800 GAGCGCTTCT TTTAAAAGCA CGTACGGTGA GC~ lll ATGATGGTGT GGATAAGAGT 28860 GAACAAATAC CATAACTTGG ATGGGTCCTT TTCATACATC CTGA~AAACA ATGTTGGGAT 29040 GTTATTGTCA TTGA~ATAAT GAACCTGGGC ATCCACCAGT TTGAGGCAAC TGGCTATGTT 29220 AATA~AACAG CTGGCGAGTT GTCCGCCTTC GACTCCAGCT GAGCGCAGTA TTGGCGTGGC 29400 GCAGCACACG TGCTGCGCAG CGAGGTAGCC A~AAACGTAC TCCACTATAG CCATCTCAGA 29460 TACAGACTTA GC~lC~l~AA TAAGGTCCCG CGCCAACCAA TACAGGCATT CATGCTCTAA 29520 GCACTGACAG GCAACA~ACA CGGAAACCCT CATAAACATT TGCGCCACGC TTTCATAGAC 29580 CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 GCCACAGTTA A~'l~l'~lCCT CGTAAGCTTT GGACCGTCTG TAGGCGCACA ACATATCTTC 29700 CAAGGCATCA Al~ll~lll"l' GAATAAACGA TTCCACCCGA TGTCCCAACA CGCCTCGAAA 29760 &AGAGATTGG GCACACATAT CAAAATCCGA CAATTGTCCC GCAGACACCT GAGACCCGCG 29940 AGGACGTGCC CTTCACGTTC AGA~lllG~l GCACCGGATG AGAATCAAAG GGAACTGTGC 30120 ACACACCGGC CACTTTGTGA ~l~l~lGGCG CTTTTGCCGC TTCCATTCCA GAGAGCATAA 30660 AG~l~ll~AT GGAATTTCAA CTCCGAGGAC TGCCGGTGCC TGCCCTCTTA AACAGCAGCA 31140 CAACAGAGCA ~lllllAAAT ACTGTTGCCC AACTGCCGAC GGACCTATCA AAATTTATAC 31200 GCGACTATCG C~l~llCGCA CTGGTTCGCG CGGCGTATTT TTTAGAACCC CCTTCTAGCA 31260 .. .. . , . , . _ .

CA 0226ll64 l999-0l-22 W O 98/04576 PCT~S97/13346 CTCCCATTCT TGCCTTCACC GACGTGGAAC TATCCACACT CAAACCCCAC TAl~l~llCA 31860 GCCTGGAATC ~lllllGAGC CGAGGTATAG ACTTCATGAC TGACCTAGGT CAGTACCTAG 31980 GCTTGGCCTT CACAGAATTG CA~AAGATAC TGACACGCGC CAGCGCGGAG CAAACGGAAC 32280 CTCTAGCATT ~llC~lCCCT CCGGCCCCCA TAAACACTTT GCAGCGCGTG TACGCCGCGC 32400 l~lll-AGAT AAGATGGCTG ATAATATTTG CTGCCGAGGC GGCAACCGGG CTCATCCCTG 32760 CCCAAGACGC CCTATACAAC ~llllGGACT GTATCCAGGA G~l~ll~ACC CACATCAGGC 32880 AGGCTGTTCC AGACGCACAG TGTCCGCACG C~~ ACA GTCCCTGTTC ~l~lllCAAT 32940 TCCGCCCTTT CGTACTCA~A CACCAGCAGG GTGTAACCTT ~lll~lAGAT GGCTTGCAGA 33000 TCGAGTACGA CAGCGAGGGC GA~llC~lGC GCGTGCCAGT TGCACCGCCA GAACAACCAC 33120 CGCACGTACA l~l~lCGCAT TTCAAGAAGA CAATACAGAC CATCGAACAG GCCACCAGGG 33180 TCCAACACCA GCTGGTCCCC ACGGCCATCG TTA~AAAACT GCTACATTTC GACGAGGCTA 33480 AGGGGGCGGC TGGCGGGTCG G~l~lC~lGA CGCAGA~AGA ACTTGAGCTC TTGAACAAAA 33600 CAAATAAATA TGACGTCCCT GAG~l~l~'AG TCGACTGGGA AACGTACTCC CGGTCTGCCT 33720 CA 0226ll64 l999-0l-22 W 098/04576 PCTrUS97/13346 l~lll~lCGA ATAGAGGCCA TGGCAGCCCA GCCTCTGTAC ATGGAGGGAA TGGCCTCCAC 33840 GTATTACATG TTTGACCCCC ACTGCATACC A~ACATCCCC AACAGTCCTG CACACGTCAT 34320 C~lC~l~ATT CCTCCTTACG ATCCGACAGA CCGCCCACGA CCGCCTCACC AAGACCGCCC 34680 CGCCACAGCG CTGCTCTCTG ACCTAACTGC CACAAGAGGG CAGAAACGCA AA~llllC~lC 35040 (2) INFORMATION FOR SEQ ID NO:20:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 32207 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

GTACCCACTA ACGGATACAC CA~ lCGA CATAACGGCG GACGTCACAC CCGACAACAC 120 _ _ . _ . , ., .. . . ... ... . . _ .. . .

W O 98/04~76 PCTrUS97/13346 ATTTATAATA AACACCGATA CACTGGCCCC ATATAAACAG CA~lllTC'AT ATCTGGGGGG 1200 TCCAAACGGC GCACACTTCC ACGCCCAGGC CGTATCAATT CCAGTGTTAG A~AACTACGT 1440 ACATAACGCG GGG~lC~llC TCAAGGGCGA AAAGAGCGAG AGGTTCTCCC GGCTGAAGAC 1500 CCTTGGAAAC CTAGTTACCA ACGTACCA~A ACTTGGTGAG GCGTTCACCG GGGGCCCGCA 1620 AGATGCCCTG GACGCCCTGG ~AAAAAAGGA TCCGGCCCTT CTTGGTGAGG GGACCACGTT 1740 GGCTACGAAA CGGAAACTAT ACAGATTGAT CCA~AGGGAC CTCAAAGAGG CTCAAAAACA 1980 CGAGACCAAT CGGGCCATGG AGGAATGGAA GCAGA~AGTA CTGGCTCTTG ACAATGCGTC 2040 AGAGAAGCAC TTCA~AATAC TACTCCCCGT ACCCGCGGAC GCCCCCGTCC AAGCGTCTCC 2160 CA 0226ll64 l999-0l-22 GCTGAACGAC ATCTACATGG ATAAGCTCCG CTC~ lG CCCGACGCGC AGCCTTTTCA 2460 CCCTGCCACA GAGTACGCGC GCATCGCCTC CAACATGAAG TCC~-l~llCA ACGACCACGG 2700 CAAGAAGCAG TATCTGGAAC ACTTTGAGGC CACCCAAAGC GTA~l~lllA CAGCCTTTCC 3660 GCTCACACAG GAGGTTACGA TCCCAGCCCT GCATTACGCG GGAC~lllCG ACAACTTGGA 3720 CCCGTTGCAC AGACAGGTGG l~ll~lCCAG ~lllllGGAG GCCCAGATCC GATTAGCGTT 3900 GGTACTGGAC AC~lllllCC ACAACGCGCC CCTTCCCGCA GAGTCTTCCT CCAATGCTTT 4080 . , . . ~ .

W O 98/04576 PCTrUS97/13346 CGCCACGCCA GAAGCGCGTC l~lllllCCC CGCGCGATGG CACCACGTCA ACGTGCAGGA 4440 ATATACCTTC GCAAATTACC AGATCCCCAG CACCGACAAC CCGATACCGA TC~ll~lG~l 5100 CGCCACGCCG CCACTCACAA TCACCCCAAA TA~ACCGACC GGAACCCCTC ACGTCTCCCC 5460 CACGCAACCC GTCACTTTAA C~lllCGl~l CCCACCTACC GCACCCACTC CCGCAACTGC 5880 CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 TCTTCCCAAC CTCGTAGAGA GATACGCGCG GG~lllC~lG GACACGCCCT CTGTAGAGGT 6360 ~ll~lGACTC CACGGTTGTC CAATCGTTGC CTAlll~lll TTGCCAGAGG GGG~lllC~l 6660 CGCGTCGGCC ACCGCGGGGG CGGC'C'~lllC CGTCGTGGAT GAGAGGGTTG TGAGAATGTC 6720 ATAGGCCTCA TAATAATGAT GGGCAATTAA GAACACGAGA TA~l~l~l~l TTTGCACGAG 7020 CA 0226ll64 l999-0l-22 W 098/04576 PCTrUS97/13346 CGTGCCCAAA CTGGGACACG GCGTCGCTTG GTCGGCACAG AAAGCACTTC AGG~ll~lGG 8940 TATGATCGCA GGCTGCAGGC TGTGCCCGCT GGACGAGAAA G~.ll.lAAA TACTGACAGT 9420 CGAGCCGGTT TGCAGAGTGG TCACCTGCCC TGCTCCACAC CCACCCCCGC ~l~l~llCCA 9660 ACTCTCAACT CACGATCCAG GGAAACCACC GTCCAGTGGC CAl~lll~ll CCCTGGCAAC 9720 CGGAGCACCC GGAGGAGAGC ~lCG~lAACC CCATAATGAC ACAGATTCAC CAGTCGCTCC 9840 AACCATCTTC CCCCTGCAGG ~lcl~l~AGc TCCTATTTTC l~lG~lCCGC GAllC~lC~A 9900 CCCCCATGGG lll~llCGAG GACTATGCCT GCCTCTGCTT ~''l"l'~'l~l~''l'A TACGCCCCAC 9960 TGCAGCTGCA ~ll~lll~ll CAAAAGTGCT TTAAGACCAC CGCCGCCGAA AAAATACTGG 10140 CA 0226ll64 l999-0l-22 19~

TAAAGCTGGT AGACAGAATT GC~lll~l~l TGGACAACCC ATTCGCCATG CCATATGTAT 10740 CAGATCCGCT ACTTAGAGAG CTGATCCGGG GCTGTACCCC ACAGGA~ATT CACAAGCACC 10800 l~ll~-GCGA CCCGCTGTGC GCCCTCAATG CTAAGGTGGT GTCAGAGGAC GTACTATTCC 10860 TCGATGCCAA CACCCTGTTC GACTGCGAGG TCGTGCAGAC TTTGGTCTTT ~l~lllAAGG 10980 GTCTCCA~AA CGCCAGGGTG GGGAAAACCA CCTCACTAGA CATTATTCGG GAGCTAACCG 11040 CGAGCTAGGA GTGGACTTTC TCCGTGAGAT GGAGACCCCG ATATGCACCT CCA~AACAGT 11400 GCTATGCTCG GTAGCGCTGG C~llllATCA CCACGCAGAC AAAGTGATCC AACACAAGAC 11640 ~lG~lllGGG AGAGCGGACG CCATTTGCGT GCAAAATGTG CTTTGCTGGA GGCCAACTTC 12360 W O98/04576 PCTrUS97/13346 A~l~l~l"l'AT GAGCCACGGG TATGCCCTCG ATACGCCTGC TCTTCAGCAT TGTATGTGTT 12540 TAATGTTGTG CTTGGTGCAA CCGTGATTGT ~l''l"l"l"l'~l'AT TTTATTTTAC TGACACTCTT 12600TGGGAGGGCA CGCTAGCTTC AGTGCGCGCC CGTTGCAACT CGTGTCCTGA ATGCTACGGG 12660 TGCCGCCGCG CGCGTAATGC GCGAGGGGGG TCGGTCTCCC ~'l~'l"l~"lll'A TAGCGTTTCC 13500 TGCGAAGGGG GCGTAACCGT AGGACA~ACT GCTTATGTAG GGGTTAGCCA CCCATTTCCC 13560 AGAGGCGAGA TTCCAGGGCC GTGACTCACT AGCTCCCCTC CCATCGAACA ACCACGCTTG ~3680 GCTTCCGGAA ATACCACCTG AGTACCCCAT TGGTTTATAC ~ AAT TGTAGAATTA 13980 GGG~lllACG CAGCTGGGTA GACCCAGCTG GGTATACCTA CTGGAATAGG GGCTGCGATG 14100 GATGGCGGCG CCCATGAAAT GGCCA~AAAT TATAATTTTT CGAGTCGCTC ACGGTCCCAC 14460 CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 CAA'll~llll GCGCCCCTCT TTGGCCAGGG GACCAAGGTC GTATGGTTCG CGCTACACTA 15000 CCATCAGCCC GACCGCCGAT GGATTAGGTG CTGCTGGACA AGT~ ~lA AACCCGCGCA 15540 GG~lll'~l'~'l' CGATCCAGAC GCTTACGAAC GCCCGCTTTA AAAACACTAT TCATAATTAA 15600 CAGAAGTTGA CACCAGCCCG CAGTTACCCA AC~ll~lATT TTTTTGGAGT GTTGACAAGT 15660 CTAAATAACG TTGTGGTTTT CGGACCCTTT CAGGGACCAA Al~llllACG TGTTGCCAAG 15900 GCGGCGTTGT GGCCACGGGC G~llC~lCGC TTGGACCTGG AGGGGTGTCA CAll~l~lGA 16260 CA 0226ll64 l999-0l-22 W O 98/04~76 PCTrUS97/13346 TCGCGCCGGG AACACGCAGG TGGCAGCGGA 1G1~1111GC CCAAACACGA GGGTTGCAGG 16560 CCCAGTAGGT CTAAGTTTTC CATACAGTAC ACCCAGTGTA AGA1~1~1~1 GGTGTGCTGC 17040 CTCTAAGAGC TCCAATTAAT TGGGCCAGTG TGGGTTGAGG TATGAACACG TTTAGGAG&A 17340 AAGTCCGAAA CAGATGTTAG GA1.1~11CC ACTGCCGCCT GTAGAACGGA AACATCGCAT 17580 ~1~111GGGT CAACTAAGGC 1111~IAATC AGG~1~11GA CCTCGTGGTG CCAAAAGTCC 17760 ACTGCCTCTG TTCGCCACGC CAA~11.1CA AGGAGTTCTT TCTCCTGGTC TATAAGTTCT 17880 CTTCTGAGCT TACTGGCCAC TAACAGGCAG GCGCTCCCTG 1~1111GAAA ~1~11~111G 18000 GACACCTGCT TTATAAGTAG GA~1.1~1CC AAAAGATTAA GGGCCAACGC GACCACGTTA 18060 CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 TTGGTTTTCC CCTGGCTGGG TTAATGGCAG GGG~lllllA AACTTAACTA TGGAAGATTG 18600 A~ll~l~lCT ll~ll~lGAC AATACACATA TACACAATAA GTTATGGGCG ACTGGTCTGG 18720 AGATAATTTT TTAAGTCCGT ATGGGTCATT GCCCCA~AAA ATCACTGCAA ACTTCCATTG 18840 ACACTTTGGA TCTC'~l~llC CATCCTTTCC CAAAAAGCGT CTATAAAAGA l~l~ll~lGG 18900 TGGATGTGGC llllllGGGT GGGTAACTGG AACGCGCCTC ATACGAACTC CAGGTCTGTG 19020 TTCCACCTCC TGCTCTTGCT CTTCCACCTC CTG~lC~l~l AACTCCTGCT CCTGCTCCTC 19500 CTCCTCTAAC TCCTGCTCCT G~lC~l~lAA CTCCTGCTCC TGATCCTCTA ACTCCTGCTC 19680 ~lG~l~ATCC TGCTGCTCCT GCTCATCCTG CTGCTCCTGC TCATCCTGCT GCTGCTCATC 19920 CTGCTGCTGT GGCTCCTGCT ~ll~lGGCTC CTG~l~ll~l GGCTCCTGCA GGGGCTCCTG 20520 .

CA 0226ll64 l999-0l-22 CTGTGGCTCC TG~ 'l"l'~l'G GCTCCTGCAG GGGCTCCTGC TGCTGTGGCT CCTGCTGCTG 20640 TGGCTCCTGC L~ll~lGGCT CCTGCTGCTG TTGTGAACTT TGGATGCTCA AC~llll~ll 20700 TCCATCGCCC CCGTCCTCCT CGTCCTCCTT CTTGTCCTCC TC~lCGl~AT C~LC~1C~1C 20760 CTCATTGTCC TCATCATCGT CATCCTCCTC GTCCTCCTCC TC~lC~lC~l CCTCCTCGTC 20820 CTCCTCCTCG TCCTCCTCCT CGTCATCCTC CTCGTCATCC TC~lC~L~AT CCTCCTCGTC 20880 ATCCTCCTCG TCATCCTCCT CGTCATCCTC CTCGTCATCC LC~lC~lCAT CCTCCTCGTC 20940 ATCCTCCTCG TCATCCTCCT CGTCATCCTC CTCGTCCTCC TCAl~l~l~l CCTGCTCCTC 21000 CTCATCATCC TTATTGTCAT TGTCATCCTT GTCAACCTGA ~lllC~llGC TAATCTCGTT 21060 GTCCCCATTA TCCTCGCCAG CCTGATTATT TTCGGAACAT T~llLllCAT TCTTGGATGC 21120 GCCATCGCTG GATGATCCCA CGTAGATCGG GGA~l~L~lG GCCCATGGGG GGTACACACT 21300 TGTAGAGGGA CCTTGGGGGG ACGATAGCCT '~ C AGGCTACGCA GGGTAGACGG 21420 TCTTTCCGGA GAC~l~LLl-C GTTTCCTACA ACTTCCTCTC GTTAAGGGCG CGCCGGTGCT 21960 CATGGCCACA GGATGTAGAT CGCAGACACT GA~ACGCTGA AACACAGCAT TAAGCTGCAA 22200 CTGCCAGGTA AACAAGGTTA AA~lGG~ll"l GCTGGCCTTG CGTTGCCATG GATGCTACCT 22320 GCGCTACTCA ~l~lllATAA GTCAGCCGGA CCAAGCTGCT GCTCTTGGGG ACGTGACTGC 22560 CTCTCGGGGG TCCATGTCTA GC~lCllCAT TTCATTACCT TGGGTGGCGT TCATCTGGCT 22860 CCAGTACCGC CACAAGGCAA ATATAACCTG TCCTGGGCTT TGGAACTCTA Cc~ll~llAT 23100 GGTAAATAGC ACAGAAGACG CAGACACCGT CAC~l~llG GCAACTGGTC GCCCACCCCC 23280 CAATGTCACC TGGGCCGCAC CCTGGAACAA CGC~l~ll'~l' ACCCAGGAGC AGTTCACTGA 23340 CCCAAAGACT ~'l'll''l''l''l'ACA GCCCGATGGC CCTTCAGGCC TCCTTGAGTG TCTAGCTGGT 23760 CCCGTGGTCA ll~l~lGGTT TGGCAGTCAC TTCCCCATTT TGGTGTCGCG TTTTGGGTTT 23820 TTG~.~l~ll GAAGGACGGA TCAGGCGGGG AGGAGGGGGT GGGGGAGACT TACTGCAGCA 24000 C~l~l~lGAG ATGACCACCG TGGTGCCTTA CACGTGGAAC GTTGGAATAC 'l'~l'~'l'~"l~AT 24240 TTTCCTCATA AAl~ll-''llG GAAATGGATT GGTCACCTAC AllllllGCA AGCACCGATC 24300 GCGGGCAGGA GCGATAGATA TACTGCTCCT GGGTATCTGC CTAAACTCGC l~l~l~llAG 24360 CATATCTCTA TTGGCAGAAG TGTTGATGTT lll~-llCCC AATATCATCT CCACAGGCTT 24420 GTGCAGACTT GAAATTTTTT TTTACTATTT ATATGTCTAC TTGGATATCT TCA~l~ll~l 24480 GTGCGTCAGT CTAGTGAGGT ACCTCCTGGT GGCATATTCT ACGC~l-C~ GGCCCAAGAA 24540 GCAGTCCCTC GGATGGGTAC TGACATCCGC TGCACTGTTA ATTGCATTGG TG~l~lCGGG 24600 GGATGCCTGT CGACACAGGA GCAGGGTGGT CGACCC'GGl-C AGCAAGCAGG CCAlGl~llA 24660 , ~

W O 98/04576 PCTrUS97/13346 TGCAGGTTTC CTGTTACCCC TGGCCCTCCT TAll~l~lll TATGCTCTCA CCTGGTGTGT 24780 GCTGCTGTTT 111~1~1111 GCTTCCCTTA CCACGTACTA AATCTACTGG ACACTCTGCT 24900 lll~llllAT TATGCGATTA AATGAGGGGT CTGATCCCAA AAGCAATGTT TAGTGGTGGT 25260 CCACCTCGCC ACAGCAGATG GAGAATGTGT CGG~l~l~ll TAGA~ACTCT GTCAGGGTGG 25860 CCTGGCAGCC l~ll~lGAGA CATGTAATCA GACCAGAGAA CCCCGACAAG GA~l~lC~lC 25980 GTTTAAGCTC TTCCACAGTC ACCGTGGCCA CCTCAAAGCC C~l~ll~lGC AACGCGGCCA 26040 TCACCAGGTT CCACATTTCG TCAGA~AAGG AGGTCCATGA GACTTGCAAG GAAGTCAGGG 26280 GAAA CACAACTGTC TCGTTCTGCA AAACCGTGAC ~ll~llGCCT TGTCCCTCGG 26340 CTGACAAGTG TACCTGGGGC ACCTCAACCA GTGCCCCAGG G~l-l~lGAA ACCATAAGTT 26460 CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 GGACCTCCAC CGTTTCTTGC l~ll~l~lGA TGCGCACATG GCGCTCCGAA AGCGTCGGAG 26880 CGAAGAGGCA ~lC~l~lAGG AGGCCGGCTT GGTGGTCCTC TGGACTCCAC GCCACGGCGC 27000 CGGTGATGGC ATAGGTGGCC CCGGTGGATA CATTAGTAGC CAl~ll~LAG GCCTGCTCCC 27120 TCACCCAGGT CTCGAAGTCC 'l"l'~'l'~lAGGA GGTTGGCCAT GGACGGAGTG ATGGCCTCCA 27240 CCGGGTCATG CGA~'l'~l"l"l' AGTCCGGAGA AGATAGGGCC CTTGGCAAGC CGCTGGACCA 27960 TCATCCTTCT CAGGGAGATG CAll~lllGG AAGTAGTGGT AGAGATGGAG CAGACTGCCA 28140 ATCGGCTGTG CGCACGGGGT CCCAGGGCCG ~llCG~lGGC ATACAGGCCG GTGAGGGCCC 28320 C~l~l~l~lG TCCGCCTGGA AACAGGGTGC TGTGAAACAG CAGGTTGCCA AGGCCGCGAA 28380 CCCTGATA~A CTGAGGTGGG TGTGGTTCTA GCAGGGTCTG TGTGATTTTG GACACCAGGT 28740 CA 0226ll64 l999-0l-22 W 098/04576 PCT~US97/13346 G~l~"l~llGA CAATGTTGTG GGCCGGTGGT GCATGTTTGG CCCGTAGCCA AAGGATACAA 28860 CCGGGGACAC C~ AGT CAGGCTGCCG AGAAACCCGC GAGATCTCTG GGGAGTAGGA 29220 AGAAACTTAG AATCCCCAAA TATGTCGCAG TCACAGGTTG TCGGGCAGAG l~l~lllCCG 29280 ATTAGAGATA CCTGATTGGT TAATACAAGC GGACGCACGC ~llG~lGGAG GC~l~ll~lC 29520 GGTCGTTACT l~l~l~llGC AAACCCTTAC TGGAGATAAT GCCATGTCTG TTGTGGAACT 29640 ATTAACATCG GGACATATCC TGCCTGCATG AGCATGTGGT ~l~lC~l~lG GTGTATATAT 29760 TGGTAATCTT ~llGllACAT TGTTGAACGA CACAAGTCTG ~ l~lCGGT AGAGATAACC 29820 CACCAGTACG GCTTGGCCAG TACCTAATAA GAAAAAATAA AATCGTTAAT ~l~l~ll'lll 29880 TTTATGACCA GGGAGCTGCT ACCCAGGTAC AAA~AATCCT TACCCAAAAA TAGAAACAGG 30120 CATACCTTCG TTAllG~l~l ~ll~llCGCG CTTTATAAAC AGTATCCCTA ll~ll~lGGl 30360 TACCACCACA TATTGCAAAC ACACATGCAG CGAGCTTGAG ACAAGGCCCA TTAl~l~l-lG 30780 CA 0226ll64 l999-0l-22 W O 98/04576 PCT~US97/13346 CAAAGATATG TATAAAA~AA ACAAGCAACA ATGTCCATAA TGGCAAAAAA AACTGGCAAT 30840 GTGTCCAGTT GTTGTAAATC TGCAATCCCA TTGAGAATAT AAGTACCAAC ACCATAACAA. 30900 GAATGGCTGA AAAACACATA CAGGGGAATT AC~L111111 AAAAAATTGG AAATATTAGA 31020 ATGGGAGGGG AAGCTTGAAA AC~L1~1111 TTTGACTGCA CATATATGTT GTTATTGTAC 31320 GAGTTAGTTT TGCACAGAAC CAGACATCCT AL~ ~111 GGAAACCTAA AATCCGGATG 31620 GTTGAAAAAA AGGCACTAAG GG~LllLllG CCAAAGGAAA AATGCCCCCG TGGGGTTAGG 31800 ~ ,

Claims (47)

What is claimed is:

1. An isolated nucleic acid encoding a Kaposi's sarcoma-associated herpesvirus polypeptide selected from the group comprising:
a. viral macrophage inflammatory protein II;
b. viral interleukin 6;
c. viral interferon regulatory factor 1;
d. complement-binding protein;
e. glycoprotein B;
f. capsid protein IV encoded by ORF 65;
g. immediate early protein encoded by ORF 73;
h. glycoprotein M; and i. glycoprotein L.

2. The synthetic DNA of claim 1.

3. The genomic DNA of claim 1.

4. The cDNA of claim 1.

5. The RNA of claim 1.

6. A replicable vector comprising the nucleic acid of claim 1.

7. A host cell comprising the vector of claim 6.

8. The eukaryotic cell of claim 7.

9. The bacterial cell of claim 7.

10. A plasmid, cosmid, .lambda. phage or YAC comprising the isolated nucleic acid of claim 1.

11. A nucleic acid of at least 14 nucleotides capable of specifically hybridizing with the isolated of specifically hybridizing with the isolated nucleic acid of claim 1.

12. The nucleic acid of claim 11 which is labeled with a detectable marker.

13. The nucleic acid of claim 12, wherein the marker is a radioactive, a colorimetric, a luminescent, or a fluorescent label.

14. An isolated polypeptide having the amino acid sequence encoded by the nucleic acid of claim 1.

15. The polypeptide of claim 14 linked to a second polypeptide to form a fusion protein.

16. The fusion protein of claim 15, wherein the second polypeptide is beta-galactosidase.

17. An antibody which specifically binds to the polypeptide of claim 14.

18. The antibody of claim 17, wherein the antibody is polyclonal antibody.

19. The antibody of claim 17, wherein the antibody is a monoclonal antibody.

20. A host cell which expresses the polypeptide of claim 14.

21. A vaccine which comprises an effective immunizing amount of the polypeptide of claim 14 and a pharmaceutically acceptable carrier.

22. An antisense molecule capable of specifically hybridizing with the nucleic acid of claim 1.

23. The antisense molecule of claim 22, wherein the molecule is a nucleic acid derivative.

24. A triplex oligonucleotide capable of specifically hybridizing with the double-stranded nucleic acid of claim 1.

25. A transgenic nonhuman mammal which comprises the nucleic acid of claim 1 introduced into the mammal at an embryonic stage.

26. A method of diagnosing a DNA virus associated with Kaposi's sarcoma in a subject which comprises:
(a) obtaining a nucleic acid sample from the subject;
(b) contacting the sample obtained in step (a) with the labeled nucleic acid of claim 12 under high stringency hybridization conditions;
(c) detecting the presence of any labeled nucleic acid hybridized in step (b), the presence of which is indicative of a DNA
virus associated with Kaposi's sarcoma, so as to thereby diagnose a DNA virus associated with Kaposi's sarcoma in the subject.

27. The method of claim 26, wherein the sample comprises a bodily fluid.

28. The method of claim 27, wherein the bodily fluid comprises serum.

29. The method of claim 26, wherein the sample comprises a tissue specimen.

30. The method of claim 29, wherein the tissue specimen comprises a tumor lesion.

31. The method of claim 26 wherein the nucleic acid is amplified before step (b).

32. A method of diagnosing a DNA virus associated with Kaposi's sarcoma in a subject which comprises:
(a) obtaining a sample from the subject;
(b) contacting the sample from step (a) with a support having already bound thereto the Kaposi's sarcoma antibody of claim 17, so as to bind the antibody to any specific Kaposi's sarcoma antigen present in the sample;
(c) removing any unbound material from the support of step (b); and (d) detecting the presence of any specific Kaposi's sarcoma antigen bound by the Kaposi's sarcoma antibody in step (c), the presence of which is indicative of the DNA
virus associated with Kaposi's sarcoma, so as to thereby diagnose the DNA virus associated with Kaposi's sarcoma in the subject.

33. The method of claim 32, wherein the sample comprises a suitable bodily fluid.

34. The method of claim 33, wherein the bodily fluid comprises serum.

35. A method of diagnosing a DNA virus associated with Kaposi's sarcoma in a subject which comprises:
(a) obtaining a suitable bodily fluid sample from the subject;

(b) contacting the sample from step (a) to a support having already bound thereto a Kaposi's sarcoma antigen encoded by the isolated nucleic acid of claim 1, so as to bind the antigen to any specific Kaposi's sarcoma antibody present in the sample;
(c) removing any unbound material from the support of step (b); and (d) detecting the presence of any specific Kaposi's sarcoma antibody bound by the Kaposi's sarcoma antigen in step (c), the presence of which is indicative of the DNA
virus associated with Kaposi's sarcoma, so as to thereby diagnose the DNA virus associated with Kaposi's sarcoma in the subject.

36. The method of claim 35, wherein the sample comprises a suitable bodily fluid.

37. The method of claim 36, wherein the bodily fluid comprises serum.

38. A method of treating a subject infected with Kaposi's sarcoma- associated herpesvirus comprising administering to the subject an effective amount of an antisense molecule of claim 22 under conditions such that the antisense molecule selectively enters an infected cell of the subject, so as to thereby treat the subject.

39. A method of treating a subject infected with Kaposi's sarcoma- associated herpesvirus comprising administering to the subject a pharmaceutically effective amount of an antiviral agent in a pharmaceutically acceptable carrier, wherein the agent specifically binds to the polypeptide of claim 14.

40. A method of prophylaxis or treatment for a subject infected with Kaposi's sarcoma-associated herpesvirus comprising administering to the subject the antibody of claim 17 in a pharmaceutically acceptable carrier.

41. A method of vaccinating a subject against Kaposi's sarcoma- associated herpesvirus comprising administering to the subject an effective amount of the polypeptide of claim 14 and a pharmaceutically acceptable carrier, so as to thereby vaccinate the subject.

42. A method of immunizing a subject against a herpesvirus associated with Kaposi's sarcoma which comprises administering to the subject an effective immunizing dose of the vaccine of claim 21 and a pharmaceutically acceptable carrier.

43. The antibody of claim 18, which antibody is specifically immunoreactive with peptides encoding an antigenic portion of viral interleukin 6.

44. The antibody of claim 43, wherein the antigenic portion of viral interleukin 6 comprises the amino acid sequences as set forth in SEQ ID NO:2 and SEQ ID NO:3.

45. The method of claim 40, wherein the antibody is a chimeric antibody.

46. The method of claim 40, wherein the antibody is a humanized antibody.

47. A method of passively immunizing a subject against a herpesvirus associated with Kaposi's sarcoma which comprises administering to the subject an effective immunizing amount of the antibody of claim 43 and a pharmaceutically acceptable carrier.