WO1995006055A1 - Hsv-2 ul26 gene, capsid proteins, immunoassays and protease inhibitors - Google Patents

Abstract

Essentially pure HSV-2 UL26 gene products and fragments thereof including mature HSV-2 protease and active fragments thereof are disclosed. Essentially pure HSV-2 UL26.5 gene products and fragments thereof including mature HSV-2 capsid protein and functional fragments are disclosed. Isolated nucleic acid molecules comprising all or part of the HSV-2 UL26 gene and/or the HSV-2 UL26.5 gene are disclosed. Expression vectors and host cells comprising such nucleic acid molecules are disclosed. Methods of identifying compounds that inhibit HSV-2 protease activity and methods of identifying compounds that inhibit HSV-2 virion assembly are disclosed. Synthetic HSV-2 substrates are disclosed. Antibodies that selectively bind to HSV-2 protease processed substrates but not unprocessed substrates or unprocessed substrates but not processed substrates are disclosed. Methods of and kits for distinguishing between HSV-1 DNA or protein and HSV-2 DNA or protein and reagents useful in such methods and kits are disclosed.

Description

HSV-2 UL26 GENE, CAPSID PROTEINS, IMMUNOASSAYS AND PROTEASE INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of copending U. S. patent application serial number 08/110,522, filed August 20, 1993, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to HSV-2 UL26 and HSV-2 UL26.5 genes; to essentially pure HSN-2 UL26 and HSN-2 UL26.5 gene products; to compositions and methods of producing and using HSN-2 UL26 and HS V-2 UL26.5 DΝA sequences and gene products.

BACKGROUND OF THE INVENTION

The herpes viruses consist of large icosahedral enveloped virions containing a linear double stranded genome. Currently, six human herpes viruses have been isolated and are known to be responsible for a variety of disease states from sub-clinical infections to fatal disease states in the immunocompromised. One human herpes virus, herpes simplex virus type 2, designated HSV-2, is usually acquired through sexual contact and gives rise to genital herpes. The frequency of recurrence of secondary genital herpes ranges between one and six times per year. It is estimated that genital HSV-2 infections occur in ten to sixty million individuals in the USA. Currently, there are no vaccines available to protect against HS V-2 infection.

Little is known regarding the genome composition of HS V-2. Nevertheless, HS V-2 presents a major public health problem. Individuals continue to become infected by the virus and no completely satisfactory anti-viral agents or vaccines are available. There is a need for a method of identifying anti-HSV-2 agents. There is a need for reagents useful in such methods. There is a need for a method of identifying compounds which modulate the activity of HSV-2 proteins and affect the ability of the virus to replicate and produce multiple infectious virions in an infected cell. There is a need for methods of and kits for distinguishing HS V-2 infections from other herpesvirus infections. SUMMARY OF THE INVENTION

The present invention relates to essentially pure HSV-2 UL26 gene products and fragments thereof including HS V-2 protease precursor protein, mature HSV-2 protease and active fragments thereof, HS V capsid precursor protein and mature HSV-2 capsid protein.

The present invention relates to essentially pure HSV-2 UL26.5 gene products and fragments thereof including HSV-2 capsid precursor protein and mature HSV-2 capsid protein.

The present invention relates to isolated nucleic acid molecules comprising the HSV-2 UL26 gene or portions thereof including isolated nucleic acid molecules that encode mature HS V-2 protease and active fragments thereof and nucleic acid molesules that encode precursor or mature HSV-2 capsid protein, regulatory, e.g., promoter regions, or functional fragments thereof.

The present invention relates to expression vectors comprising the HSV-2 UL26 gene or portions thereof including nucleotide sequences that encode mature HS V-2 protease and active fragments thereof and nucleotide sequences that encode precursor or mature HSV-2 capsid protein or functional fragments thereof.

The present invention relates to host cells that contain expression vectors comprising the HS V-2 UL26 gene or portions thereof including nucleotide sequences that encode mature HS V-2 protease and active fragments thereof and nucleotide sequences that encode precursor or mature HS V-2 capsid protein or functional fragments thereof.

The present invention relates to isolated nucleic acid molecules comprising the HS V-2 UL26.5 gene or portions thereof including isolated nucleic acid molecules that encode mature HSV-2 capsid protein, regulatory, e.g., Promoter regions or fragments thereof and nucleotide sequences that encode precursor or mature HSV-2 capsid protein or functional fragments thereof.

The present invention relates to expression vectors comprising the HSV-2 UL26.5 gene or portions thereof including nucleotide sequences that encode mature HS V-2 capsid protein or fragments thereof and nucleotide sequences that encode precursor or mature HSV-2 capsid protein or functional fragments thereof.

The present invention relates to host cells that contain expression vectors comprising the HS V-2 UL26.5 gene or portions thereof including nucleotide sequences that encode mature HSV-2 capsid protein or fragments thereof and nucleotide sequences that encode precursor or mature HS V-2 capsid protein or functional fragments thereof. The present invention relates to methods of identifying compounds that inhibit HSV-2 protease activity comprising contacting HSV-2 protease or active fragments thereof with an HS V-2 protease substrate in the presence of a test compound, detecting the level of proteolytic cleavage of the substrate and comparing that level to the level that occurs in the absence of the test compound. The present invention relates to methods of identifying compounds that inhibit HS V-2 virion assembly by contacting HS V-2 capsid proteins in the presence of a test compound, detecting the level of capsid-capsid association and comparing that level to the level that occurs in the absence of the test compound. The present invention relates to HSV-2 protease substrates produced by means of chemical synthesis or recombinantly produced and predicated on fragments or all of the UL26 gene product.

The present invention relates to antibodies that selectively bind to HSV-2 protease processed substrates but not unprocessed substrates or that selectively bind to unprocessed substrates but not to processed substrates.

The present invention relates to methods of distinguishing between HSV-1 DNA and HS V-2 DNA comprising PCR amplification of DNA using primers which will amplify HSV-1 DNA but not HSV-2 DNA and/or PCR amplification of DNA using primers which will amplify HSV-2 DNA but not HSV-1 DNA. The present invention relates to PCR primers which will amplify HSV-1

DNA but not HS V-2 DNA and PCR primers which will amplify HS V-2 DNA but not HSV-l DNA.

The present invention relates to kits for distinguishing between HSV-1 DNA and HS V-2 DNA comprising a container comprising PCR primers which will amplify HSV-1 DNA but not HSV-2 DNA and a positive control and size marker to determine if HSV-1 DNA has been amplified by the primers and/or a container comprising PCR primers which will amplify HSV-2 DNA but not HSV-1 DNA and a positive control and size marker to determine if HS V-2 DNA has been amplified by the primers. The present invention relates to methods of distinguishing between HS V- 1 protein and HSV-2 protein comprising an immunoassay using antibodies that selectively bind to HSV-1 protein but not HSV-2 protein and/or an immunoassay using antibodies that selectively bind to HSV-2 protein but not HSV-1 protein.

The present invention relates to antibodies which selectively bind to HS V- 1 protein but not HSV-2 protein or antibodies which selectively bind to HSV-2 protein but not HSV-1 protein. The present invention relates to kits for distinguishing between HSV-1 protein and HS V-2 protein. Said kit comprising a carrier being compartmented to receive a series of containers in close confinement which comprises a first container comprising antibodies which selectively bind to HSV-1 protein but not HSV-2 protein and a means to detect whether the antibodies are bound to HS V- 1 protein and/or a second container comprising antibodies which selectively bind to HSV-2 protein but not HSV-1 protein and a means to detect whether the antibodies are bound to HS V-2 protein.

The present inventional relateds to the HSV-2 protease promoter and/or enhancer elements and their uses.

The present invention relates to the HS V-2 capsid protein promoter and/or enhancer elements and their uses.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates the HSV-2 UL26 gene. The symbol < > denotes the limits of the

HSV-2 UL26 gene product. A putative termination codon is underlined.

The symbol [[ ]] denotes the limits of the HS V-2 UL26.5 gene product.

The symbol [ ] denotes the limits of two major proteolytic sites. The cissile bond is indicated by the *. The symbol | | denotes the promoter region of the HS V-2 UL26.5 gene, a putative

"TATA box" is underlined.

Figure 2 illustrates the expression of chloramphenicol acetyltransferase (CAT) when regulated in the HS V-2 UL26.5 promoter.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term UL26 gene refers to a DNA molecule comprising a nucleotide sequence that encodes the HS V-2 protease and a form of the HS V-2 capsid protein. The UL26 gene is disclosed in SEQ ID NO:l. The coding region of the UL26 gene consists of nucleotides 534-2447 of SEQ ID NO: 1. When expressed, the UL26 gene encodes a 638 amino acid active protease precursor disclosed in SEQ ID NO: l and SEQ ID NO:2.

As used herein, the term "active protease precursor" refers to the unprocessed UL26 translation product. The active protease precursor is an active HSV-2 protease. When produced, the active protease precursor autocleaves at an internal protease cleavage site between amino acid residues 247 and 248. The amino terminal 247 amino acid portion retains protease activity. As us-εd herein, the term "mature protease" refers to the amino terminal 247 amino acid protein that is produced by autocleavage of the active protease precursor. The amino acid sequence of the mature protease is disclosed as amino acids 1-247 of SEQ ID NO:l and SEQ ID NO:2. As used herein, the term "HS V-2 protease" is meant to refer to, interchangeably, active protease precursor, mature protease or active fragments thereof.

As used herein, the term "UL26.5" gene refers to a DNA molecule comprising a nucleotide sequence that encodes the HSV-2 capsid protein. The UL26.5 gene is an internal sequence within the UL26 gene which is separately transcribed. The UL26.5 gene is disclosed in SEQ ID NO:l and includes the coding region from nucleotide 1461-2447. When expressed, the UL26.5 gene encodes a 329 amino acid capsid precursor disclosed in SEQ ID NO:l and SEQ ID NO:2 as amino acids 310-638. As used herein, the term "capsid precursor" refers to the unprocessed UL26.5 translation product. While not wishing to be bound by any particular mechanistic theory regarding the function of the gene products of this invention, but based in part on the literature concerning HSV-1, it is believed that after it is produced, the capsid precursor is cleaved by the HS V-2 protease at an internal protease cleavage site between amino acid residues 613 and 614 of SEQ ID NO.l and SEQ ID NO:2. The 304 amino acid portion is the capsid protein used in viral assembly and viral DNA packaging. It is the C-terminal processing of UL26.5 that enables packaging of viral DNA into mature capsids. Inhibition of this processing event results in the inability to package DNA into mature capsids. As used herein, the term "mature capsid protein" refers to the 304 amino acid protein that is produced by cleavage of the capsid precursor by the HSV-2 protease. The amino acid sequence of the mature capsid protein is disclosed as amino acids 310-613 of SEQ ID NO:l and SEQ ID NO:2.

As used herein, the term "HSV-2 capsid protein" is meant to refer to, interchangeably, capsid precursor and mature capsid protein.

As used herein the term "functional fragments" when used to modify a specific gene or gene product means a less than full length portion of the gene or gene product which retains substantially all of the biological function associat-ed with the full length gene or gene product to which it relates. To determine whether a fragment of a particular gene or gene product is a functional fragment one merely generates the fragments by well-known nucleolytic or proteolytic techniques and tests the thus generated fragments for the described biological function.

The present invention relates to essentially pure HSV-2 protease, to compositions and methods for producing and using HSV-2 protease, to nucleic acid molecules that encode HSV-2 protease and to methods for producing and using nucleic acid molecules that encode HSV-2 protease. The present invention relates to essentially pure HSV-2 capsid protein, to compositions and methods for producing and using HSV-2 capsid protein, to nucleic acid molecules that encode HS V-2 capsid protein, to methods for producing and using nucleic acid molecules that encode HS V-2 capsid protein. The present invention relates to substrates which are cleaved by HS V-2 protease, to methods of identifying compounds that inhibit HSV-2 protease activity, to methods of identifying compounds which inhibit HSV-2 capsid assembly, to methods of distinguishing between samples containing HSV-1 DNA and samples containing HSV-2 DNA, to methods of distinguishing between samples containing HSV-1 protein and samples containing HSV-2 protein, and to reagents, including oligonucleotides and antibodies, for performing such methods.

Some embodiments of the present invention provide methods for identifying compounds which inhibit or otherwise modulate the activity of HS V-2 protease. Thus, the present invention provides methods for identifying compounds useful as anti-HSV-2 agents since the activity of the HSV-2 protease is essential for the viral life cycle. According to the present invention, HSV-2 protease is contacted with an HS V-2 protease substrate (substrate) in the presence of a test compound to determine whether or not the test compound affects proteolytic activity. The effect of the test compound on the HS V-2 protease may be determined by comparing the proteolytic activity in the presence of the test compound to the proteolytic activity that would be observed in the absence of the compound.

Proteolytic activity refers to the ability of the HS V-2 protease to enzymatically process the substrate into products, i.e. cleave a single substrate peptide molecule into two or more peptide molecules (proteolytic products). In the viral life cycle, protease precursor is processed into mature protease and capsid precursor is processed into mature capsid by such proteolytic cleavage. This conversion is necessary for virion assembly and viral DNA packaging. The level of proteolytic activity may be determined by a variety of means well known by those having ordinary skill in the art. Essentially, a means is provided to distinguish unprocessed substrate from proteolytic product. Thus, after the substrate, HSV-2 protease and test compound are contacted, the level of proteolytic activity can be observed by detecting the amount of unprocessed substrate remaining, the amount of unprocessed substrate depleted, or the amount of proteolytic product generated. The present invention provides essentially pure HS V-2 protease which is useful in an assay to identify compounds which modulate HSV-2 protease activity. The present invention provides methods of producing essentially pure HS V-2 protease. The amino acid sequence of HSV-2 protease is disclosed in SEQ ID NO:l and SEQ ID NO:2. As described above, the 638 amino acid active protease precursor is disclosed in SEQ ID NO:l and SEQ ID NO:2. The active protease precursor is an active HS V-2 protease which is processed by autocleavage at an internal protease cleavage site between amino acid residues 247 and 248 to produce a 247 amino acid protein referred to as mature protease. Purified active protease precursor, mature protease and active fragments thereof may be produced by routine peptide synthesis methods or by using recombinant DNA technology using the information provided in SEQ ID NO:l. Using standard procedures and readily available starting materials, one having ordinary skill in the art can produce HSV-2 protease. Furthermore, using standard procedures and readily available starting materials, one having ordinary skill in the art can determine whether a fragment and/or derivative of the active protease precursor or mature protease is an active fragment. Assays for determining whether or not a protein or peptide is capable of cleaving a specific substrate is disclosed herein. To determine if an HS V-2 protease fragment has proteolytic activity, one having ordinary skill in the art can perform protease activity assays as described herein without test compounds and using the fragment or derivative of the protease instead of the protease identical to SEQ ID NO:2. If the fragment or derivative cleaves the substrate, it is active, i.e. the fragment or derivative possesses proteolytic activity. Thus, one having ordinary skill in the art can routinely determine if a fragment or derivative of the protease is an active fragment or derivative.

The present invention relates to nucleotide sequences that encode HSV-2 protease and to nucleotide sequences that encode HSV-2 capsid protein. The UL26 gene including a nucleotide sequence which encodes HSV-2 protease and a precursor form of HSV-2 capsid protein is disclosed in SEQ ID NO:l. The UL26.5 gene including a nucleotide sequence which encodes HSV-2 capsid protein is also disclosed in SEQ ID NO:l. One having ordinary skill in the art can, using standard techniques and readily available starting materials, use the information disclosed herein including SEQ ID NO: 1 to obtain or synthesize a nucleic acid molecule that encodes HSV-2 protease or a nucleic acid molecule that encodes HSV-2 capsid protein. Further, using standard techniques, readily available starting materials and the information disclosed herein including SEQ ID NO:l, one having ordinary skill in the art can produce essentially pure HSV-2 protease including, active precursor protease, mature protease or active HS V-2 protease fragments. Likewise, using standard techniques, readily available starting materials and the information disclosed herein including SEQ ID NO:l, one having ordinary skill in the art can produce essentially pure HSV-2 capsid protein including capsid precursor, mature capsid, or HS V-2 capsid fragments capable of assembly functional fragments. One having ordinary skill in the art can, using standard techniques and readily available starting materials, use the information disclosed herein including SEQ ID NO: 1 to obtain or synthesize a nucleic acid molecule that encodes HSV-2 protease or HSV-2 capsid protein using codons which provide optimum protein production in a given host cell used in an expression system. Nucleic acid molecules encoding HSV-2 protease or HSV-2 capsid protein may be generated by those having ordinary skill in the art without undue experimentation using a variety of techniques. Using, for example, Polymerase Chain Reaction (PCR) methodology, primers may be designed and used to produce multiple copies of the nucleotide sequences that encode the HS V-2 protease or HSV-2 capsid protein. The entire nucleotide sequence encoding active protease precursor may be obtained routinely by amplifying the viral DNA. Similarly, the nucleotide sequence encoding mature protease may be obtained routinely by amplifying the viral DNA. Likewise, the nucleotide sequence encoding an active HS V-2 protease fragment may be obtained routinely by amplifying the viral DNA. In a similar manner, the entire nucleotide sequence encoding capsid precursor, mature capsid or functional fragments thereof may be obtained routinely by amplifying the viral DNA. Alternatively, using restriction enzymes, DNA encoding HS V-2 protease, including the active protease precursor, the mature protease, or active fragments thereof or HSV-2 capsid protein including capsid precursor, mature capsid or functional fragments thereof may be obtained from viral DNA cloned into vectors and identified by hybridization using probes designed from the disclosed nucleotide sequence. Moreover, nucleic acid molecules that encode the HSV-2 protease or the HS V-2 capsid protein may also be synthesized using techniques well known to those having ordinary skill in the art. Codons which encode HSV-2 protease or HSV-2 capsid protein may be selected to optimize protein production in a host cell selected for recombinant production of the HS V-2 protease or HS V-2 capsid protein. The HSV-2 genome is highly rich in G+C nucleotides. This is particularly true for the UL26 gene which encodes HSV-2 protease. Such high G+C character poses a problem in overexpressing genes in E. coli because of codon usage and an increased chance of frame-shift mutations. In an effort to improve expression of UL26 in E. coli, the UL26 gene and fragments thereof were changed to provide codons preferred in E. coli yet maintaining the authentic amino acid sequence of the protease. The reference for preferred codon usage is: Wada et al, (1992) "Codon Usage Tabulated from the GenBank Genetic Sequence Data", Nucleic Acid Research, Vol. 20 Supplement, pages 2111-2118, which is incorporated herein by reference. Optimization of codon usage is well known and can be employed to design nucleic acid molecules according to the present invention which can be expressed at an improved level of efficiency in a selected host.

One having ordinary skill in the art can, using well known techniques, insert such DNA molecules into vectors such as commercially available expression vectors for use in well known expression systems. For example, commercially available plasmids such as pSΕ420 (Invitrogen, San Diego, CA) or pET- 16(b) (Novagen, Madison W.I.) may be used for production of HS V-2 protease in E. coli. The commercially available plasmid pYES2 (Invitrogen, San Diego, CA) may, for example, be used for production in S. cerevisiae strains of yeast. The commercially available MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, CA) may, for example, be used for production in insect cells. The commercially available plasmid pcDNA I (Invitrogen, San Diego, CA) may, for example, be used for production in mammalian cells such as Chinese Hamster Ovary cells. One having ordinary skill in the art can use these commercial expression vectors and systems or others to produce the HS V-2 protease or HS V-2 capsid protein using routine techniques and readily available starting materials. (See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989) which is incorporated herein by reference.) Thus, the desired proteins can be prepared in both prokaryotic and eukaryotic systems, resulting in a spectrum of processed forms of the protein.

The particulars for the construction of expression systems suitable for desired hosts are known to those in the art. Briefly, for recombinant production of the protein, the DNA encoding the polypeptide is suitably ligated into the expression vector of choice. The DNA is operably linked to all regulatory elements which are necessary for expression of the DNA in the selected host. One having ordinary skill in the art can, using well known techniques, prepare expression vectors for recombinant production of the polypeptide.

The expression vector including the DNA that encodes the HSV-2 protease or HS V-2 capsid protein is used to transform or transfect the compatible host which is then cultured and maintained under conditions wherein expression of the foreign DNA takes place. The protein of the present invention thus produced is recovered from the culture, either by lysing the cells or from the culture medium as appropriate and known to those in the art. One having ordinary skill in the art can, using well known techniques, isolate the protein that is produced using such expression systems.

According to one embodiment of the invention, protein may be produced and purified as follows. A DNA molecule that comprises a nucleotide sequence that encodes the HSV-2 protease or the HSV-2 capsid protein is produced which includes a nucleotide sequence that encodes multiple histidine residues at a terminal portion of the protein. This DNA molecule is incorporated into an expression vector which is introduced into suitable host cells. The DNA is expressed and the protein, including the terminal histidine residues, which are referred to herein as the histidine tag or His-tag, is produced. The cells are collected and maintained on ice in phosphate buffered saline at pH 8.5. The cells are then lysed by sonication. The sonicated cellular material is centrifuged at 30,000 x g. The supernatant is then filtered through a .2 micron filter. The filtered supernatant is incubated with a metal chelating resin (e.g., a nitrilo triacetic acid nickel resin is one of many such resins useful for such a purpose) for 2 hours at room temperature, after which time the resin is separated from unbound material by centrifugation. The resin is then packed into a column and washed with 50 mM imidazole to eliminate non specifically bound proteins. The His-tagged protease is then eluted from the Ni column with 150 mM imidazole buffer. The eluate from the column is further purified by column chromatography using Pharmacia Superdex 75 sizing column in phosphate buffered saline. The DNA molecule may be engineered to include a specific cleavage site between the histidine tag and authentic HSV-2 protease to enable removal of the histidine tag from the expressed protein. Removal of the histidine tag may be accomplished as follows: The (asparte)4 lysine sequence can be engineered to follow the histidine tag and precede the HSV-2 sequence when the histine tag is placed at the amino-terminus of the HS V-2 protease. Enterokinase specifically cleaves after the (aspartate)4lysine sequence thereby generating authentic HSV-2 protease.

In addition to producing these proteins by recombinant techniques, automated peptide synthesizers may also be employed to produce the HSV-2 protease or the HS V-2 capsid protein. Such techniques are well known to those having ordinary skill in the art.

The present invention provides essentially pure substrates for HS V-2 protease cleavage activity including synthetic substrates. An HSV-2 protease substrate is a peptide which can be cleaved at least into two separate peptides by HSV-2 protease mediated proteolysis. In some embodiments, the size differential between cleaved and uncleaved substrates may be used to detect whether or not the protease is active. In some embodiments, the substrates of the present invention are labelled so that they may be detected. In some embodiments, the substrates are fixed to a solid phase. In some embodiments of the invention, either the substrate or a proteolytic product has a biologically or chemical activity not present in the other which can be used to distinguish one from the other. Examples of biological activities include enzyme activity and the ability to bind with specific antibodies.

Two amino acid sequences are contained in UL26 that have been identified as natural cleavage sites. The first is LQAS (SEQ ID NO:3) wherein the HSV-2 protease cleaves the peptide between the A and the S. The second is VNAS (SEQ ID NO:4) wherein the HSV-2 protease cleaves the peptide between the A and the S. Natural or synthetic substrates may be produced which contain either of these two cleavage sites. Accordingly, a substrate according to the present invention have either the formula R, - SEQ ID NO:3 - R₂ or the formula

wherein R_x and R₂ are, independently, hydrogen or one or more amino acids. In some embodiments, the substrate is the UL26 gene product which contains two protease cleavage sites: one comprising SEQ ID NO:3 and one comprising SEQ ID NO:4. In some embodiments, the substrate is the UL26.5 gene product which contains a protease cleavage sites comprising SEQ ID NO:4. In some embodiments, Rj is preferably 1-20 amino acids, more preferably 1-10, and most preferably 3, 4, 5, 6, 7, 8 or 9 amino acids. In some embodiments, R₂ is preferably 1-20 amino acids, more preferably 1-10, and most preferably 3, 4, 5, 6, 7, 8 or 9 amino acids. One having ordinary skill in the art can readily design substrates according to the above formula. The following peptides have been designed as substrates.

1. Peptides including the internal cleavage site SEQ ID NO:3 (LQA*S): AHTYLQA*SEKFK SEQ ID NO:5

AGIAGHTYLQA*SEKFK SEQ ID NO:6

GIAGHTYLQA*SEKFK SEQ ID NO:7

IAGHTYLQA*SEKFK SEQ ID NO:8

GHTYLQA*SEKFK SEQ ID NO:9 HTYLQA*SEKFKM SEQ ID NO:10

HTYLQA*SEKFKMW SEQ ID NO: 11

HTYLQA*SEKFKMWG SEQ ID NO: 12

HTYLQA*SEKFKMWGA SEQ ID NO: 13

HTYLQA*SEKFKMWGAE SEQ ID NO: 14 2. Peptides including the terminal cleavage site SEQ ID NO:4

(VNA*S): ALVNA*SS AAHVDVD SEQ ID NO: 15

The asterisk (*) indicates the scissile bond where cleavage by HSV-2 protease occurs. The substrates may be obtained from proteolytic cleavage of the UL26 or

UL26.5 protein product. They may be produced recombinantly by expression of UL26 or UL26.5 gene or fragment thereof containing the cleavage site or may be made by means of synthetic organic chemical means using standard peptide synthetic procedures well known in the art such as Merrifield synthesis. One having ordinary skill in the art can readily design assays using the HSV-

2 protease and substrate to identify compounds that modulate HSV-2 protease activity. As used herein, the term "test assay" refers to assays that include a mixture of HSV-2 protease, substrate and test compound; and the term "control assay" refers to assays that include a mixture of HSV-2 protease and substrate without test compound. To determine whether or not a test compound modulates HSV-2 protease activity, the level of HSV-2 protease activity in a test assay may be compared to the level of HSV-2 protease activity in a control assay.

In some embodiments of the present invention, the size differential between cleaved and uncleaved substrate is used to determine whether or not substrates are cleaved when contacted with HSV-2 protease in the presence of a test compound. In some embodiments, an HPLC assay is performed. Sample containing protease is incubated with a substrate, for example HTYLQASEKFKMWGAE (SEQ ID NO: 14), for 4 hrs at 37° C in phosphate buffered saline after which the reaction is terminated with trifluoroacetic acid. The reaction is then run on an HPLC column, showing activity manifested by the peptide cleavage products. In some embodiments of the present invention, immunoassays are used to detect whether or not substrates are cleaved when contacted with HS V-2 protease in the presence of a test compound. In some embodiments, antibodies are provided which specifically bind to uncleaved substrate but not HS V-2 protease cleavage products. Such antibodies are referred to herein as "substrate- specific antibodies". In some embodiments, antibodies are provided which specifically bind to HS V-2 protease cleavage products but not uncleaved substrate. Such antibodies are referred to herein as "product-specific antibodies". Antibodies which react to either a product or a substrate but not both (i.e. substrate-specific antibodies and product- specific antibodies collectively) are referred to herein as "non-crossreactive antibodies". In some embodiments, antibodies are fixed to a solid phase. In some embodiments, antibodies are labelled.

For example, a mixture containing HS V-2 protease, substrate and test compound is maintained under appropriate conditions and for a sufficient amount of time to allow the proteolytic reaction to occur unless the test compound affects the reaction. The mixture can be added to a container which has non-crossreactive antibodies attached to the inner surface. If the non-crossreactive are substrate- specific antibodies, any uncleaved substrate remaining in the mixture will bind to the antibodies. If the substrate is labelled, the contained may be rinsed and the amount of label present may be detected. The level of HSV-2 protease activity is determined accordingly. If the non-crossreactive are product- specific antibodies, any HSV-2 protease products in the mixture will bind to the antibodies. If the substrate is labelled at a portion which is liberated as the product, the contained may be rinsed and the amount of label present may be detected. The level of HS V-2 protease activity is determined accordingly. ICP35 antibodies (Catalog No.: 13-118-100; Rivers Park, 9108 Gulford Rd.

Columbia, Maryland) may be used to detect cleaved substrate. Such antibodies are product specific and only bind to capsid protein after it has been proteolytically processed by the HSV-2 protease.

Alternatively, instead of using labelled substrates, the exemplified immunoassays may be modified as sandwich assays in which antibodies specific for the bound antigen complex are detected. Such antibodies are referred to herein as complex-specific antibodies. The container is again rinsed and sufficient time is allowed for the binding of the complex specific antibody to any complex present. The level of complex specific antibody is detected and indicative of the level of HS V-2 protease activity. In some embodiments of immunoassays, unlabelled substrate is used in the reaction mixture. After the reaction mixture is added to a container comprising a non-crossreactive antibody and maintained for a sufficient time for the non- crossreactive antibody to bind to either substrate br product, either labelled substrate or labelled product, respectively, is added and will bind to any non-crossreactive antibody not bound with substrate or product from the reaction mixture. Detecting the amount of labelled substrate or labelled product indicates the level of proteolytic cleavage.

In some embodiments, the substrate is labeled and the label is released when the substrate is converted to proteolytic products. Detecting the release of the label, which indicates the HSV-2 protease activity, may be accomplished by a variety of well known means. In some embodiments, labelled substrate is fixed to a solid phase. Upon cleavage by HSV-2 protease, the label attached to the portion of the substrate that becomes an unattached product, is released. Comparing the level of label present before and after the reaction mixture indicates how much label is released and thus the level of HSV-2 protease activity. Alternatively, detecting the amount of label freed from the solid phase indicates the level of HSV-2 protease activity.

In another embodiment, methods of detecting HSV-2 protease activity include fluorescence liberation assays in which substrate contains fluorescent label adjacent to the scissile bond. At such a location, the label is not detectable in uncleaved substrate. However, when the substrate is cleaved by HSV-2 protease at the cleavage site, the fluorescent group becomes exposed and the fluorescence becomes detectable. Thus, the level of proteolytic activity may be measured by measuring detectable fluorescence after contacting the substrate with HSV-2 protease in the presence of a test compound.

In another embodiment, methods of detecting HSV-2 protease activity include scintillation proximity assays in which radiolabelled substrate is conjugated to solid beads which, when in close proximity to the radiolabel, are excited and become detectable by scintillation. When the substrate is cleaved, the radiolabel is no longer in close proximity to the beads and the beads are not excited and not detectable by scintillation. Thus, the level of proteolytic activity may be measured by measuring the excitation of the beads by scintillation after contacting the conjugated substrate with HSV-2 protease in the presence of a test compound.

In addition to these embodiments, one having ordinary skill in the art can apply well known techniques to devise other methods of identifying compounds that modulate HS V-2 protease activity using various means of detecting the HS V-2 protease cleavage or the lack thereof.

The present invention relates to kits for identifying compounds that modulate HSV-2 protease activity. Such kits include separate containers which comprise HSV-2 protease, substrate, and optionally, antibodies or other reagents for detecting HSV-2 protease activity or distinguishing between uncleaved substrate and products. The substrate or antibodies may be fixed to the inner surface of a container. The substrate or antibodies may be labelled.

Some embodiments of the present invention also provide methods of identifying compounds which inhibit or otherwise modulate HSV-2 capsid assembly using a multimerization assay. The present invention provides methods of identifying compounds useful as anti-HSV-2 agents since capsid assembly is essential for viral replication and infectivity. According to the present invention, chimeric genes are provided which comprise either a sequence including the HSV-2 UL26.5 gene or a portion thereof which encodes an HSV-2 capsid protein linked to a sequence encoding the yeast GALA DNA-binding protein or a sequence including the HSV-2 UL26.5 gene or a portion thereof which encodes an HS V-2 capsid protein linked to a sequence encoding the yeast GALA activation protein. While it is preferred that the portion of the chimeric gene that encodes the HSV-2 capsid protein encodes the mature capsid, the capsid precursor protein may also be usefully employed. Chimeric genes are inserted into Saccharomyces cerevisiae plasmids and the plasmids are introduced in S. cerevisiae which contains an integrated GALA- responsive lacZ indicator gene. When the chimeric genes on the plasmids are expressed, fusion proteins are produced. The portions of the fusion proteins comprising the HSV-2 capsid protein will, under selected condition bind to each other and thereby bring together the DNA-binding domain and activation domain of GALA. When the two GALA domains which are in close proximity interact with the GAL4-responsive lacZ indicator gene, the indicator gene is expressed and, under the proper conditions a detectable blue color is observed. If the fusion proteins are prevented from binding, the two GALA domains will not be present in proximity to each other and the indicator gene will not be activated. Thus, no blue color will be present to observe. Thus, this yeast system provides a rapid and specific assay for the interaction of HSV-2 capsid proteins that occur^' during virion assembly. In the presence of compounds which interrupt or inhibit HSV-2 capsid protein interaction, the GALA domains in the fusion proteins produced by expression of the chimeric genes will not associate and thereby will not activate the lacZ gene in the yeast system.

Accordingly, compounds may be identified by the absence of activation of the lacZ gene in transformed yeast which inhibit HS V-2 capsid assembly and therefore possess anti-viral properties.

Some embodiments of the present invention provides methods of distinguishing between samples containing HSV-1 DNA and samples containing HSV-2 DNA or samples containing HSV-1 proteins and samples containing HSV-2 proteins. Accordingly, the present invention provides a method of diagnosing whether an individual is infected with HSV-1 and/or HSV-2. Methods are disclosed for identifying whether an individual is infected with HSV-1 and/or HSV-2 wherein HSV-1 infection can be distinguished from HSV-2 infection.

According to some embodiments of the invention, PCR technology is used to distinguish between samples containing HSV-1 DNA and samples containing HSV- 2 DNA. Such methods provide a means for distinguishing between HSV-1 and HS V-2 infections and allow for the diagnosis of the type of HS V infection an individual has. Specific primers are designed that will provide for amplification of HSV-1 DNA but not HSV-2 DNA and/or HSV-2 DNA but not HSV-1 DNA. Accordingly, by performing amplification techniques using such primers with biological samples taken from individuals such as cell, serum or tissue samples, especially samples taken at sites where blisters or other manifestations of viral shedding are observed, one can determine whether or not the DNA in the sample is derived from HSV-1 or HSV-2 and therefore whether the individual from which the sample was taken is infected with HSV-1 or HSV-2.

The nucleotide sequence of the UL26 gene including the nucleotide sequence which encodes the HSV-2 protease and the HSV-2 capsid protein is disclosed in SEQ ID NO: 1. The nucleotide sequence encoding HS V- 1 protease and HSV-1 capsid protein are disclosed in SEQ ID NO: 16. A set of PCR primers were designed which amplify HSV-2 sequences but not HSV-1 sequences. Thus, detection of amplified DNA indicates that HSV-2 is present. Similarly, a set of PCR primers were designed which amplify HSV-1 sequences but not HSV-2 sequences. Thus, detection of amplified DNA indicates that HSV-1 is present. It is preferred that both sets of primers are provided and used in separate amplification protocols with material from the same sample in order to provide an additional control. Other optional controls include positive controls which contain DNA sequences that will be amplified and/or negative controls that cannot be amplified by the primers. Amplified DNA may be detected by running the material on an electrophoresis gel after the amplification protocol is complete. DNA molecules of the expected length of an amplification product may be provided as size markers. Present invention also relates to kits for distinguishing whether a sample contains DNA from HSV-1 or HSV-2. The kits of the present invention are useful to diagnose whether an individual is infected with HSV- 1 and/or HS V-2. The kits contain containers which comprise primers that will amplify HSV-1 DNA but not HSV-2 DNA or containers that will amplify HSV-2 DNA but not HSV-1 DNA. Kits may optionally contain both sets of primers in separate containers for running separate amplification procedures using different portions of the same sample. Kits may optionally contain positive and/or negative controls in separate containers. Kits may optionally contain DNA molecules in a separate container which can serve as a size marker. The DNA molecule may be of the expected length of a DNA molecule amplified using the primers.

According to some embodiments of the invention, immunoassays are used to distinguish between samples containing HSV-1 protein and samples containing HSV-2 protein. The immunoassays are used to distinguish between HSV-1 and HS V-2 infections and diagnose the type of HSV infection an individual has. Such immunoassays are based upon differences between UL26 gene products of HSV- 1 and HSV-2 or between UL26.5 gene products of HSV- 1 and HSV-2. Immunoassays may be based upon differences in proteases and/or capsid proteins of HSV-1 and HS V-2. Specific antibodies are provided which selectively bind to epitopes on HSV-1 antigens not present on HSV-2 antigens or which selectively bind to epitopes on HSV-2 antigens not present on HSV-1 antigens. For example, specific antibodies are provided which selectively bind to HSV-1 protease but. not HSV-2 protease or which selectively bind to HSV-2 protease but not HSV-1 protease. Likewise, specific antibodies are provided which selectively bind to HSV- 1 capsid but not HSV-2 capsid or which selectively bind to HSV-2 capsid but not HSV-1 capsid.

Accordingly, by performing antibody binding assays, using specific antibodies with biological samples taken from individuals such as cell, serum or tissue samples, especially samples taken from sites where blisters or other manifestations of viral shedding are observed, one can determine whether or not the HSV- 1 -specific antibodies or the HSV-2-specific antibodies bind to proteins in the sample and therefore whether the individual from which the sample was taken is infected with HSV-1 and/or HSV-2. The amino acid sequence of HSV- 2 active protease precursor spans amino acids 1-638 in SEQ ID NO:l and SEQ ID NO:2. The amino acid sequence of HSV-2 mature protease spans amino acids 1-247 of SEQ ID NO:l and SEQ ID NO:2. The amino acid sequence of HSV-2 capsid precursor spans amino acids 310-638 in SEQ ID NO:l and SEQ ID NO:2. The amino acid sequence of HSV-2 mature capsid spans amino acids 310-613 of SEQ ID NO:l and SEQ ID NO:2. The amino acid sequence of HSV-1 protease and capsid are disclosed in SEQ ID NO:17. The amino acid sequence of HSV-1 active protease precursor spans amino acids 1-635 in SEQ ID NO: 17. The amino acid sequence of HSV-1 mature protease spans amino acids 1-247 of SEQ ID NO:17. The amino acid sequence of HSV- 1 capsid precursor spans amino acids 307-635 in SEQ ID NO: 17. The amino acid sequence of HSV- 1 mature capsid spans amino acids 307-610 of SEQ ID NO: 17.

Antibodies which specifically bind to HSV-2 protease but not HSV-1 protease may be produced by those having ordinary skill in the art using routine methods and widely available starting materials. Likewise, antibodies which specifically bind to HSV-2 capsid but not HSV-1 capsid may be produced by those having ordinary skill in the art using routine methods and widely available starting materials. Either of these HSV-2 specific antibodies are used to detect HSV-2 in an immunoassay which can distinguish HSV-1 from HSV-2. Similarly, antibodies which specifically bind to HSV-1 protease but not HSV-2 protease may be produced by those having ordinary skill in the art using routine methods and widely available starting materials. Likewise, antibodies which specifically bind to HSV-1 capsid but not HSV-2 capsid may be produced by those having ordinary skill in the art using routine methods and widely available starting materials. These HSV-1 specific antibodies are used to detect HSV-1 in an immunoassay which can distinguish HSV-1 from HSV-2. It is preferred that both immunoassays be performed using material from the same sample in order to provide an additional control. Other optional controls include positive controls which include peptides which will bind to the antibody used in the immunoassay and/or negative controls which include peptides which will not bind to the antibody used in the immunoassay. Antibodies may be labelled. Alternatively, an antibody that specifically binds to the HSV specific antibodies may be used. One having ordinary skill in the art can readily produce immunoassays including all necessary reagents using the information provided herein.

HSV-1 protease antibody produced by Serotech as Antibody 45KD and commercially available from Bioproducts for Science Inc. as catalog number MCA406 (P.O. Box 29176, Indianapolis, IN) can be used in immunoassays to distinguish HSV-2 from HSV-1. The Serotech antibody binds to HSV-1 precursor or mature capsid protein but not HS V-2 precursor or mature capsid protein. Accordingly, an immunoassay using the Serotech antibodies may be performed to determine if a sample contains HSV-1 or HSV-2 and thus if the individual from whom the sample was taken is infected with HSV- 1 or HS V-2.

Present invention also relates to kits for diagnosing whether an individual is infected with HSV-1 or HSV-2. The kits of the present invention may comprise a container comprising antibodies which bind to HSV-1 protease but not HSV-2 protease and/or a container comprising antibodies which bind to HS V-2 protease but not HSV-1 protease. It is preferred that the kit comprises both types of antibodies in separate containers. Antibodies used in the kits may be labelled. The kits contain all other reagents and materials for performing an immunoassay with the antibodies. Kits may optionally contain positive and/or negative controls in separate containers. Kits may optionally contain means to detect the antibody including, for example a second antibody which specifically binds to the anti-HSV protease antibody. The kits of the present invention may comprise a container comprising antibodies which bind to HSV-1 capsid but not HSV-2 capsid and/or a container comprising antibodies which bind to HSV-2 capsid but not HSV-1 capsid. It is preferred that the kit comprises both types of antibodies in separate containers. Antibodies used in the kits may be labelled. The kits contain all other reagents and materials for performing an immunoassay with the antibodies. Kits may optionally contain positive and/or negative controls in separate containers. Kits may optionally contain means to detect the antibody including, for example a second antibody which specifically binds to the anti-HSV capsid antibody. Kits may comprise the Serotech antibody.

Another aspect of the present invention relates to the HSV-2 protease promoter and/or enhancer elements and their uses. The HSV-2 protease promoter may be synthesized or isolated and linked to coding sequences which encode proteins other than HSV-2 protease. Accordingly, the present invention relates to recombinant DNA molecules which comprise at least a portion of the nucleotide sequence between nucleotides 1-534 of SEQ ID NO:l operably linked to a nucleotide sequence that encodes a protein other than HS V-2 protease. The present invention relates to cells which comprise DNA molecules which comprise at least a portion of the nucleotide sequence between 1 and 534 of SEQ ID NO:l operably linked to a nucleotide sequence that encodes a protein other than HS V-2 protease. Another aspect of the invention applies to bacteriophage lambda clones which harbor HSV-2 UL26 gene (SEQ. I.D. No.:l) and sequences upstream and downstream of the gene. Accordingly, the linked sequences can be used to screen for UL26 promoter regulatory and/or enhancer regions.

Another aspect of the present invention relates to the HSV-2 capsid protein promoter and its uses. The HSV-2 capsid protein promoter is located upstream of nucleotide 1461 of SEQ ID NO:l. It may be synthesized or isolated and linked to coding sequences which encode proteins other than HSV-2 capsid protein. Accordingly, the present invention relates to recombinant DNA molecules which comprise at least a portion of the nucleotide sequence upstream of nucleotide 1461 of SEQ ID NO: 1 operably linked to a nucleotide sequence that encodes a protein other than HSV-2 capsid protein. The present invention relates to cells which comprise DNA molecules which comprise at least a portion of the nucleotide sequence upstream of nucleotide 1461 of SEQ ID NO:l operably linked to a nucleotide sequence that encodes a protein other than HS V-2 capsid protein. Nucleotides 1191 to 1461 (SEQ ID NO: 1 ), for example, were linked to the chloramphenicol acetyl transferase gene and shown to possess significant promoter activity when transfected into VERO cells

EXAMPLES Example ϊ

A proteolytic activity essential to the virion maturation of herpes viruses has been characterized for HSV-2. The HSV-2 protease, also referred to as HSV-2 UL26, has a molecular weight (Apparent) of about 67,028 Da and a pi = 6.94. The HSV-2 protease can be employed using molecular and biochemical technology in in vitro assays identify inhibitors of this activity by rational design and screening and to test these inhibitors for antiviral activity in infected cells.

The HSV-2 UL26 gene was cloned as an Ncol-EcoRI fragment (1938 base pairs) which contained the start codon, the entire open reading frame, the stop codon, and 22 base pairs of 3'-unrranslated sequence. Full-length HSV-2 UL26 was expressed in E. coli using the pOTS vector system in which the gene is inserted downstream of the strong and tightly regulated P_L promoter from bacteriophage lambda of the pOTS-207 vector. Tight regulation of the promoter is essential when expressing genes that are likely to be toxic to the cells, such as proteases. The 27 KD protease domain corresponding to one of the autoproteolytic products derived from the HSV-2 UL26 primary translation product was produced in E. coli using the tightly regulated expression vector pΕT-16(b) (Novagen, Madison W.I.) which contains the T7 promoter.

Each construct was designed to include six histidine codons and the (aspartate)4lysine codons preceding the HS V-2 UL26 start codon so that the expressed protein will contain a cleavable histidine tag at the N-terminus for purification of the protein on Nickel columns. Other chelating columns may be used. The His-tagged protein is eluted from the column by addition of imidazole Alternatively, it can be eluted by other means such as pH change. Columns and technical protocols useful to purify protein may be obtained from commercially available sources such as Qiagen. For the PL promoter vectors, the recombinant constructs are then introduced into E. coli AR120 (nalidixic acid inducible strain) and E. coli AR58 (heat inducible strain) for expression and processing/ purification studies. For the T7 promoter vectors, the recombinant constructs are introduced into E. coli BL21, an IPTG inducible strain. The proteins can be readily purified by chromatography on nickel chelate column.

The p27 protease fragment is active as shown by its ability to remove the last 25 amino acids from a construct comprising most of the UL26.5 coding region.

Example 2

The p27 protease gene was synthesized to contain codons characteristic of highly expressed E. coli genes, yet maintaining the amino acid sequence of p27 protease. The synthesized gene was placed downstream of the tightly regulated T7 promoter in the expression vector pET-16(b). Following IPTG (ImM) induction the 27 k Da protein domain was highly expressed in E. coli.

Example 3

The above HSV-2 UL26 gene (Nα?l-EcøRI fragment) and the p27 protease is cloned into the insect cell expression vector pVL1392. The recombinant construct is then introduced into insect cells derived from Spodopterafrugiperda. High titer viral stocks are then prepared for protease activity analysis and subsequent scale up for protein production.

Example 4 Oligonucleotide PCR primers were designed to the DNA region of HSV- 1

UL26 gene and HSV-2 UL26 gene that shared the least amount of identity to ensure the specificity of the assay. Such a region can easily be viewed by computer analysis comparing the two DNA sequences disclosed in SEQ ID NO:l and SEQ ID NO: 16, respectively. The region of least identity between the two homologs lies within the UL26.5 domain, i.e. the portion of the gene that encodes the capsid. The following provides the sequences of the primers used and the locations of the primers are given based on the nucleotide numbers given in the nucleotide sequence comparison provided in the enclosed computer analysis. As shown below it is helpful to design a system to generate HSV-1 and HSV-2 specific products of different sizes to improve the analysis.

5'-PCR primers (sense-strand sequence):

SEQIDNO:18 HSV-1:5^*-CCGGTGCCCAATCGTCCGT-3'(#864-882) SEQIDNO:19 HSV-2:5'-GTCCGTGCGCGTCAAGTCG-3'(#1397-1416)

3'-PCR primers (antisense-strand sequence):

SEQ ID NO:20 HSV-1 : S'-πCCGGCTCCCCCACCTGA-S' (#1560-1542)

SEQ ID NO:21 HSV-2: S'-ATTCGGATCCTGGAGGCGA-S' (#2470-2452)

Expected PCR product sizes using these sets of primers: HSV-1: 696 base pairs HSV-2: 1073 base pairs.

Separate PCR amplification protocols are performed on samples suspected of containing either HSV-1 or HSV-2 DNA using SEQ ID NO: 18 and SEQ ID NO:20 in the HSV-1 assay or SEQ ID NO: 19 and SEQ ID NO:21 in the HSV-2 assay. If a DNA fragment of 696 base pairs is generated in the HSV-1 assay, the presence of HSV-1 DNA in the sample is indicated. To detect the presence of a 696 base pair fragment, the amplification product is migrated through an electrophoresis matrix. A size marker of DNA of about 696 base pairs is run through the same matrix simultaneously. If a DNA fragment of 1073 base pairs is generated in the HSV-2 assay, the presence of HSV-2 DNA in the sample is indicated. To detect d e presence of a 1073 base pair fragment, the amplification product is migrated through an electrophoresis matrix. A size marker of DNA of about 1073 base pairs is run through the same matrix simultaneously.

A kit is provided which comprises a container comprising SEQ ID NO: 18 and SEQ ID NO:20 in the HSV-1 assay. A kit is provided which comprises a container comprising SEQ ID NO: 19 and SEQ ID NO:21 in the HSV-2 assay. A kit is provided which comprises both a container comprising SEQ ID NO: 18 and SEQ ID NO:20 in the HSV-1 assay and a container comprising SEQ ID NO: 19 and SEQ ID NO:21 in the HSV-2 assay. Size marker DNA may optionally be provided. In some kits, a size marker of 696 base pairs is provided. In some kits, a size marker of 1073 base pairs is provided. In some kits, a size marker of 696 base pairs and a size marker of 1073 base pairs are provided.

Example 5

A region of the putative HSV-2 UL26.5 promoter contained in the HS V-2 UL26 gene was cloned to test for promoter activity. The 256 base pair region that was analyzed spanned nucleotides #1191 to # 1447 of SEQ ID NO:l. The DNA fragment was cloned by the polymerase chain reaction using the sense strand primer (5'-AACATGAGCTGCGTGACC-3') spanning nucleotide # 1191 to # 1209 of SEQ ID NO: 1 and the antisense strand primer (5'-AAAGAAGAAGAAGAAGAC-3') spanning nucleotides #1447 to # 1429 of SEQ ID NO: 1 Promoter activity is tested by cloning the 256 base pair PCR fragment upstream of the chloramphenicol acetyltransferase (CAT) reporter gene in the commercially available vector pCAT Basic (Promega). The resulting construct can then be introduced into a suitable mammalian cell line, e.g., Vero cells, to test for promoter activity by analyzing the levels of CAT activity. The cell line is devoid of endogenous CAT activity; hence, after introducing the promoter construct into such a cell line, the levels of CAT activity is a direct measure of HSV-2 UL26.5 promoter activity. Vero cells were grown in DMEM+10% FCS containing Gentamicine

(lOug/ml). 15 micograms of the HSV-2 UL26.5/pCAT construct was electroporated into 5 million Vero cells using standard protocols. 48 hrs after electroporation cells were harvested in 100 microliters of 0.25 M Tris buffer pH 8.0. Cells were lysed by repeated freeze-thaw, spun down at 15,000 rpm and the supernatants were transferred to fresh tubes. Total protein concentration was determined using Bio- Rad Protein Assay Dye Reagent Kit (Cat. # 500-0006). 5 Microliters of D- Threo[dichloroacetyl-l-1 "C] Chloramphenicol (Amersham, 56 mCi mmol) and 5 microliters of n-Butyral CoA (5 mg/ml) was added to an aliquot of cell extract supernatant in a 100 microliter final volume to assay for CAT activity by ethyl acetate extraction followed by thin layer chromatography. In addition to the above construct, the 256 base pair HSV-2 UL 26.5 fragment was also cloned upstream of the CAT reporter gene in the pCAT Enhancer vector, which contains an SV40 enhancer element. This construct was also tested for CAT activity in Vero cells by the same methods described above.

The control vector pCAT control (contains the S V40 promoter and enhancer) was used as a comparison of HSV-2 UL26.5 promoter strength.

Figure 2 summarizes the results of four experiments. Column 1 is a negative control and represents CAT expression in the absense of promoter and enhancer transcriptional control elements. Column 2, a positive control, employs SV40 promoter and SV40 enhancer elements to drive CAT gene expression. Column 3 represents CAT gene expression driven by UL26.5 promoter alone and Column 4 represents CAT gene expression when the UL26.5 promoter is used in combination with the S V40 enhancer element.

Having established a basal UL26.5 expression level (Column 3), additional fragments of the gene sequence within figure 1 can be used to identify the UL26.5 enhancer elements merely by isolating fragments of convenient length upstream from nucleotide 1191 back to nucleotide 1, introducing the fragments into the basal expression construct oriented operatively with respect to the promoter region and testing their ability to enhance CAT expression over the basal level.

The promoter described here are useful for regulating the expression of heterologous genes when operably linked thereto.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: DiLella, Anthony G. Debouck, Christine

(ii) TITLE OF INVENTION: Novel Gene

(iii) NUMBER OF SEQUENCES: 21

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: SmithKline Beecham Corporation Corporate Patents - US UW2220

(B) STREET: P.O. Box 1539

(C) CITY: King of Prussia

(D) STATE: Pennsylvania

(E) COUNTRY: USA (F) ZIP: 19406-0939

(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 #1.25, mmd

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Jervis, Herbert H. (B) REGISTRATION NUMBER: 31,171

(C) REFERENCE/DOCKET NUMBER: P50188

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 215-270-5019 (B) TELEFAX: 215-270-5090

(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2472 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: genomic DNA

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 534..2447

(ix) FEATURE:

(A) NAME/KEY: misc_feature (B) LOCATION: 1461..2447 (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

GTCGACGAGG CGCGTGGTGG ATATGTCGTC GGGCGCCCGC CAGGCGGCGC TCGTGCGCCT 60 CACCGCGCTG GAGCTCATCA ACCGCACCCG CACAAACACC ACCCCTGTGG GGGAGATTAT 120

TAACGCCCAC GATGCCTTGG GGA ACAATA CGAACAGGGC CTGGGGCTGC TCGCCCAGCA 180

GGCACGCATC GGCTTGGCGT CGAACGCCAA GCGATTCGCC ACGTTCAACG TGGGCAGCGA • 240

CTACGACCTG TTGTACTTTT TGTGTCTCGG GTTCATTCCC CAGTACCTGT CCGTGGCCTA 300

GGGAAGGGTG GGGGTGGTGG TGGTGGGGTG TTTTTCTGTT GTTGTTGTTT CTGGTCCGCC 360 TGGTCACAAA AGGCACGGCG CCCCGAAACG CGGGCTTTAG TCCCGGCCCG GACGTCGGCG 420

GACACACAAC AACGGCGGGC CCCGTGGGTG GGTAAGTTGG TTCGGGGGCA TCGCTGTATT 480

CCCTTGCCCG CTTCCACCCC CCCTTCCCGT TTGGTTTGTT TGTGCGGGTG CCC ATG 536 Met

1

GCG TCG GCG GAA ATG CGC GAG CGG TTG GAG GCG CCT CTG CCC GAC CGG 584 Ala Ser Ala Glu Met Arg Glu Arg Leu Glu Ala Pro Leu Pro Asp Arg 5 10 15

GCG GTG CCC ATC TAC GTG GCC GGG TTT TTG GCC CTG TAC GAC AGC GGG 632 Ala Val Pro lie Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser Gly 20 25 30

GAC CCG GGC GAG CTG GCC CTG GAC CCA GAC ACG GTG CGT GCG GCC CTG 680 Asp Pro Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala Leu 35 40 45 CCT CCG GAG AAC CCC CTG CCG ATC AAC GTA GAC CAC CGC GCT CGG TGC 728

Pro Pro Glu Asn Pro Leu Pro lie Asn Val Asp His Arg Ala Arg Cys

50 55 60 65

GAG GTG GGC CGG GTG CTC GCC GTG GTC AAC GAC CCT CGG GGG CCG TTT 776 Glu Val Gly Arg Val Leu Ala Val Val Asn Asp Pro Arg Gly Pro Phe

70 75 80

TTT GTG GGG CTG ATC GCG TGC GTG CAG CTG GAG CGC GTC CTC GAG ACG 824 Phe Val Gly Leu lie Ala Cys Val Gin Leu Glu Arg Val Leu Glu Thr 85 90 95

GCC GCC AGC GCC GCT ATT TTT GAG CGC CGC GGA CCC GCG CTC TCC CGG 872 Ala Ala Ser Ala Ala lie Phe Glu Arg Arg Gly Pro Ala Leu Ser Arg 100 105 110

GAG GAG CGT CTG CTG TAC CTG ATC ACC AAC TAC CTG CCA TCG GTC TCG 920 Glu Glu Arg Leu Leu Tyr Leu lie Thr Asn Tyr Leu Pro Ser Val Ser 115 120 125 CTG TCC ACA AAA CGC CGG GGG GAC GAG GTT CCG CCC GAC CGC ACC CTG 968 Leu Ser Thr Lys Arg Arg Gly Asp Glu Val Pro Pro Asp Arg Thr Leu 130 135 140 145

TTT GCG CAC GTG GCC CTG TGC GCC ATC GGG CGG CGC CTT GGA ACC ATC 1016 Phe Ala His Val Ala Leu Cys Ala lie Gly Arg Arg Leu Gly Thr lie

150 155 160 GTC ACC TAC GAC ACC AGC CTA GAC GCG GCC ATC GCT CCG TTT CGC CAC 1064 Val Thr Tyr Asp Thr Ser Leu Asp Ala Ala lie Ala Pro Phe Arg His 165 170 175

CTG GAC CCG GCG ACG CGC GAG GGG GTG CGA CGC GAG GCC GCC GAG GCC 1112 Leu Asp Pro Ala Thr Arg Glu Gly Val Arg Arg Glu Ala Ala Glu Ala 180 185 190

GAG CTC GCG CTG GCC GGG CGC ACC TGG GCC CCC GGC GTG GAG GCG CTC 1160 Glu Leu Ala Leu Ala Gly Arg Thr Trp Ala Pro Gly Val Glu Ala Leu 195 200 205

ACA CAC ACG CTG CTC TCC ACC GCC GTC AAC AAC ATG ATG CTG CGT GAC 1208 Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg Asp 210 215 220 225

CGC TGG AGC CTC GTG GCC GAG CGG CGG CGG CAG GCC GGG ATC GCC GGA 1256

Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gin Ala Gly lie Ala Gly

230 235 240

CAC ACG TAC CTT CAG GCG AGC GAA AAA TTT AAA ATA TGG GGG GCG GAG 1304 His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys lie Trp Gly Ala Glu 245 250 255 TCT GCC CCT GCG CCG GAG CGT GGG TAT AAA ACC GGC GCC CCG GGT GCC 1352 Ser Ala Pro Ala Pro Glu Arg Gly Tyr Lys Thr Gly Ala Pro Gly Ala 260 265 270

ATG GAC ACA TCC CCC GCC GCG AGC GTT CCC GCG CCG CAG GTC GCC GTC 1400 Met Asp Thr Ser Pro Ala Ala Ser Val Pro Ala Pro Gin Val Ala Val 275 280 285

CGT GCG CGT CAA GTC GCG TCG TCG TCG TCT TCT TCT TCT TCT TTT CCG 1448 Arg Ala Arg Gin Val Ala Ser Ser Ser Ser Ser Ser Ser Ser Phe Pro 290 295 300 305

GCA CCG GCC GAT ATG AAC CCC GTT TCG GCA TCG GGC GCC CCG GCC CCT 1496 Ala Pro Ala Asp Met Asn Pro Val Ser Ala Ser Gly Ala Pro Ala Pro 310 315 320

CCG CCG CCC GGC GAC GGG AGT TAT TTG TGG ATC CCC GCC TCT CAT TAC 1544 Pro Pro Pro Gly Asp Gly Ser Tyr Leu Trp lie Pro Ala Ser His Tyr 325 330 335 AAT CAG CTC GTC ACC GGG CAA TCC GCG CCC CGC CAC CCG CCG CTG ACC 1592 Asn Gin Leu Val Thr Gly Gin Ser Ala Pro Arg His Pro Pro Leu Thr 340 345 350

GCG TGC GGC CTG CCG GCC GCG GGG ACG GTG GCC TAC GGA CAC CCC GGC 1640 Ala Cys Gly Leu Pro Ala Ala Gly Thr Val Ala Tyr Gly His Pro Gly 355 ^' 360 365

GCC GGC CCG TCC CCG CAC TAC CCG CCT CCT CCC GCC CAC CCG TAC CCG ' 1688 Ala Gly Pro Ser Pro His Tyr Pro Pro Pro Pro Ala His Pro Tyr Pro 370 375 380 385

GGT ATG CTG TTC GCG GGC CCC AGT CCC CTG GAG GCC CAG ATC GCC GCG 1736 Gly Met Leu Phe Ala Gly Pro Ser Pro Leu Glu Ala Gin lie Ala Ala 390 395 400

CTG GTG GGG GCC ATC GCC GCC GAC CGC CAG GCG GGT GGG CTT CCG GCG 1784

Leu Val Gly Ala lie Ala Ala Asp Arg Gin Ala Gly Gly Leu Pro Ala

405 410 415 GCC GCC GGA GAC CAC GGG ATC CGG GGG TCG GCG AAG CGC CGC CGA CAC 1832 Ala Ala Gly Asp His Gly He Arg Gly Ser Ala Lys Arg Arg Arg His 420 425 430

GAG GTG GAG CAG CCG GAG TAC GAC TGC GGC CGT GAC GAG CCG GAC CGG 1880 Glu Val Glu Gin Pro Glu Tyr Asp Cys Gly Arg Asp Glu Pro Asp Arg 435 440 445

GAC TTC CCG TAT TAC CCG GGC GAG GCC CGC CCC GAG CCG CGC CCG GTC 1928 Asp Phe Pro Tyr Tyr Pro Gly Glu Ala Arg Pro Glu Pro Arg Pro Val

450 455 460 465

GAC TCC CGG CGC GCC GCG CGC CAG GCT TCC GGG CCC CAC GAA ACC ATC 1976 Asp Ser Arg Arg Ala Ala Arg Gin Ala Ser Gly Pro His Glu Thr He 470 475 480

ACG GCG CTG GTG GGG GCG GTG ACG TCC CTG CAG CAG GAA CTG GCG CAC 2024 Thr Ala Leu Val Gly Ala Val Thr Ser Leu Gin Gin Glu Leu Ala His 485 490 495

ATG CGC GCG CGT ACC CAC GCC CCC TAC GGG CCG TAT CCG CCG GTG GGG 2072 Met Arg Ala Arg Thr His Ala Pro Tyr Gly Pro Tyr Pro Pro Val Gly 500 505 510 CCC TAC CAC CAC CCC CAC GCA GAC ACG GAG ACC CCC GCC CAA CCA CCC 2120 Pro Tyr His His Pro His Ala Asp Thr Glu Thr Pro Ala Gin Pro Pro 515 520 525

CGC TAC CCC GCC GAG GCC GTC TAT CTG CCG CCG CCG CAC ATC GCC CCC 2168 Arg Tyr Pro Ala Glu Ala Val Tyr Leu Pro Pro Pro His He Ala Pro 530 535 540 545

CCG GGG CCT CCT CTA TCC GGG GCG GTC CCC CCA CCC TCG TAT CCC CCA 2216 Pro Gly Pro Pro Leu Ser Gly Ala Val Pro Pro Pro Ser Tyr Pro Pro 550 555 560

GTT GCG GTT ACC CCC GGT CCC GCT CCC CCG CTA CAT CAG CCC TCC CCC 2264 Val Ala Val Thr Pro Gly Pro Ala Pro Pro Leu His Gin Pro Ser Pro 565 570 575

GCA CAC GCC CAC CCC CCT CCG CCG CCG CCG GGA CCC ACG CCT CCC CCC 2312 Ala His Ala His Pro Pro Pro Pro Pro Pro Gly Pro Thr Pro Pro Pro 580 585 590 GCC GCG AGC TTA CCC CAA CCC GAG GCG CCC GGC GCG GAG GCC GGC GCC 2360 Ala Ala Ser Leu Pro Gin Pro Glu Ala Pro Gly Ala Glu Ala Gly Ala 595 600 605

TTA GTT AAC GCC AGC AGC GCG GCC CAC GTG AAC GTG GAC ACG GCC CGG 2408 Leu Val Asn Ala Ser Ser Ala Ala His Val Asn Val Asp Thr Ala Arg 610 615 620 625

GCC GCC GAT CTG TTT GTG TCA CAG ATG ATG GGG TCC CGC TAACTCGCCT 2457 Ala Ala Asp Leu Phe Val Ser Gin Met Met Gly Ser Arg 630 635

CCAGGATCCG AATTC 2472 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 638 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ala Ser Ala Glu Met Arg Glu Arg Leu Glu Ala Pro Leu Pro Asp 1 5 10 15 Arg Ala Val Pro He Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser

20 25 30

Gly Asp Pro Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala 35 40 45

Leu Pro Pro Glu Asn Pro Leu Pro He Asn Val Asp His Arg Ala Arg 50 55 60

Cys Glu Val Gly Arg Val Leu Ala Val Val Asn Asp Pro Arg Gly Pro 65 70 75 80

Phe Phe Val Gly Leu He Ala Cys Val Gin Leu Glu Arg Val Leu Glu

85 90 95 Thr Ala Ala Ser Ala Ala He Phe Glu Arg Arg Gly Pro Ala Leu Ser 100 105 110

Arg Glu Glu Arg Leu Leu Tyr Leu He Thr Asn Tyr Leu Pro Ser Val 115 120 125

Ser Leu Ser Thr Lys Arg Arg Gly Asp Glu Val Pro Pro Asp Arg Thr 130 135 140

Leu Phe Ala His Val Ala Leu Cys Ala He Gly Arg Arg Leu Gly Thr 145 150 155 160

He Val Thr Tyr Asp Thr Ser Leu Asp Ala Ala He Ala Pro Phe Arg 165 170 175 His Leu Asp Pro Ala Thr Arg Glu Gly Val Arg Arg Glu Ala Ala Glu 180 185 190

Ala Glu Leu Ala Leu Ala Gly Arg Thr Trp Ala Pro Gly Val Glu Ala 195 200 205

Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg 210 215 220

Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gin Ala Gly He Ala 225 230 235 240

Gly His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys He Trp Gly Ala 245 250 255 Glu Ser Ala Pro Ala Pro Glu Arg Gly Tyr Lys Thr Gly Ala Pro Gly 260 265 270

Ala Met Asp Thr Ser Pro Ala Ala Ser Val Pro Ala Pro Gin Val Ala 275 280 285 Val Arg Ala Arg Gin Val Ala Ser Ser Ser Ser Ser Ser Ser Ser Phe

290 295 300

Pro Ala Pro Ala Asp Met Asn Pro Val Ser Ala Ser Gly Ala Pro Ala

305 310 315 320

Pro Pro Pro pro Gly Asp Gly Ser Tyr Leu Trp He Pro Ala Ser His

325 330 335

Tyr Asn Gin Leu Val Thr Gly Gin Ser Ala Pro Arg His Pro Pro Leu 340 345 350

Thr Ala Cys Gly Leu Pro Ala Ala Gly Thr Val Ala Tyr Gly His Pro 355 360 365

Gly Ala Gly Pro Ser Pro His Tyr Pro Pro Pro Pro Ala His Pro Tyr 370 375 380 Pro Gly Met Leu Phe Ala Gly Pro Ser Pro Leu Glu Ala Gin He Ala 385 390 395 400

Ala Leu Val Gly Ala He Ala Ala Asp Arg Gin Ala Gly Gly Leu Pro 405 410 415

Ala Ala Ala Gly Asp His Gly He Arg Gly Ser Ala Lys Arg Arg Arg 420 425 430

His Glu Val Glu Gin Pro Glu Tyr Asp Cys Gly Arg Asp Glu Pro Asp 435 440 445

Arg Asp Phe Pro Tyr Tyr Pro Gly Glu Ala Arg Pro Glu Pro Arg Pro 450 455 460

Val Asp Ser Arg Arg Ala Ala Arg Gin Ala Ser Gly Pro His Glu Thr

465 470 475 480

He Thr Ala Leu Val Gly Ala Val Thr Ser Leu Gin Gin Glu Leu Ala 485 490 495

His Met Arg Ala Arg Thr His Ala Pro Tyr Gly Pro Tyr Pro Pro Val 500 505 510

Gly Pro Tyr His His Pro His Ala Asp Thr Glu Thr Pro Ala Gin Pro 515 520 525

Pro Arg Tyr Pro Ala Glu Ala Val Tyr Leu Pro Pro Pro His He Ala

530 535 540 Pro Pro Gly Pro Pro Leu Ser Gly Ala Val Pro Pro Pro Ser Tyr Pro- 545 550 555 560

Pro Val Ala Val Thr Pro Gly Pro Ala Pro Pro Leu His Gin Pro Ser 565 570 575

Pro Ala His Ala His Pro Pro Pro P^'ro Pro Pro Gly Pro Thr Pro Pro

580 585 590

Pro Ala Ala Ser Leu Pro Gin Pro Glu Ala Pro Gly Ala Glu Ala Gly 595 600 605

Ala Leu Val Asn Ala Ser Ser Ala Ala His Val Asn Val Asp Thr Ala 610 615 620 Arg Ala Ala Asp Leu Phe Val Ser Gin Met Met Gly Ser Arg 625 630 635

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Leu Gin Ala Ser 1

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Val Asn Ala Ser 1

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Ala His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 6 :

Ala Gly He Ala Gly His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Gly He Ala Gly His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

He Ala Gly His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 aminc acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Gly His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys 1 5 10 (2 ) INFORMATION FOR SEQ ID NO : 10 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

( i) SEQUENCE DESCRIPTION: SEQ ID NO:10:

His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys Met 1 5 10

(2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

( i) SEQUENCE DESCRIPTION: SEQ ID NO:11:

His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys Met Trp 1 5 10

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys Met Trp Gly 1 5 10

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys Met Trp Gly Ala 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys Met Trp Gly Ala Glu 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Ala Leu Val Asn Ala Ser Ser Ala Ala His Val Asp Val Asp 1 5 10

(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1908 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 1..1908

(ix) FEATURE: (A) NAME/KEY: misc_feature

(B) LOCATION: 919..1908 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

ATG GCA GCC GAT GCC CCG GGA GAC CGG ATG GAG GAG CCC CTG CCC GAC 48 Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp 1 5 10 15

AGG GCC GTG CCC ATT TAC GTG GCT GGG TTT TTG GCC CTG TAT GAC AGC 96 Arg Ala Val Pro He Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser 20 25 30

GGG GAC TCG GGC GAG TTG GCA TTG GAT CCG GAT ACG GTG CGG GCG GCC 144 Gly Asp Ser Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala 35 40 45 CTG CCT CCG GAT AAC CCA CTC CCG ATT AAC GTG GAC CAC CGC GCT GGC 192 Leu Pro Pro Asp Asn Pro Leu Pro He Asn Val Asp His Arg Ala Gly 50 55 60

TGC GAG GTG GGG CGG GTG CTG GCC GTG GTC GAC GAC CCC CGC GGG CCG 240 Cys Glu Val Gly Arg Val Leu Ala Val Val Asp Asp Pro Arg Gly Pro

65 70 75 80

TTT TTT GTG GGG CTG ATC GCC TGC GTG CAG CTG GAG CGC GTC CTC GAG 288 Phe Phe Val Gly Leu He Ala Cys Val Gin Leu Glu Arg Val Leu Glu 85 90 95

ACG GCC GCC AGC GCT GCG ATT TTC GAG CGC CGC GGG CCG CCG CTC TCC 336 Thr Ala Ala Ser Ala Ala He Phe Glu Arg Arg Gly Pro Pro Leu Ser 100 105 110

CGG GAG GAG CGC CTG TTG TAC CTG ATC ACC AAC TAC CTG CCC TCG GTC 384 Arg Glu Glu Arg Leu Leu Tyr Leu He Thr Asn Tyr Leu Pro Ser Val 115 120 125 TCC CTG GCC ACA AAA CGC CTG GGG GGC GAG GCG CAC CCC GAT CGC ACG 432 Ser Leu Ala Thr Lys Arg Leu Gly Gly Glu Ala His Pro Asp Arg.Thr 130 135 140

CTG TTC GCG CAC GTC GCG CTG TGC GCG ATC GGG CGG CGC CTC GGC ACT 480 Leu Phe Ala His Val Ala Leu Cys Ala He Gly Arg Arg Leu Gly Thr 145 150 155 160

ATC GTC ACC TAC GAC ACC GGT CTC GAC GCC GCC ATC GCG CCC TTT CGC 528 He Val Thr Tyr Asp Thr Gly Leu Asp Ala Ala He Ala Pro Phe Arg 165 170 175

CAC CTG TCG CCG GCG TCT CGC GAG GGG GCG CGG CGA CTG GCC GCC GAG 576 His Leu Ser Pro Ala Ser Arg Glu Gly Ala Arg Arg Leu Ala Ala Glu 180 185 190

GCC GAG CTC GCG CTG TCC GGG CGC ACC TGG GCG CCC GGC GTG GAG GCG 624 Ala Glu Leu Ala Leu Ser Gly Arg Thr Trp Ala Pro Gly Val Glu Ala 195 200 205 CTG ACC CAC ACG CTG CTT TCC ACC GCC GTT AAC AAC ATG ATG CTG CGG 672 Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg 210 215 220

GAC CGC TGG AGC CTG GTG GCC GAG CGG CGG CGG CAG GCC GGG ATC GCC 720 Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gin Ala Gly He Ala 225 230 235 240 GGA CAC ACC TAC CTC CAG GCG AGC GAA AAA TTC AAA ATG TGG GGG GCG 768

Gly His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys Met Trp Gly Ala

245 250 255 GAG CCT GTT TCC GCG CCG GCG CGC GGG TAT AAG AAC GGG GCC CCG GAG 816

Glu Pro Val Ser Ala Pro Ala Arg Gly Tyr Lys Asn Gly Ala Pro Glu

260 265 270

TCC ACG GAC ATA CCG CCC GGC TCG ATC GCT GCC GCG CCG CAG GGT GAC . 864 Ser Thr Asp He Pro Pro Gly Ser He Ala Ala Ala Pro Gin Gly Asp

275 280 285

CGG TGC CCA ATC GTC CGT CAG CGC GGG GTC GCC TTG TCC CCG GTA CTG 912

Arg Cys Pro He Val Arg Gin Arg Gly Val Ala Leu Ser Pro Val Leu 290 295 300

CCC CCC ATG AAC CCC GTT CCG ACA TCG GGC ACC CCG GCC CCC GCG CCG 960

Pro Pro Met Asn Pro Val Pro Thr Ser Gly Thr Pro Ala Pro Ala Pro

305 310 315 320

CCC GGC GAC GGG AGC TAC CTG TGG ATC CCG GCC TCC CAT TAC AAC CAG 1008 Pro Gly Asp Gly Ser Tyr Leu Trp He Pro Ala Ser His Tyr Asn Gin 325 330 335 CTC GTC GCC GGC CAT GCC GCG CCC CAA CCC CAG CCG CAT TCC GCG TTT 1056 Leu Val Ala Gly His Ala Ala Pro Gin Pro Gin Pro His Ser Ala Phe 340 345 350

GGT TTC CCG GCT GCG GCG GGG TCC GTG GCC TAT GGG CCT CAC GGT GCG 1104 Gly Phe Pro Ala Ala Ala Gly Ser Val Ala Tyr Gly Pro His Gly Ala 355 360 365

GGT CTT TCC CAG CAT TAC CCT CCC CAC GTC GCC CAT CAG TAT CCC GGG 1152 Gly Leu Ser Gin His Tyr Pro Pro His Val Ala His Gin Tyr Pro Gly 370 375 380

GTG CTG TTC TCG GGA CCC AGC CCA CTC GAG GCG CAG ATA GCC GCG TTG 1200 Val Leu Phe Ser Gly Pro Ser Pro Leu Glu Ala Gin He Ala Ala Leu 385 390 395 400

GTG GGG GCC ATA GCC GCG GAC CGC CAG GCG GGC GGT CAG CCG GCC GCG 1248 Val Gly Ala He Ala Ala Asp Arg Gin Ala Gly Gly Gin Pro Ala Ala 405 410 415 GGA GAC CCT GGG GTC CGG GGG TCG GGA AAG CGT CGC CGG TAC GAG GCG 1296 Gly Asp Pro Gly Val Arg Gly Ser Gly Lys Arg Arg Arg Tyr Glu Ala 420 425 430

GGG CCG TCG GAG TCC TAC TGC GAC CAG GAC GAA CCG GAC GCG GAC TAC 1344 Gly Pro Ser Glu Ser Tyr Cys Asp Gin Asp Glu Pro Asp Ala Asp Tyr 435 440 445

CCG TAC TAC CCC GGG GAG GCT CGA GGC GCG CCG^'CGC GGG GTC GAC TCC 1392 Pro Tyr Tyr Pro Gly Glu Ala Arg Gly Ala Pro Arg Gly Val Asp Ser 450 455 460

CGG CGC GCG GCC CGC CAT TCT CCC GGG ACC AAC GAG ACC ATC ACG GCG 1440 Arg Arg Ala Ala Arg His Ser Pro Gly Thr Asn Glu Thr He Thr Ala 465 470 475 480

CTG ATG GGG GCG GTG ACG TCT CTG CAG CAG GAA CTG GCG CAC ATG CGG 1488 Leu Met Gly Ala Val Thr Ser Leu Gin Gin Glu Leu Ala His Met Arg 485 490 495 GCT CGG ACC AGC GCC CCC TAT GGA ATG TAC ACG CCG GTG GCG CAC TAT 1536 Ala Arg Thr Ser Ala Pro Tyr Gly Met Tyr Thr Pro Val Ala His Tyr 500 505 510

CGC CCT CAG GTG GGG GAG CCG GAA CCA ACA ACG ACC CAC CCG GCC CTT 1584 Arg Pro Gin Val Gly Glu Pro Glu Pro Thr Thr Thr His Pro Ala Leu 515 520 525

TGT CCC CCG GAG GCC GTG TAT CGC CCC CCA CCA CAC AGC GCC CCC TAC 1632 Cys Pro Pro Glu Ala Val Tyr Arg Pro Pro Pro His Ser Ala Pro Tyr 530 535 540

GGT CCT CCC CAG GGT CCG GCG TCC CAT GCC CCC ACT CCC CCG TAT GCC 1680 Gly Pro Pro Gin Gly Pro Ala Ser His Ala Pro Thr Pro Pro Tyr Ala 545 550 555 560

CCA GCT GCC TGC CCG CCA GGC CCG CCA CCG CCC CCA TGT CCT TCC ACC 1728

Pro Ala Ala Cys Pro Pro Gly Pro Pro Pro Pro Pro Cys Pro Ser Thr

565 570 575

CAG ACG CGC GCC CCT CTA CCG ACG GAG CCC GCG TTC CCC CCC GCC GCC 1776 Gin Thr Arg Ala Pro Leu Pro Thr Glu Pro Ala Phe Pro Pro Ala Ala 580 585 590 ACC GGA TCC CAA CCG GAG GCA TCC AAC GCG GAG GCC GGG GCC CTT GTC 1824 Thr Gly Ser Gin Pro Glu Ala Ser Asn Ala Glu Ala Gly Ala Leu Val 595 600 605

AAC GCC AGC AGC GCA GCA CAC GTG GAC GTT GAC ACG GCC CGC GCC GCC 1872 Asn Ala Ser Ser Ala Ala His Val Asp Val Asp Thr Ala Arg Ala Ala 610 615 620

GAT TTG TTC GTC TCT CAG ATG ATG GGG GCC CGC TGA 1908

Asp Leu Phe Val Ser Gin Met Met Gly Ala Arg 625 630 635

(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 635 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp 1 5 10 15

Arg Ala Val Pro He Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser 20 25 30 Gly Asp Ser Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala 35 40 45

Leu Pro Pro Asp Asn Pro Leu Pro He Asn Val Asp His Arg Ala Gly 50 55 60

Cys Glu Val Gly Arg Val Leu Ala Val Val Asp Asp Pro Arg Gly Pro 65 70 75 80 Phe Phe Val Gly Leu He Ala Cys Val Gin Leu Glu Arg Val Leu Glu 85 90 95

Thr Ala Ala Ser Ala Ala He Phe Glu Arg Arg Gly Pro Pro Leu Ser 100 105 110

Arg Glu Glu Arg Leu Leu Tyr Leu He Thr Asn Tyr Leu Pro Ser Val 115 120 125 Ser Leu Ala Thr Lys Arg Leu Gly Gly Glu Ala His Pro Asp Arg Thr 130 135 140

Leu Phe Ala His Val Ala Leu Cys Ala He Gly Arg Arg Leu Gly Thr 145 150 155 160

He Val Thr Tyr Asp Thr Gly Leu Asp Ala Ala He Ala Pro Phe Arg 165 170 175

His Leu Ser Pro Ala Ser Arg Glu Gly Ala Arg Arg Leu Ala Ala Glu 180 185 190

Ala Glu Leu Ala Leu Ser Gly Arg Thr Trp Ala Pro Gly Val Glu Ala 195 200 205 Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg 210 215 220

Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gin Ala Gly He Ala 225 230 235 240

Gly His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys Met Trp Gly Ala 245 250 255

Glu Pro Val Ser Ala Pro Ala Arg Gly Tyr Lys Asn Gly Ala Pro Glu 260 265 270

Ser Thr Asp He Pro Pro Gly Ser He Ala Ala Ala Pro Gin Gly Asp 275 280 285 Arg Cys Pro He Val Arg Gin Arg Gly Val Ala Leu Ser Pro Val Leu 290 295 300

Pro Pro Met Asn Pro Val Pro Thr Ser Gly Thr Pro Ala Pro Ala Pro

305 310 315 320

Pro Gly Asp Gly Ser Tyr Leu Trp He Pro Ala Ser His Tyr Asn Gin

325 330 335

Leu Val Ala Gly His Ala Ala Pro Gin Pro Gin Pro His Ser Ala Phe 340 345 350

Gly Phe Pro Ala Ala Ala Gly Ser Val Ala Tyr Gly Pro His Gly Ala 355 360 365 Gly Leu Ser Gin His Tyr Pro Pro His Val Ala His Gin Tyr Pro Gly 370 375 380

Val Leu Phe Ser Gly Pro Ser Pro Leu Glu Ala Gin He Ala Ala Leu 385 390 395 400

Val Gly Ala He Ala Ala Asp Arg Gin Ala Gly Gly Gin Pro Ala Ala 405 410 415 Gly Asp Pro Gly Val Arg Gly Ser Gly Lys Arg Arg Arg Tyr Glu Ala 420 425 430

Gly Pro Ser Glu Ser Tyr Cys Asp Gin Asp Glu Pro Asp Ala Asp Tyr

435 440 445

Pro Tyr Tyr Pro Gly Glu Ala Arg Gly Ala Pro Arg Gly Val Asp Ser

450 455 460 Arg Arg Ala Ala Arg His Ser Pro Gly Thr Asn Glu Thr He Thr Ala 465 470 475 480

Leu Met Gly Ala Val Thr Ser Leu Gin Gin Glu Leu Ala His Met Arg 485 490 495

Ala Arg Thr Ser Ala Pro Tyr Gly Met Tyr Thr Pro Val Ala His Tyr 500 505 510

Arg Pro Gin Val Gly Glu Pro Glu Pro Thr Thr Thr His Pro Ala Leu 515 520 525

Cys Pro Pro Glu Ala Val Tyr Arg Pro Pro Pro His Ser Ala Pro Tyr 530 535 540 Gly Pro Pro Gin Gly Pro Ala Ser His Ala Pro Thr Pro Pro Tyr Ala 545 550 555 560

Pro Ala Ala Cys Pro Pro Gly Pro Pro Pro Pro Pro Cys Pro Ser Thr 565 570 575

Gin Thr Arg Ala Pro Leu Pro Thr Glu Pro Ala Phe Pro Pro Ala Ala 580 585 590

Thr Gly Ser Gin Pro Glu Ala Ser Asn Ala Glu Ala Gly Ala Leu Val 595 600 605

Asn Ala Ser Ser Ala Ala His Val Asp Val Asp Thr Ala Arg Ala Ala 610 615 620 Asp Leu Phe Val Ser Gin Met Met Gly Ala Arg 625 630 635

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

CCGGTGCCCA ATCGTCCGT 19 (2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

GTCCGTGCGC GTCAAGTGG 19

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE^' DESCRIPTION: SEQ ID NO:20:

TTCCGGCTCC CCCACCTGA 19

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

( i) SEQUENCE DESCRIPTION: SEQ ID NO:21:

ATTCGGATCC TGGAGGCGA 19

Claims

1. An essentially pure protein encoded by an HSN-2 UL26 gene and functional fragments thereof.

2. The essentially pure protein of claim 1 wherein said protein is selected from the group consisting of HSN-2 protease precursor protein, mature HSN-2 protease and functional fragments of said mature HSN-2 protease.

3. The essentially pure protein of claim 1 wherein said protein is mature HS V-2 protease.

4. An essentially pure protein encoded by HSV-2 UL26.5 gene or fragments thereof.

5. The essentially pure protein of claim 4 wherein said protein is selected from the group consisting of HSV-2 capsid precursor protein, mature HSV- 2 capsid protein and functional fragments thereof.

6. The essentially pure protein of claim 1 wherein said protein is mature

HS V-2 capsid protein.

7. An isolated nucleic acid molecules comprising an HSV-2 UL26 gene or functional fragments thereof.

8. The isolated nucleic atid molecule of claim 7 comprising a nucleotide sequence of SEQ ID NO: 1 or a functional fragment thereof.

9. The isolated nucleic acid molecule of claim 7 comprising a nucleotide sequence that encodes mature HS V-2 protease.

10. The isolated nucleic acid molecule of claim 7 comprising an HSV-2 UL26.5 gene or a functional fragment thereof.

11. The isolated nucleic acid molecule of claim 10 comprising a nucleotide sequence that encodes mature HS V-2 capsid protein.

12. The isolated nucleic acid molecule of claim 10 comprising the HSV- 2 UL26.5 promoter.

13. An expression vector comprising an HSV-2 UL26 gene or functional fragment thereof.

14. The expression vector of claim 13 wherein said UL26 gene is disclosed in SEQ ID NO:l.

15. The expression vector of claim 13 wherein said fragment of said UL26 gene is selected from the group consisting of: a nucleotide sequence that encodes mature HSV-2 protease, a nucleotide sequence that encodes mature HSV-2 capsid protein, a nucleotide sequence that encodes an HSV-2 UL26.5 gene, a nucleotide sequence that encodes mature HSV-2 capsid protein and the HSV-2 UL26.5 promoter.

16. A host cell that has been transformed with an expression vector of claim 13, said host cell being capable of expressing said UL26 gene or functional fragment thereof.

17. A method of identifying compounds that inhibit HS V-2 protease activity comprising the steps of: a) contacting HSV-2 protease or functional fragment thereof with an HS V-2 protease substrate in the presence of a test compound; b) detecting the level of proteolytic cleavage of said substrate; and c) comparing that level to the level of proteolytic activity that occurs when HS V-2 protease or functional fragment thereof is contacted with an HS V-2 protease substrate in the absence of a test compound.

18. A method of identifying compounds that inhibit HSV-2 virion assembly comprising a) in the presence of a test compound, contacting two or more proteins that comprise at least portions of HSV-2 capsid protein in the presence of a test compound; b) detecting the level of capsid-capsid association; and c) comparing said level of capsid-capsid association to the level of capsid-capsid association that occurs when two or more proteins that comprise at least portions of HSV-2 capsid protein are contacted in the absence of the test compound.

19. A synthetic HSV-2 protease substrate having the formula R, - SEQ

ID NO:3 - R₂ or R, - SEQ ID NO:4 - R₂.

20. The synthetic HSV-2 protease substrate of claim 19 selected from the group consisting of: SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15.

21. An antibody that selectively binds to an unprocessed HSV-2 protease wherein said antibody is incapable of binding to a processed HSV-2 substrate.

22. A method of distinguishing between HSV- 1 DNA and HSV-2 DNA comprising the steps of: a) amplifying DNA in a sample using primers which amplify

HSV-1 DNA but which do not amplify HSV-2 DNA and/or amplifying DNA in a sample using primers which amplify HSV-2 DNA but which do not amplify HSV-1 DNA; b) detecting the presence of amplified DNA.

23. A set of PCR primers comprising nucleotide sequences which can be used to amplify HSV-1 DNA but cannot be used to amplify HSV-2 DNA or comprising nucleotide sequences which can be used to amplify HSV-2 DNA but cannot be used to amplify HSV-1 DNA.

24. A kit for distinguishing between HSV- 1 DNA and HS V-2 DNA comprising a container comprising a set of PCR primers of claim 23 and a container comprising a DNA size marker molecule.

25. A method of distinguishing between HS V-l protein and HS V-2 protein comprising the steps of: a) performing an immunoassay using antibodies capable of selectively binding to HSV-1 protein and incapable of binding to HSV-2 protein and/or performing an immunoassay using antibodies capable of selectively binding to HSV-2 protein incapable of binding to HSV-1 protein; and b) detecting the presence of bound antibodies.

26. An antibody capable of selectively binding to HS V-2 protein and incapable of binding to HSV-1 protein and an antibody capable of selectively binding to HSV- 1 protein and incapable of binding to HS V-2 protein.

27. A kit for distinguishing between HSV-1 protein and HSV-2 protein comprising a container comprising an antibody of claim 26 and/or a container comprising an antibody capable of selectively binding to HSV-1 protein and incapable of binding to HS V-2 protein and/or antibody capable of selectively binding to HS V-2 protein and incapable of binding to HSV- 1 protein.

28. HSV-2 protease inhibitor compounds identified by the method of claim 17.

29. HSV-2 virion assembly inhibiting compounds identified by the method of claim 18.