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Retroviral Enzymes
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Retroviral Enzymes

A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Animal Viruses".

Deadline for manuscript submissions: closed (30 December 2009) | Viewed by 239727

Special Issue Editor

Dr. Luis Menéndez-Arias

E-Mail Website
Guest Editor
Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, c/ Nicolás Cabrera 1, Campus de Cantoblanco, 28049 Madrid, Spain
Interests: HIV; reverse transcription; drug resistance; genetic variation; proteolytic processing; HIV protease
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The retroviral RNA genome encodes for three enzymes essential for virus replication: (i) the viral protease (PR), that converts the immature virion into a mature virus through the cleavage of precursor polypeptides; (ii) the reverse transcriptase (RT), responsible for the conversion of the single-stranded genomic RNA into double-stranded proviral DNA; and (iii) the integrase (IN) that inserts the proviral DNA into the host cell genome. All of them are important targets for therapeutic intervention. Knowledge on their structure and mechanism of action should help us to design better drugs against AIDS and other diseases caused by retroviruses.

Dr. Luis Menéndez-Arias
Guest Editor

Keywords

  • aspartyl-proteases
  • reverse transcription
  • integration
  • virus maturation
  • antiretroviral drugs
  • drug targets
  • viral mutation and variability

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Editorial

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152 KiB  
Editorial
Special Issue: Retroviral Enzymes
by Luis Menéndez-Arias
Viruses 2010, 2(5), 1181-1184; https://doi.org/10.3390/v2051181 - 7 May 2010
Cited by 4 | Viewed by 8279
Abstract
The retroviral RNA genome encodes for three enzymes essential for virus replication: (i) the viral protease (PR), that converts the immature virion into a mature virus through the cleavage of precursor polypeptides; (ii) the reverse transcriptase (RT), responsible for the conversion of the [...] Read more.
The retroviral RNA genome encodes for three enzymes essential for virus replication: (i) the viral protease (PR), that converts the immature virion into a mature virus through the cleavage of precursor polypeptides; (ii) the reverse transcriptase (RT), responsible for the conversion of the single-stranded genomic RNA into double-stranded proviral DNA; and (iii) the integrase (IN) that inserts the proviral DNA into the host cell genome. All of them are important targets for therapeutic intervention. This Special Issue provides authoritative reviews on the most recent research towards a better understanding of structure-function relationships in retroviral enzymes. The Issue includes three reviews on retroviral PRs, seven on RT and reverse transcription, and four dedicated to viral integration. [...] Full article
(This article belongs to the Special Issue Retroviral Enzymes)

Review

Jump to: Editorial

254 KiB  
Review
Interactions of Host Proteins with the Murine Leukemia Virus Integrase
by Barbara Studamire and Stephen P. Goff
Viruses 2010, 2(5), 1110-1145; https://doi.org/10.3390/v2051110 - 5 May 2010
Cited by 11 | Viewed by 11375
Abstract
Retroviral infections cause a variety of cancers in animals and a number of diverse diseases in humans such as leukemia and acquired immune deficiency syndrome. Productive and efficient proviral integration is critical for retroviral function and is the key step in establishing a [...] Read more.
Retroviral infections cause a variety of cancers in animals and a number of diverse diseases in humans such as leukemia and acquired immune deficiency syndrome. Productive and efficient proviral integration is critical for retroviral function and is the key step in establishing a stable and productive infection, as well as the mechanism by which host genes are activated in leukemogenesis. Host factors are widely anticipated to be involved in all stages of the retroviral life cycle, and the identification of integrase interacting factors has the potential to increase our understanding of mechanisms by which the incoming virus might appropriate cellular proteins to target and capture host DNA sequences. Identification of MoMLV integrase interacting host factors may be key to designing efficient and benign retroviral-based gene therapy vectors; key to understanding the basic mechanism of integration; and key in designing efficient integrase inhibitors. In this review, we discuss current progress in the field of MoMLV integrase interacting proteins and possible roles for these proteins in integration. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Early events in MLV infection leading to the production of the integrated provirus. Virions deliver a viral core particle into the cytoplasm, which carries out the process of reverse transcription to form the Pre-Integration Complex or PIC, containing the Integrase enzyme (red) bound to the termini of the Long Terminal Repeats or LTRs (grey boxes) of the viral DNA (green helix). The PIC enters the nucleus, likely via the dispersion of the nuclear membrane during mitosis. The incoming DNA is then integrated into the host genome (blue helix). DNAs that fail to integrate are often circularized by host repair enzymes to form circular DNAs with one or two copies of the viral LTRs.</p> Full article ">
414 KiB  
Review
Implications of the Nucleocapsid and the Microenvironment in Retroviral Reverse Transcription
by Marylène Mougel, Andrea Cimarelli and Jean-Luc Darlix
Viruses 2010, 2(4), 939-960; https://doi.org/10.3390/v2040939 - 2 Apr 2010
Cited by 14 | Viewed by 13225
Abstract
This mini-review summarizes the process of reverse-transcription, an obligatory step in retrovirus replication during which the retroviral RNA/DNA-dependent DNA polymerase (RT) copies the single-stranded genomic RNA to generate the double-stranded viral DNA while degrading the genomic RNA via its associated RNase H activity. [...] Read more.
This mini-review summarizes the process of reverse-transcription, an obligatory step in retrovirus replication during which the retroviral RNA/DNA-dependent DNA polymerase (RT) copies the single-stranded genomic RNA to generate the double-stranded viral DNA while degrading the genomic RNA via its associated RNase H activity. The hybridization of complementary viral sequences by the nucleocapsid protein (NC) receives a special focus, since it acts to chaperone the strand transfers obligatory for synthesis of the complete viral DNA and flanking long terminal repeats (LTR). Since the physiological microenvironment can impact on reverse-transcription, this mini-review also focuses on factors present in the intra-cellular or extra-cellular milieu that can drastically influence both the timing and the activity of reverse-transcription and hence virus infectivity. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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Figure 1

Figure 1
<p>Schematic representation of the replication of a simple retrotransposon. a. The genomic RNA is a unique RNA synthesized by transcription of the integrated retrotransposon DNA. <b>b.-c.</b> The RNA copy is exported from the nucleus and translated by the cellular translation machinery – ribosomes are illustrated here - to produce the Gag and GagPol like polyprotein precursors. <b>d.-e.</b> During formation of a ribonucleoparticle (RNP) called VLP (virus-like particle or VLP) the Gag and GagPol precursors undergo maturation by a Pol-encoded protease. At the same time the RNA copy of the retrotransposon, together with the replication primer tRNA are incorporated into the VLP. Note that the VLP’s remain in the cytoplasm and are not exported (cross) contrary to replication-competent retroviruses. <b>f.</b> Reverse transcription of the RNA copy is carried out by the RT and is chaperoned by NC-like proteins in the VLP nucleoprotein structure to generate a new copy of retrotransposon DNA. <b>g.-h.</b> The new DNA copy is imported into the nucleus and integrated into the host cell genome by the Pol-encoded integrase to complete the copy-and-paste process.</p> Full article ">Figure 2
<p>In vitro model systems to study retroviral reverse transcription. Flexible <span class="html-italic">in vitro</span> model systems have been set up to study in detail the process of retrovirus reverse transcription [21,23,24,25,30]. Such models include (i) <span class="html-italic">in vitro</span> generated RNA (vRNA) representing the 5’ and 3’ UTR domains containing the <span class="html-italic">cis</span>-acting elements essential for cDNA synthesis, namely the PBS, the binding site for the replication primer tRNA, the untranslated 5’ and 3’ regions (U5 and U3), the repeats (R in blue) and the polypurine tract (PPT); (ii) Replication primer tRNA of natural origin (P-tRNA) or generated by <span class="html-italic">in vitro</span> transcription, or a synthetic oligonucleotide complementary to the PBS; (iii) the RT enzyme (not shown); (iv) NC protein (not shown); (v) if required, the IN enzyme, VIF, VPR and cellular factors such as SEVI [31].<span class="html-italic">In vitro</span> models such as this have rendered possible a detailed investigation of the essential steps of reverse transcription, following tRNA annealing to the PBS by NC: a.initiation of ss-cDNA synthesis (see large orange arrow);the first strand transfer which corresponds to an annealing reaction chaperoned by NC and requiring the R sequences (white arrow) [31,32];b. minus-strand cDNA elongation (double orange arrow);c. initiation of plus-strand DNA synthesis and transfer (not shown here for the sake of clarity; see also Figure 3);d. the fidelity of the strand transfer and of cDNA synthesis by RT and the influence of RT mutations;e. the role of the RT-associated RNase H activity on the strand transfer;f. the role of NC on DNA strand transfer and the fidelity of reverse transcription via its interaction with RT and the vRNA;g. the influence of vRNA mutations, incubation conditions (ions, temperature, nucleotides <span class="html-italic">etc.</span>) and viral and cellular factors such as VIF, SEVI.</p> Full article ">Figure 3
<p>Illustration of the reverse transcription process. The individual steps are as follows. <b>a.-b.</b> annealing of the replication primer tRNA by NC. Stars correspond to modified nucleotides in the primer tRNA, notably m6A at position 58 important for the fidelity of the plus-strand DNA transfer and in the anti-codon loop recognized by RT. <b>c.</b> Initiation of cDNA synthesis by RT by extension of the –CCA 3’ terminal nucleotides. <b>d.</b> SscDNA(-) transfer to the RNA 3’ R sequences by NC. <b>e.</b> minus -trand DNA transfer by RT. <b>f. </b>Initiation of plus-strand DNA by extension of the PPT RNA by RT. <b>g.-h.</b> Plus-strand DNA transfer at the level of the PBS sequences by NC and elongation of viral DNA strands by RT that includes ds DNA unwinding to complete LTR DNA synthesis. <b>i.</b> The linear ds DNA is shown here with the LTR’s and the terminal TG/CA nucleotides.</p> Full article ">Figure 4
<p>Molecular interactions in the course of reverse transcription. This scheme illustrates essential molecular interactions taking place prior to and during reverse transcription. (i) The genomic RNA is in a dimeric form where there are many RNA-RNA interactions, in addition to the Dimer Linkage Structure (DLS). (ii) Several hundred NC molecules, in a poorly characterized oligomeric form [72] (see top arrow pointing to NC-NC interactions), coat the genomic RNA providing protection against cellular nucleases and UV irradiation; (iii) A number of small cellular RNAs are incorporated into virions via interactions with Gag-NC and Pol-RT and Pol-IN (not illustrated here); except for the primer tRNA the function, if any, of the other cellular RNAs is poorly understood. (iv) The RT and IN enzymes interact with the genomic RNA-NC complex ensuring reverse transcription and ultimately integration of the newly made viral DNA. (v) In the absence of the viral factor VIF, APOBEC restriction factors are incorporated into virions via interactions with the viral RNA and NC, which results in the production of highly mutated viral DNA molecules. (vi) The reverse transcription machinery is housed within the incoming virion core where capsid protein molecules provide protection against host restriction factors such as TRIM proteins (see also text). (vii) Small amounts of the viral transactivator TAT have been found in the virion core. Tat may counteract the negative impact of cellular miRNA on the stability of the viral RNA prior to virion formation [124].</p> Full article ">
814 KiB  
Review
HIV-1 Ribonuclease H: Structure, Catalytic Mechanism and Inhibitors
by Greg L. Beilhartz and Matthias Götte
Viruses 2010, 2(4), 900-926; https://doi.org/10.3390/v2040900 - 30 Mar 2010
Cited by 68 | Viewed by 16517
Abstract
Since the human immunodeficiency virus (HIV) was discovered as the etiological agent of acquired immunodeficiency syndrome (AIDS), it has encouraged much research into antiviral compounds. The reverse transcriptase (RT) of HIV has been a main target for antiviral drugs. However, all drugs developed [...] Read more.
Since the human immunodeficiency virus (HIV) was discovered as the etiological agent of acquired immunodeficiency syndrome (AIDS), it has encouraged much research into antiviral compounds. The reverse transcriptase (RT) of HIV has been a main target for antiviral drugs. However, all drugs developed so far inhibit the polymerase function of the enzyme, while none of the approved antiviral agents inhibit specifically the necessary ribonuclease H (RNase H) function of RT. This review provides a background on structure-function relationships of HIV-1 RNase H, as well as an outline of current attempts to develop novel, potent chemotherapeutics against a difficult drug target. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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Figure 1

Figure 1
<p>Schematic of the process of reverse transcription. <b>A</b> The viral RNA genome is shown as a thick red line. Reverse transcription is initiated by the binding of an endogenous tRNA<sup>lys3</sup> molecule to the primer binding site (PBS) on the genome. <b>B</b> RT elongates the tRNA primer to the 5’ end of the genome, creating a fragment called (-)-strand strong stop DNA ((-)ssDNA). The RNase H activity of RT concomitantly degrades the RNA genome during DNA synthesis. The degradation of the 5’ end of the genome is necessary for (-)-strand transfer, and failure to degrade the RNA at this point results in the arrest of reverse transcription. <b>C</b> First or (-)-strand transfer. The (-)ssDNA fragment dissociates from the PBS sequence and re-associates with the repeat (R) sequence at the 3’ end of the genome. This step is capable of both intrastrand and interstrand transfer. <b>D </b>Continuation of (-)-strand DNA synthesis. RT extends the 3’ end of the (-)ssDNA fragment toward the PBS sequence, while the RNase H activity concomitantly degrades the RNA genome, which the exception of the polypurine tract (PPT). <b>E</b> The PPT is used as the primer for the initiation of (+)-strand DNA synthesis. The PPT primer is extended by the RT polymerase activity. <b>F</b> After approximately 12 nucleotides have been added, the PPT primer is removed by RNase H activity. The nascent (+)-strand DNA is extended to the 5’ end of the (-)-strand DNA, copying the PBS sequence from the tRNA that is still associated with the (-)-strand DNA. Here, the tRNA is removed by the RNase H activity, leaving a single ribonucleotide (rA) at the 3’ end of the U5 sequence (shown in red). <b>G</b> In the second, or (+)-strand transfer, the PBS sequences on both strands associate. This step occurs predominantly in an intrastrand fashion. <b>H</b> Both DNA strands are extended to the ends of their templates, forming the provirus that is ready to be integrated into the host genome by integrase. The long terminal repeats (LTRs) that are formed as a result of reverse transcription are shown.</p> Full article ">Figure 2
<p><b>A</b> Crystal structure of HIV-1 RT (PDB code: 1RTD. The p51 subunit is shown in orange, while the p66 subunit is divided into the fingers (cyan), connection (blue) and RNase H (grey) subdomains. The residues of the conserved DEDD motif are shown and red and marked with arrows. <b>B</b> Crystal structures of the RNase H domain of HIV-1 RT (PDB code:1RTD) [<a href="#B5" class="html-bibr">5</a>], human RNase H1 (PDB code: 2QKB) [<a href="#B12" class="html-bibr">12</a>] and <span class="html-italic">E. coli</span> RNase H1 (PBD code: 1WSJ). All three show the same mixed beta-sheet with asymmetric alpha helices, while the human and <span class="html-italic">E. coli</span> RNases H contain the C-helix, or basic loop.</p> Full article ">Figure 3
<p>The chemistry of RNase H cleavage is believed to be a two-metal ion mechanism. <b>A</b> Two divalent metal ions (red spheres, marked A and B) are coordinated by the active site residues D549, D443, D498 and E478 approximately 4Å apart. Metal ion A activates a water molecule. <b>B </b>The activated water molecule carries out a nucleophilic attack (blue arrow) driving the phosphoryl transfer reaction. <b>C </b>In the putative transition state, the metal ions move toward each other to bring the nucleophile within range of the scissile phosphate. <b>D</b> The reaction products consist of a 3’ OH group and a 5’ phosphate group, and the metal ions are again likely to be re-positioned.</p> Full article ">Figure 4
<p>HIV RT can exist in two distinct binding modes when bound to a nucleic acid substrate. The polymerase-dependent mode is characterized by the polymerase active site being in contact with the 3’ primer terminus. All other possible conformations are considered polymerase-independent.</p> Full article ">Figure 5
<p>Polymerase-dependent binding can occur in two distinct positions. Post-translocation, where the 3’ primer terminus occupies the P site, leaving the N site available for nucleotide binding, or pre-translocation, where the N-site is occupied by the 3’ primer terminus and the incoming nucleotide is blocked by the primer terminus. An RT enzyme bound in a polymerase-dependent mode is in thermodynamic equilibrium between both pre- and post-translocational positions. The equilibrium is sequence-dependent.</p> Full article ">Figure 6
<p>During (-)-strand DNA synthesis, the PPT region of the RNA genome is resistant to RNase H cleavage, while a portion of the RNA genome is degraded concomitantly with (-)-strand DNA synthesis. Here, a specific cleavage is made to create the PPT primer. <b>B</b> The RNase H-resistant PPT sequence forms the primer for (+)-strand DNA synthesis when the rest of the genome is completely degraded by RNase H. <b>C</b> The RNA primer is extended 12 nucleotides, <b>D </b>then RT pauses and changes orientations to a polymerase-independent binding mode in order to cleave at the DNA:RNA junction, and remove the PPT primer. <b>E</b> The 12-mer fragment is extended toward the 5’ end of the (-)-strand DNA (see <a href="#figure1" class="html-fig">Figure 1</a>F). After the second strand transfer event, the (+)-strand DNA is fully synthesized resulting in a fully double-stranded provirus. Adapted from [<a href="#B49" class="html-bibr">49</a>].</p> Full article ">Figure 7
<p>Examples of the main classes of small molecule inhibitors against HIV-1 RT-associated RNase H activity. <b>A</b> N-hydroxyimides. Developed from influenza inhibitors and among the first to use the 3-oxygen pharmacophore [<a href="#B84" class="html-bibr">84</a>]. <b>B </b>Pyrimidinol carboxylic acid derivatives [<a href="#B85" class="html-bibr">85</a>]. Potent inhibitors based on the successful scaffold of the metal-chelating active site inhibitors. <b>C</b> Diketo acid derivatives are also active in some cases against the viral integrase. RDS1643 shown here is the only diketo acid to have antiviral activity <span class="html-italic">in vivo</span> [<a href="#B86" class="html-bibr">86</a>]. <b>D</b> The vinylogous ureas (NSC727447 pictured here) represent a different kind of inhibitor that binds allosterically near the p51 thumb [<a href="#B87" class="html-bibr">87</a>]. <b>E</b> N-acyl hydrazones appear to bind to multiple binding sites depending on the specific inhibitor, including the RNase H domain and a site that overlaps the NNRTI binding pocket (DHBNH picture above) [<a href="#B88" class="html-bibr">88</a>]. <b>F</b> Hydroxylated tropolones (β-thujaplicinol pictured here) are the subject of several studies that have provided the basis for a biochemical mechanism of inhibition by active site RNase H inhibitors [<a href="#B39" class="html-bibr">39</a>,<a href="#B89" class="html-bibr">89</a>,<a href="#B90" class="html-bibr">90</a>].</p> Full article ">Figure 8
<p>A schematic of possible models of active site inhibitor binding, based on studies with the tropolone derivative β-thujaplicinol (blue sphere) [<a href="#B39" class="html-bibr">39</a>,<a href="#B90" class="html-bibr">90</a>]. Evidence suggests that the inhibitor is unable to bind to an enzyme-substrate (E-S) complex (top left), only to free enzyme forming (top right) and enzyme-inhibitor (E-I) complex (bottom right). However, the substrate might be able to bind to this E-I complex, forming an E-S-I complex that is not productive with respect to RNase H cleavage (bottom left). As suggested by Himmel <span class="html-italic">et al.</span>, the inhibitor occupies the position normally claimed by the scissile phosphate [<a href="#B90" class="html-bibr">90</a>]. As such, it is possible that the substrate undergoes a change in trajectory in relation to the scissile phosphate and the RNase H active site [<a href="#B39" class="html-bibr">39</a>]. Then eventually, the inhibitor dissociates and RT is allowed to cleave the uninhibited substrate (E-S complex, top left).</p> Full article ">
2554 KiB  
Review
Structural Aspects of Drug Resistance and Inhibition of HIV-1 Reverse Transcriptase
by Kamalendra Singh, Bruno Marchand, Karen A. Kirby, Eleftherios Michailidis and Stefan G. Sarafianos
Viruses 2010, 2(2), 606-638; https://doi.org/10.3390/v2020606 - 11 Feb 2010
Cited by 70 | Viewed by 20216
Abstract
HIV-1 Reverse Transcriptase (HIV-1 RT) has been the target of numerous approved anti-AIDS drugs that are key components of Highly Active Anti-Retroviral Therapies (HAART). It remains the target of extensive structural studies that continue unabated for almost twenty years. The crystal structures of [...] Read more.
HIV-1 Reverse Transcriptase (HIV-1 RT) has been the target of numerous approved anti-AIDS drugs that are key components of Highly Active Anti-Retroviral Therapies (HAART). It remains the target of extensive structural studies that continue unabated for almost twenty years. The crystal structures of wild-type or drug-resistant mutant HIV RTs in the unliganded form or in complex with substrates and/or drugs have offered valuable glimpses into the enzyme’s folding and its interactions with DNA and dNTP substrates, as well as with nucleos(t)ide reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTIs) drugs. These studies have been used to interpret a large body of biochemical results and have paved the way for innovative biochemical experiments designed to elucidate the mechanisms of catalysis and drug inhibition of polymerase and RNase H functions of RT. In turn, the combined use of structural biology and biochemical approaches has led to the discovery of novel mechanisms of drug resistance and has contributed to the design of new drugs with improved potency and ability to suppress multi-drug resistant strains. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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Figure 1

Figure 1
<p>The crystal structure of HIV-1 RT bound to double stranded DNA (PDB code 2hmi). HIV-1 RT functions as heterodimer of p66 and p51 subunits. Due to the resemblance of p66 to a closed right hand, subdomains of p66 have been named as the ‘palm’ (red), fingers (blue), and thumb (green). The p66 subdomain contains two active sites, the polymerase and the RNase H active sites (orange). The region between the RNase H and polymerase active sites is known as the connection (yellow) subdomain. The p51 (dark brown) subunit is derived from the proteolytic cleavage of RNase H from p66 and has identical primary and secondary structure. However, the tertiary structure of p51 is markedly different than p66 leading to a non-functional arrangement of catalytic residues. The template/primer (white/gray) is seen in the DNA-binding cleft formed primarily by the p66 subunit of the enzyme. Figures 1, 2, 3, 7, 9 and 11 were generated using PyMOL [<a href="#" class="html-">4</a>].</p> Full article ">Figure 2
<p>Interactions of RNA/DNA and DNA/DNA template/primers with HIV-1 RT. The two different template/primers (cyan/red) bind in the nucleic acid binding cleft of RT in a similar way. The RNA/DNA (panel A; PDB code 1hys) maintains the protein contacts seen in the complex with DNA/DNA (panel B, PDB code 2hmi) (these contacts are not shown in panel A. and has thirteen additional contacts. Nine of these contacts are through the 2’-OH group of the RNA sugar backbone (blue), whereas four are mediated through phosphate-backbone of RNA/DNA (plum). In panel B, the template-protein contacts are colored yellow and the primer-protein contacts are colored magenta.</p> Full article ">Figure 3
<p>A. The effect of sugar ring conformation on catalysis of DNA synthesis by HIV-1 RT. Sugar-ring conformation of the nucleotide at the 3’-primer end (panel A). For efficient DNA synthesis to occur, the sugar ring conformation of the nucleotide at the 3’-primer end should be in the north (2’-exo/3’-endo) conformation (panel A, shown in yellow). The south (2’-endo/3’-exo) conformation (panel A, shown in cyan) of the sugar ring at the primer terminus mispositions the primer 3’-OH away for an in-line nucleophilic attack on the α-phosphate of the incoming dNTP (green), thereby resulting in inefficient catalysis. <b>B.</b> The sugar-ring conformation of the incoming dNTP should be north (panel B, green). If the incoming dNTP or nucleotide analog were to have a south conformation (panel B, cyan) this would result in steric hindrance with the aromatic ring of Y115 (shown in red). Thus, the favored conformation of the incoming dNTP is the north conformation (panel B, green). The software Coot [<a href="#" class="html-">55</a>] was used to prepare various sugar ring conformations of the primer terminus and incoming dNTP, starting from the structural coordinates of the HIV-1 RT/DNA/dNTP ternary complex (PDB Code 1rtd).</p> Full article ">Figure 4
<p>Stereo-view of the polymerase active site of HIV-1 RT. The primer strand is shown in gray, the incoming dNTP in orange, and the RT active-site residues interacting with substrates or metal ions in cyan. Metal ions A and B are shown as magenta bullets. The Cα-traces of the p66 palm and fingers subdomains are shown in red and blue, respectively. The yellow dotted lines depict the coordination geometry of the metal ions and the interactions of p66 fingers-subdomain residues with the incoming dNTP. The coordination geometry of metal ion B (also known as structural metal ion) is octahedral. Due to lack of the primer 3’OH group in the crystal structure (PDB file 1rtd), the coordination of metal ion A (known as catalytic metal) is incomplete. Interactions of fingers-subdomain residues K65, R72, and Q151 with dNTP are also shown in yellow dotted lines. Figures 4, 5, and 7 were generated by MolMol [<a href="#" class="html-">58</a>].</p> Full article ">Figure 5
<p>Conformational changes of p66 fingers and thumb subdomains during DNA synthesis by HIV-1 RT. Similar to other nucleic acid polymerases, HIV-1 RT undergoes conformational changes at various steps of the catalytic cycle. In the unliganded HIV-1 RT (E, shown as red tracing), the fingers and thumb subdomains fold over the active site to render it inaccessible. Binding of the template/primer opens up the fingers and thumb subdomains to accommodate the DNA/DNA or RNA/DNA substrates (green tracing). The binding of incoming dNTP causes the p66 fingers subdomain to move to a closed form and trap dNTP in a catalytically competent conformation (cyan tracing). After incorporation of dNMP, release of PPi, and translocation of the elongated template/primer, HIV-1 RT assumes the conformation seen in enzyme-DNA bound structure (green).</p> Full article ">Figure 6
<p>Nucleoside inhibitors (NRTIs) of HIV-1 RT: Chemical structures of NRTIs. With the exception of EFdA, all NRTIs lack a 3’ OH moiety. After their incorporation into DNA by HIV-1 RT, they act as chain terminators. In contrast, EFdA contains a 3’-OH and acts as a translcoation-defective reverse transcriptase inhibitor. FTC is 5-fluoro derivative of 3TC; the latter has not been included in this figure. Chemical structures were drawn with Chem Sketch 3.5 [<a href="#" class="html-">82</a>].</p> Full article ">Figure 7
<p>Stereo-view of the NNRTI binding site. NNRTIs bind in a pocket formed primarily by hydrophobic residues, which are rendered as cyan sticks with N and O atoms colored blue and red, respectively. A recently approved NNRTI, etravirine, is shown in gray. The p66 palm, thumb, and connection subdomains are shown in red, green, and yellow, respectively. Structural coordinates are from PDB code 1sv5.</p> Full article ">Figure 8
<p>Mechanism of NNRTI inhibition: NNRTI-binding alters the geometry of the p66 thumb subdomain, changes the position of the ‘primer grip’, and causes misalignment of important components at the polymerase active site. The structure of RT in complex with TIBO (cyan and gray ribbons) is superposed onto the DNA-bound RT (red) to demonstrate the shift in the position of ‘primer grip’ as a result of NNRTI binding. Residues Y181 and D185 are shown as reference points for the NNRTI binding pocket and YMDD loop, respectively. The TIBO inhibitor is shown as orange balls and sticks, whereas the template/primer (gray/blue) is shown as ribbons and plates.</p> Full article ">Figure 9
<p>Structural components of NRTI resistance by the excision mechanism: Molecular model showing the binding of ATP (red) to AZT-resistant HIV-1 RT and unblocking of AZTMP-terminated primer. Van der Waals surfaces are drawn for polymerase active site residues (magenta) and residues involved in ATP binding and AZT resistance (yellow). Mutated amino acids M41L, K70R, L210W, and T215Y are shown with black labels. The two terminal nucleotide base pairs of the template/primer are shown. The 3′ end of the primer is AZTMP and is positioned at the N site. AZT-resistant enzyme has the amino acid substitutions M41L, D67N, K70R, T215Y, and K219Q. Y215 is likely to have aromatic interactions with the purine ring of ATP (red). R70 is likely to interact with the ribose ring and/or the α-phosphate of ATP. The wild-type amino acids at K219 and D67 were retained in the figure to show a potential salt bridge between the residues.</p> Full article ">Figure 10
<p>The NNRTI binding pocket (NNIBP) in various RT/NNRTI complexes. Clockwise from top left: RT/Etravirine (PDB Code 1sv5), RT/nevirapine (1vrt), RT/delavirdine (1klm), and RT/efavirenz (1ikw). Mutations conferring resistance to these drugs occur primarily in and around the NNIBP. Residue D185 of the YMDD loop is labeled as a polymerase active site reference point. The p66 palm, thumb, and connection subdomains are shown in red, green, and yellow, respectively.</p> Full article ">Figure 11
<p>Binding of TMC-278 at the NNIBP results in a conformational change of residues Y181 and Y188. In panel A, comparison of unliganded RT (green ribbon and side chains) and RT/TMC-278 complex (blue ribbon and white side chains) shows that the aromatic rings of Y181 and Y188 “flip” as a result of TMC-278 binding to HIV-1 RT. Panel B shows the comparison of WT (blue ribbon, white side chains, and gray TMC-278) and Y181C/K103N (cyan ribbon, yellow side chains, and orange TMC-278) RT complexes with TMC-278. The yellow side chains belong to the TMC-278 bound K103N mutant RT. This figure also shows that the loss of interaction due to Y181C mutation is compensated by the interaction between cyanovinyl group and conserved Y183 (based on structures from pdb codes: 1dlo, 2zd1 and 3bgr).</p> Full article ">
623 KiB  
Review
Initiation of HIV Reverse Transcription
by Catherine Isel, Chantal Ehresmann and Roland Marquet
Viruses 2010, 2(1), 213-243; https://doi.org/10.3390/v2010213 - 18 Jan 2010
Cited by 40 | Viewed by 16011
Abstract
Reverse transcription of retroviral genomes into double stranded DNA is a key event for viral replication. The very first stage of HIV reverse transcription, the initiation step, involves viral and cellular partners that are selectively packaged into the viral particle, leading to an [...] Read more.
Reverse transcription of retroviral genomes into double stranded DNA is a key event for viral replication. The very first stage of HIV reverse transcription, the initiation step, involves viral and cellular partners that are selectively packaged into the viral particle, leading to an RNA/protein complex with very specific structural and functional features, some of which being, in the case of HIV-1, linked to particular isolates. Recent understanding of the tight spatio-temporal regulation of reverse transcription and its importance for viral infectivity further points toward reverse transcription and potentially its initiation step as an important drug target. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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Figure 1
<p>Secondary <b>(a)</b> and tertiary <b>(b)</b> structures of tRNA<sub>3</sub><sup>Lys</sup>.</p> Full article ">Figure 2
<p>Partners and model for the tRNA<sub>3</sub><sup>Lys</sup> packaging complex <b>(a)</b> Organisation of the Gag-Pol and Gag precursors. MA: matrix ; CA: capsid ; NC: nucleocapsid ; p6: HIV-1 p6 protein ; PR: protease; RT: reverse transcriptase, with its Fingers, Palm, Thumb, Connection (Conn.) and RNase H domains; IN: integrase. The dark blue rectangle in the capsid domain of the precursors corresponds to the C-terminal helix 4 that was shown to interact with lysyl-tRNA synthetase (LysRS) <b>(b)</b>Organisation of LysRS: the anticodon (AC) binding domain is located between positions 125 and 207 and the LysRS dimerisation (Dz) domain between positions 238 and 266. The two boxes toward the C-terminus of the protein, between positions 314 and 343 and 544 and 559 are important for amino acid recognition. The area highlighted in red (208-259), overlapping the dimerisation domain, is involved in binding to the capsid.<b> (c) </b>Model for the packaging complex. The purple patch corresponds to the anticodon binding domain and the red patch to amino acids 208-259 that interact with the CA domain of Gag.</p> Full article ">Figure 3
<p>Schematic representation of HIV-1 specific initiation<b> (a)</b><span class="html-italic">versus</span> unspecific elongation <b>(b)</b> of reverse transcription. The vRNA template is represented by a thin grey line. The natural tRNA<sub>3</sub><sup>Lys</sup> primer or an 18 mer DNA primer are in black and the newly synthesized DNA is represented by thick blue and red lines representative of the initiation and elongation steps of reverse transcription, respectively. In the presence of the natural tRNA primer, transition between initiation and elongation occurs after the addition of the first 6 nucleotides to the 3’ end of the primer. In the case of the HIV-1 MAL isolate, transition is facilitated by the anticodon/A-rich loop interaction upstream of the PBS, represented by the close contact between tRNA<sub>3</sub><sup>Lys</sup> and the vRNA.</p> Full article ">Figure 4
<p>Secondary structure of the RNA partners of the HIV-1 initiation complex of reverse transcription and of the binary primer/template complexes, in the case of the HIV-1 MAL (representative of a subtype A PBS domain) and subtype B isolates.The regions undergoing intra- or intermolecular rearrangements upon formation of the primer/template complex are highlighted in various colours. Boxes or sequences of the same color represent areas that are base-paired in the binary complex.<b> (a)</b> The human tRNA<sub>3</sub><sup>Lys</sup>. <b>(b)</b> The PBS sub-domain in the free form of the HIV-1 MAL vRNA. <b>(c) </b>The HIV-1 MAL vRNA/tRNA<sub>3</sub><sup>Lys</sup> complex. <b>(d)</b> The PBS sub-domain in the free form of the HIV-1 NL-4.3 (subtype B) isolate. The PAS and mutations 2L and 2R are indicated. <b>(e)</b> Localization of the anti-PAS region of tRNA<sub>3</sub><sup>Lys</sup>.</p> Full article ">Figure 5
<p>Secondary structure models of the HIV-2 vRNA (<b>a</b>) and of the vRNA/tRNA<sub>3</sub><sup>Lys</sup> complex (<b>b</b>). The tRNA is in red and the vRNA in black.</p> Full article ">Figure 6
<p>Schematic representation of the temporal regulation of reverse transcription in producer and target cells.</p> Full article ">
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Review
Comparative Studies on Retroviral Proteases: Substrate Specificity
by József Tözsér
Viruses 2010, 2(1), 147-165; https://doi.org/10.3390/v2010147 - 14 Jan 2010
Cited by 37 | Viewed by 15120
Abstract
Exogenous retroviruses are subclassified into seven genera and include viruses that cause diseases in humans. The viral Gag and Gag-Pro-Pol polyproteins are processed by the retroviral protease in the last stage of replication and inhibitors of the HIV-1 protease are widely used in [...] Read more.
Exogenous retroviruses are subclassified into seven genera and include viruses that cause diseases in humans. The viral Gag and Gag-Pro-Pol polyproteins are processed by the retroviral protease in the last stage of replication and inhibitors of the HIV-1 protease are widely used in AIDS therapy. Resistant mutations occur in response to the drug therapy introducing residues that are frequently found in the equivalent position of other retroviral proteases. Therefore, besides helping to understand the general and specific features of these enzymes, comparative studies of retroviral proteases may help to understand the mutational capacity of the HIV-1 protease. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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<p>Phylogenetic relationship of retroviral proteases. Sequence alignment and the phylogenetic tree was made by ClustalW and Phylip programs, respectively. Abbreviations used: BLV, bovine leukemia virus; BFV, bovine foamy virus; EIAV, equine infectious anemia virus; EFV, equine foamy virus; FeLV, feline leukemia virus; FFV, feline foamy virus; FIV, feline immunodeficiency virus; GALV, gibbon-ape leukemia virus; HIV, human immunodeficiency virus; HTLV, human T-lymphotropic virus; HFV, human foamy virus; JSRV, jaagsiekte sheep retrovirus; MAV, myeloblastosis associated virus; MMLV, Moloney murine leukemia virus; MMTV, mouse mammary tumor virus; MPMV, Mason-Pfizer monkey virus; SIV, simian immunodeficiency virus; SFV, simian foamy virus; WDSV, walleye dermal sarcoma virus; WEHV, walleye epidermal hyperplasia virus; Proteases reviewed in this paper are in larger bold.</p> Full article ">Figure 2
<p>Naturally occurring cleavage sites in retroviral Gag and Gag-Pro-Pol polyproteins. The site cleaved by the cognate retroviral protease is indicated by an arrow. Type 1 cleavage sites are in red. Abbreviations used: CA, capsid; NC, nucleocapsid; TF, transframe protein; PR, protease; RH, RNaseH; IN, integrase; DU, dUTPase. Proteins and peptides with unidentified functions are abbreviated with the size of the protein in kDa (e.g., p12 is a protein having 12 kDa, while pp refers to phosphoprotein), as pX, or by the letter n.</p> Full article ">Figure 3
<p>Organization of the Gag-Pro-Pol proteins in lentiviral HIV-1, deltaretroviral HTLV-1, alpharetroviral MAV and spumaretroviral HFV. Sites of PR processing are indicated by arrows.</p> Full article ">Figure 4
<p>Structure-based sequence alignment of the retroviral proteases. Active site aspartate residues are in red, amino acid residues involved in substrate binding are in blue and underlined.</p> Full article ">Figure 5
<p>Schematic representation of the HIV-1 MA↓CA cleavage site substrate in the S4 S3' subsites of HIV-1 PR. The indicated substrate sequence was modeled into the binding site of the crystallographic structure of the PR. The relative size of each subsite is indicated approximately by the area enclosed by the curved line.</p> Full article ">Figure 6
<p>Sequence polymorphism and drug-resistant mutations of HIV-1 PR. Sequence of the HIV-1<sub>HXB2 </sub>PR is in green, natural variations are in blue, resistant mutations are in red. Those residues that can be observed at the equivalent position of another retroviral PR are shown in italics. Residues that are involved in ligand binding are marked with blue background.</p> Full article ">Figure 7
<p>Cross-activity of retroviral proteases assayed on oligopeptide substrates representing naturally occurring cleavage sites. For simplicity, the k<sub>cat</sub>/K<sub>m</sub> values (mM<sup>-1</sup>s<sup>-1</sup>) were indicated as follows: &lt; 0. 1: +; 0.1 – 1: ++; 1-10: +++; 10-100: ++++; &gt; 100: +++++. Parenthesis indicates shift in the site of cleavage.</p> Full article ">
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Review
Retroviral Integration Site Selection
by Sébastien Desfarges and Angela Ciuffi
Viruses 2010, 2(1), 111-130; https://doi.org/10.3390/v2010111 - 12 Jan 2010
Cited by 56 | Viewed by 18264
Abstract
The stable insertion of a copy of their genome into the host cell genome is an essential step of the life cycle of retroviruses. The site of viral DNA integration, mediated by the viral-encoded integrase enzyme, has important consequences for both the virus [...] Read more.
The stable insertion of a copy of their genome into the host cell genome is an essential step of the life cycle of retroviruses. The site of viral DNA integration, mediated by the viral-encoded integrase enzyme, has important consequences for both the virus and the host cell. The analysis of retroviral integration site distribution was facilitated by the availability of the human genome sequence, revealing the non-random feature of integration site selection and identifying different favored and disfavored genomic locations for individual retroviruses. This review will summarize the current knowledge about retroviral differences in their integration site preferences as well as the mechanisms involved in this process. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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<p>Overview of the early steps of HIV-1 life cycle. <b>(A)</b> To enter a target cell, HIV-1 gp120 binds to specific cellular receptors,<span class="html-italic"> i.e.,</span> CD4 and a chemokine coreceptor (CCR5 or CXCR4), triggering the gp41-mediated fusion between the viral and the cellular membrane, and releasing the viral core in the cytoplasm of the host cell. The viral single stranded, positive, RNA genome (black line, flanked by open black squares depicting R-U5 and U3-R in its 5’ and 3’ termini respectively) is reverse transcribed into a linear double stranded cDNA copy (red line, flanked by open red squares representing the LTR = U3-R-U5), which is a component of the preintegration complex (PIC), also containing the viral integrase (IN), as well as other viral and cellular proteins. The PIC is translocated to the nucleus and the viral cDNA is either integrated through the action of IN or remains unintegrated (linear, 1-LTR circles, 2-LTR circles). From this point on, the cellular machinery of the host is recruited to transcribe the viral genome in order to produce all the components required to generate newly infectious particles. <b>(B)</b> The integration process is divided into three major steps: the 3’ processing and the strand transfer reaction, both catalyzed by IN, and the repair of the integrated viral DNA by the DNA repair machinery of the host cell. The PIC-containing viral DNA (red line, with 5’ ends depicted by filled circles) is first processed by the IN-mediated removal of a dinucleotide (GT) at each 3’ end of the viral DNA, leaving a protruding (AC) dinucleotide at the 5’ ends. IN then catalyzes the stable insertion of the processed viral DNA into a target DNA (black line), by simultaneously and asymmetrically breaking the target DNA 5 bp apart (blue bonds) (4 to 6 bp depending on the retrovirus) and joining it to the 3’ recessed ends of the viral DNA, leaving an integration intermediate with unpaired bases at each viral-target DNA junction. The DNA repair machinery of the host cell fills in the five nucleotide gap at each side of the viral DNA and removes the two 5’ overhang nucleotides from the viral DNA, resulting in the duplication of 5 bp of the target DNA at both sides of the proviral DNA. (C and D) Schematic concepts of <span class="html-italic">in vitro</span> integration assays showing half-site integration <b>(C)</b> and concerted or full-site integration <b>(D)</b>.</p> Full article ">Figure 2
<p>Dynamic model depicting the mechanism of LEDGF/p75-mediated HIV integration. LEDGF/p75 (green oval) associates with PC4 (red protein) and the RNA polymerase II machinery (yellow ovals) at promoter regions, but steric hindrance may prevent successful recruitment of preintegration complexes (gray oval with viral DNA in red). In this proposed model, LEDGF/p75 remains associated with the RNA pol II transcription elongation complex, potentially interacting with PC4 and menin/MLL complex. While this complex displaces nucleosomes (not depicted) and unwinds DNA to allow RNA polymerization, LEDGF/p75 may recruit HIV PIC and promote integration. This model is consistent with LEDGF/p75-captured DNA sequences and HIV integration sites being present throughout the transcription unit, without specific DNA binding consensus motif.</p> Full article ">
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Review
Current and Novel Inhibitors of HIV Protease
by Jana Pokorná, Ladislav Machala, Pavlína Řezáčová and Jan Konvalinka
Viruses 2009, 1(3), 1209-1239; https://doi.org/10.3390/v1031209 - 11 Dec 2009
Cited by 100 | Viewed by 23546
Abstract
The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in [...] Read more.
The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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<p>Trends in annual rates of death due to 7 leading causes among persons 25-44 years old in the United States during period 1987-2004. Dramatic decrease in the rate of death due to AIDS coincides with the introduction of HIV protease inhibitors (source: National Vital Statistics, Centers for Disease Control and Prevention, Atlanta).</p> Full article ">Figure 2
<p>The three-dimensional crystal structure of HIV PR dimer depicting mutations associated with resistance to clinically used protease inhibitors [<a href="#B7" class="html-bibr">7</a>]. Mutated residues are represented with their Cα atoms (spheres) and colored in the shades of red and blue for major and minor mutations, respectively. For major mutations, the semi-transparent solvent accessible surface is also shown in red. Active site aspartates and PI darunavir bound to the active site are represented in stick models. The figure was generated using the structure of highly mutated patient derived HIV-1 PR (PDB code 3GGU [<a href="#B8" class="html-bibr">8</a>]) and program PyMol [<a href="#B9" class="html-bibr">9</a>].</p> Full article ">Figure 3
<p>Chemical structures of the first generation HIV protease inhibitors.</p> Full article ">Figure 4
<p>Chemical structures of the second generation HIV protease inhibitors</p> Full article ">Figure 5
<p>Chemical structures of inhibitors HIV protease in the pipeline.</p> Full article ">Figure 6
<p>Chemical structure of DMP450 (<span class="html-italic">Mozenavir (DuPont)</span>.</p> Full article ">Figure 7
<p>Crystal structure of metallacarborane inhibitor bound to HIV PR. <b>(a) </b>Two metallacarborane clusters bind to the flap-proximal part of the active site. The HIV PR is represented by a ribbon diagram and colored by rainbow from blue to red (N- to C- termini), the atoms of the metallacarborane cluster are represented by spheres and colored orange for boron atoms, gray for carbon atoms, and blue for cobalt. The structural formula is depicted in <b>(b)</b>. Hydrogens are omitted for clarity.</p> Full article ">Figure 8
<p></p> Full article ">Figure 9
<p>HIV PR flap conformations. <b>(a)</b><b> </b>Overall structure of the apo-form of the HIV PR. The flaps (residues 43-58) in semi-open conformation are highlighted in red, residues 37-42, so called flap elbows are also indicated. The figure was generated using the structure of free HIV-1 PR (PDB code 1HHP [<a href="#B145" class="html-bibr">145</a>]) and program PyMol [<a href="#B9" class="html-bibr">9</a>]. <b>(b)</b><b> </b>Overall structure of the HIV PR with flaps (in dark green) in closed conformation. Residues 37-42, so called flap elbows are also indicated. Inhibitor bound in the enzyme active site is omitted from the figure. The figure was generated using the structure of a highly mutated patient derived HIV-1 PR (PDB code 3GGU [<a href="#B8" class="html-bibr">8</a>]) and program PyMol [<a href="#B9" class="html-bibr">9</a>].</p> Full article ">
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Review
Mutation Rates and Intrinsic Fidelity of Retroviral Reverse Transcriptases
by Luis Menéndez-Arias
Viruses 2009, 1(3), 1137-1165; https://doi.org/10.3390/v1031137 - 4 Dec 2009
Cited by 91 | Viewed by 18298
Abstract
Retroviruses are RNA viruses that replicate through a DNA intermediate, in a process catalyzed by the viral reverse transcriptase (RT). Although cellular polymerases and host factors contribute to retroviral mutagenesis, the RT errors play a major role in retroviral mutation. RT mutations that [...] Read more.
Retroviruses are RNA viruses that replicate through a DNA intermediate, in a process catalyzed by the viral reverse transcriptase (RT). Although cellular polymerases and host factors contribute to retroviral mutagenesis, the RT errors play a major role in retroviral mutation. RT mutations that affect the accuracy of the viral polymerase have been identified by in vitro analysis of the fidelity of DNA synthesis, by using enzymological (gel-based) and genetic assays (e.g., M13mp2 lacZ forward mutation assays). For several amino acid substitutions, these observations have been confirmed in cell culture using viral vectors. This review provides an update on studies leading to the identification of the major components of the fidelity center in retroviral RTs. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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<p>Major steps of the retroviral replication cycle and host factors that could influence the viral mutation rate.</p> Full article ">Figure 2
<p>Proposed models and mutational intermediates leading to the generation of base substitutions and frameshift errors [<a href="#B86" class="html-bibr">86</a>].</p> Full article ">Figure 3
<p>View of the nucleic acid binding cleft of HIV-1 RT showing the location of amino acids that influence fidelity of DNA synthesis (blue and orange CPK models). Ribbon diagrams are used for the representation of p66 (blue) and p51 (green). A stick representation is used for the template (red) and primer (magenta) strands of the DNA/DNA complex. The incoming dNTP is represented with a yellow CPK. Atomic coordinates were taken from Protein Data Bank file 1RTD [<a href="#B114" class="html-bibr">114</a>].</p> Full article ">
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Review
HIV-1 Protease: Structural Perspectives on Drug Resistance
by Irene T. Weber and Johnson Agniswamy
Viruses 2009, 1(3), 1110-1136; https://doi.org/10.3390/v1031110 - 3 Dec 2009
Cited by 122 | Viewed by 12539
Abstract
Antiviral inhibitors of HIV-1 protease are a notable success of structure-based drug design and have dramatically improved AIDS therapy. Analysis of the structures and activities of drug resistant protease variants has revealed novel molecular mechanisms of drug resistance and guided the design of [...] Read more.
Antiviral inhibitors of HIV-1 protease are a notable success of structure-based drug design and have dramatically improved AIDS therapy. Analysis of the structures and activities of drug resistant protease variants has revealed novel molecular mechanisms of drug resistance and guided the design of tight-binding inhibitors for resistant variants. The plethora of structures reveals distinct molecular mechanisms associated with resistance: mutations that alter the protease interactions with inhibitors or substrates; mutations that alter dimer stability; and distal mutations that transmit changes to the active site. These insights will inform the continuing design of novel antiviral inhibitors targeting resistant strains of HIV. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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<p>Structures of HIV-1 protease dimer. Superposition of unliganded protease (PR<sub>WT</sub> in blue, PDB ID: 1HHP [<a href="#B29-viruses-01-01110" class="html-bibr">29</a>]), unliganded protease with F53L mutation (PR<sub>F53L</sub> in green, PDB ID: 2G69, [<a href="#B28-viruses-01-01110" class="html-bibr">28</a>]) and protease complex with darunavir (red, PDB ID: 2IEN, [<a href="#B30-viruses-01-01110" class="html-bibr">30</a>] inhibitor is removed for clarity). The unliganded structures exhibit opened flap conformation, while the protease flaps form the closed conformation with darunavir.</p> Full article ">Figure 2
<p>Hydrogen bond interactions of the peptide tetrahedral intermediate with HIV-1 protease (PDB ID: 3B7V [<a href="#B32-viruses-01-01110" class="html-bibr">32</a>]). The peptide intermediate is in yellow bonds and the protease in grey. Hydrogen bonds are indicated by broken lines. Interactions with the catalytic Asp25 and 25’ are omitted for clarity.</p> Full article ">Figure 3
<p>Sites of the resistance mutations on protease dimer. The protease dimer is in pink ribbons with darunavir in green sticks. Major and minor resistance mutations are colored as red and blue spheres, respectively. Mutations are distributed on both the monomers to increase visibility.</p> Full article ">Figure 4
<p>Resistance mutations showing loss of direct interactions with the inhibitor. (a) The I84V substitution results in loss of van der Waals contacts between residue 84 and darunavir. Wild type Ile84 (PDB ID: 2IEN) and mutant Val84 (PDB ID: 2IEO) are shown in green and magenta sticks, respectively. Only part of darunavir (grey bonds) is shown for clarity [<a href="#B30-viruses-01-01110" class="html-bibr">30</a>]. (b) Drug resistant mutation I50V is accompanied by loss of several interactions with indinavir [<a href="#B61-viruses-01-01110" class="html-bibr">61</a>]. Wild type Ile50 (PDB ID: 1SDT) and mutant Val (PDB ID: 2AVS) are shown as green and magenta sticks. Only the central portion of indinavir is shown in grey. The interatomic distances are given in Å.</p> Full article ">Figure 5
<p>The V82A mutation shows a shift in the main chain atoms of residues 81 and 82 that partially compensates for the loss of interactions due to substitution of a smaller side chain [<a href="#B64-viruses-01-01110" class="html-bibr">64</a>]. The wild type Val82 (PDB ID: 1SDT) and Ala mutant (PDB ID: 1SDV) are shown as green and magenta sticks, respectively.</p> Full article ">Figure 6
<p>(a)The I50V mutation at the tip of the flap results in loss of intersubunit interactions with Ile47’ and Ile84’ [<a href="#B61-viruses-01-01110" class="html-bibr">61</a>]. Ile50 (PDB ID: 1SDT) and Val50 (PDB ID: 2AVS) are represented as green and magenta sticks, respectively. (b) The F53L variant eliminates intersubunit hydrophobic interactions between residues 53 and Ile50’ [<a href="#B28-viruses-01-01110" class="html-bibr">28</a>]. This loss of interaction is accompanied by a wider separation of the flaps. The wild type (PDB ID: 1HHP) and F53L flaps (PDB ID: 2G69) are shown in green and magenta sticks. The separation between the flaps is indicated in Å.</p> Full article ">Figure 7
<p>(a) The longer methionine side chain of Met90 in the L90M variant forms shorter van der Waals interactions with the main chain of catalytic Asp25, unlike the Leu90 in the wild type protease [<a href="#B68-viruses-01-01110" class="html-bibr">68</a>]. The wild type (PDB ID: 2IEN) and mutant protease (PDB ID: 2F81) are shown in green and magenta colored sticks, respectively. Part of darunavir is shown colored by element type. (b) The protease variant with G73S (PDB ID: 2AVV) substitution forms new hydrogen bond interactions of Ser73 with Thr74 and Asn88 (magenta dashed lines) [<a href="#B61-viruses-01-01110" class="html-bibr">61</a>]. The hydrogen bond network of Thr74, Asn88, Asp29 and Thr31 propagates the effects to the active site cavity.</p> Full article ">Figure 8
<p>The variant I54V (PDB ID: 2B80) with peptide tetrahedral intermediate has lost the water mediated interactions with Ile50 and Ile50’, as indicated by red circle, in comparison to the wild type protease interactions in <a href="#viruses-01-01110-f002" class="html-fig">Figure 2</a> [<a href="#B32-viruses-01-01110" class="html-bibr">32</a>]. The hydrogen bond interactions between the protease and the intermediate are shown as broken lines.</p> Full article ">Figure 9
<p>Chemical structures of darunavir and the new antiviral inhibitor GRL-02031.</p> Full article ">
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Review
Reverse Transcriptase and Cellular Factors: Regulators of HIV-1 Reverse Transcription
by Kylie Warren, David Warrilow, Luke Meredith and David Harrich
Viruses 2009, 1(3), 873-894; https://doi.org/10.3390/v1030873 - 10 Nov 2009
Cited by 38 | Viewed by 17114
Abstract
There is ample evidence that synthesis of HIV-1 proviral DNA from the viral RNA genome during reverse transcription requires host factors. However, only a few cellular proteins have been described in detail that affect reverse transcription and interact with reverse transcriptase (RT). HIV-1 [...] Read more.
There is ample evidence that synthesis of HIV-1 proviral DNA from the viral RNA genome during reverse transcription requires host factors. However, only a few cellular proteins have been described in detail that affect reverse transcription and interact with reverse transcriptase (RT). HIV-1 integrase is an RT binding protein and a number of IN-binding proteins including INI1, components of the Sin3a complex, and Gemin2 affect reverse transcription. In addition, recent studies implicate the cellular proteins HuR, AKAP149, and DNA topoisomerase I in reverse transcription through an interaction with RT. In this review we will consider interactions of reverse transcription complex with viral and cellular factors and how they affect the reverse transcription process. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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Figure 1
<p>RT Structure.<b> </b>The p66 subunit is shown in yellow and p51 subunit in purple. A vRNA:tRNA structure is juxtaposed on the RT molecule, where the vRNA is green and the tRNA is brown. The model was generated and adapted using RasMol and the PBD file ID# 1R0A [<a href="#B2" class="html-bibr">2</a>].</p> Full article ">Figure 2
<p>Model of the HIV-1 RNA structure from +1 to +540.<b> </b>Proposed interactions between tRNA<sup>Lys,3</sup> and the U5 Stem-loop are indicated. The viral RNA sequence is colored black and the tRNA<sup>Lys,3</sup> sequence is in red. TAR: transactivation response element. Poly-A Stem loop contains the poly-adenylation signal AAUAAA functions on the 3’ long terminal repeat. The ψ indicates the vRNA packaging signal. The PAS on the vRNA and the anti-PAS on tRNA<sup>Lys,3</sup> are boxed in green. Adapted from Wilkinson <span class="html-italic">et al. </span>(2008) [<a href="#B24" class="html-bibr">24</a>].</p> Full article ">Figure 3
<p>Schematic of RTC interacting cellular factors. Known direct (—) and indirect (---) associations between RTC (blue) and host factors that affect reverse transcription. Cellular factors are incorporated into virion particles (green), not incorporated (yellow) or conditionally incorporated (red).</p> Full article ">
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Review
The Interaction Between Lentiviral Integrase and LEDGF: Structural and Functional Insights
by Stephen Hare and Peter Cherepanov
Viruses 2009, 1(3), 780-801; https://doi.org/10.3390/v1030780 - 6 Nov 2009
Cited by 20 | Viewed by 16504
Abstract
Since its initial description as an HIV-1 integrase (IN) interactor seven years ago, LEDGF has become one of the best-characterized host factors involved in viral replication. Results of intensive studies in several laboratories indicated that the protein serves as a targeting factor for [...] Read more.
Since its initial description as an HIV-1 integrase (IN) interactor seven years ago, LEDGF has become one of the best-characterized host factors involved in viral replication. Results of intensive studies in several laboratories indicated that the protein serves as a targeting factor for the lentiviral DNA integration machinery, and accounts for the characteristic preference of Lentivirus to integrate within active transcription units. The IN-LEDGF interaction has been put forward as a promising target for antiretroviral drug development and as a potential tool to improve safety of lentiviral vectors for use in gene therapy. Additionally, as a natural ligand of lentiviral IN proteins, LEDGF has been successfully used in structural biology studies of retroviral DNA integration. This review focuses on the structural aspects of the IN-LEDGF interaction and their functional consequences. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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Figure 1

Figure 1
<p>The retroviral DNA integration pathway. The PIC, formed following reverse transcription of the viral RNA genome in the host cell cytoplasm, contains viral DNA (light blue), IN (grey oval), along with other viral and host cell proteins (not shown). Within the PIC, the active sites of IN (red ovals) activate water molecules for nucleophilic attacks on the phosphodiester backbone close to the 3° ends of the viral DNA. This 3°-processing reaction (1) results in the removal of a di- or tri-nucleotide from both 3’ ends of the viral DNA, exposing the reactive 3’ hydroxyl groups attached to invariant CA dinucleotides. Following nuclear import, the PIC comes into contact with host chromosomal DNA (orange) (2). Whereupon, the IN active sites activate the hydroxyl groups at the 3° viral DNA ends to cut a pair of phosphodiester bonds in the opposing strands of chromosomal DNA, 4-6 bp apart (the exact separation depends on the retroviral genus, and equals 5 bp for lentiviruses) (3). The resulting intermediate (4) contains viral DNA joined at each 3’ end to chromosomal DNA, flanked by short gaps and 5’-overhangs. The final DNA repair step (5) that joins the 5° viral DNA ends to the host DNA is presumably carried out by host proteins.</p> Full article ">Figure 2
<p>The role of LEDGF in lentiviral biology. Representations of IN and viral and host DNA are conserved from <a href="#figure1" class="html-fig">Figure 1</a>. LEDGF (pink) interacts with the PIC via its C-terminal IBD, with host DNA via its AT-hooks and with an unidentified component of the chromatin (grey rectangle) via its N-terminal PWWP domain, tethering the PIC to select loci of host cell chromatin.</p> Full article ">Figure 3
<p>The solution structure of the LEDGF IBD. The helical bundle is shown in cartoon representation, with individual helices labeled (PDB ID 1z9e). Side chains contributing to the hydrophobic area at the left side of the helical bundle as drawn and the positive face on the underside are shown as sticks and labeled.</p> Full article ">Figure 4
<p>The primary IN:LEDGF interface. <b>A.</b> Cartoon representation of the co-crystal structure of the IN<sub>CCD</sub>:LEDGF<sub>IBD</sub> complex (PDB ID 2b4j). IN chains are colored green (chain A) and cyan (chain B) and a pair of LEDGF chains interacting at either end of the IN CCD dimer are pink. <b>B.</b> Stereo close-up view of the region enclosed by a black rectangle in <b>A</b>, showing details of the CCD:IBD interface. The protein backbone and side chains, shown in ribbon and stick representations, respectively, are colored by atom. Side chains of residues involved in interactions are shown, as well as a water molecule coordinated between main chain carbonyls of IN Thr-125 and LEDGF Ile-365.</p> Full article ">Figure 5
<p>The interaction between the IBD and IN NTD. <b>A.</b> Cartoon representation of the HIV-2 IN<sub>NTD+CCD</sub> and LEDGF<sub>IBD</sub> co-crystal structure (PDB ID 3f9k) with the IN dimer colored green (chain A) and cyan (chain B) and LEDGF colored pink. Red spheres represent magnesium ions in the IN active sites and dark grey spheres represent Zn<sup>2+</sup> ions coordinated by the HHCC motif of the NTDs. <b>B.</b> Details of the NTD:IBD interface, showing the area enclosed by a black rectangle in <b>A</b>. The charge-charge interactions are shown as black dashed lines between the stick representations of the side chains involved.</p> Full article ">Figure 6
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336 KiB  
Review
HIV-1 Integrase-DNA Recognition Mechanisms
by Jacques J. Kessl, Christopher J. McKee, Jocelyn O. Eidahl, Nikolozi Shkriabai, Ari Katz and Mamuka Kvaratskhelia
Viruses 2009, 1(3), 713-736; https://doi.org/10.3390/v1030713 - 5 Nov 2009
Cited by 13 | Viewed by 14948
Abstract
Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been [...] Read more.
Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been recapitulated in vitro using recombinant integrase and synthetic DNA substrates. Biochemical and biophysical studies of these model reactions have been pivotal in advancing our understanding of mechanistic details for how IN interacts with viral and target DNAs, and are the focus of the present review. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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<p>Schematic presentation of HIV-1 IN activity assays <span class="html-italic">in vitro</span>. <b>(A)</b> These reactions are typically performed with purified recombinant IN and 21-mer double-stranded DNA mimicking the U5 sequence. The enzyme first removes the GT dinucleotide from the 3’-terminal, and then covalently joins the recessed 3’-end to the target DNA. In these reactions the U5 sequence serves as both viral and target DNA. The strand transfer products result from integration of only one viral DNA end into the target DNA, while pair-wise integration products are not observed. <b>(B)</b> The concerted integration assays and critical nucleoprotein intermediates. Selective interaction of IN with viral DNA ends results in a highly stable nucleoprotein complex termed the stable synaptic complex (SSC). Next, IN in the context of SSC engages with the target DNA to form the strand transfer complex (STC), which carries out the concerted integration reaction. These nucleoprotein complexes are readily separated by native agarose gel electrophoresis. Deproteinization of the STC leads to the formation of the concerted integration product. The asterisks in A and B indicate the P<sup>32</sup> labeled 5’-end of viral DNA.</p> Full article ">Figure 2
<p>(A) Schematic presentation of the three domain structure of HIV-1 IN. The NTD residues (H12, H14, C40 and C43) coordinate Zn and contribute to the functional multimerization. It is not entirely clear whether the NTD directly binds viral or target DNA. The CCD contains the catalytic DDE motif. This domain interacts with both viral and target DNA. A number of residues (Y143, Q148 and K159) selectively interact with terminal U5 bases, while S119 has been implicated in direct interactions with the target DNA. The CCD is also critical for the functional multimerization. The CTD is highly basic and non-specifically interacts with viral DNA. Several CTD residues implicated in viral DNA binding are indicated. It remains to be determined whether the CTD could also coordinate the target DNA. <b>(B)</b> Sequences of U3 and U5 termini of viral DNA. The base-pairs that are identical in U3 and U5 sequences are in bold. A majority of IN-viral DNA mapping experiments used the U5 sequence and the interacting sites are indicated with circles. Note color coordination between the residues in A and respective nucleotide positions in B. The catalytic residues in A and the arrow pointing to the specific cleavage sites at U3 and U5 termini are in red. The CCD amino acids Y143, Q148 and K159 (colored green) have been shown to selectively crosslink with the terminal nucleotides marked with green circles [<a href="#" class="html-">60</a>]. The E246C mutant is colored blue and its multiple crosslinking sites [<a href="#" class="html-">61</a>] in viral DNA are depicted by blue circles.</p> Full article ">Figure 3
<p>Molecular models for two distinct forms of HIV-1 IN tetramers. <b>(A)</b> An “open” conformation of tetrameric IN. This model is based on the HIV-1 IN two domain (NTD-CCD) structure [<a href="#" class="html-">41</a>] and our MS footprinting results [<a href="#" class="html-">48</a>] indicating that such a conformation is stabilized by LEDGF/p75 in the absence of viral DNA. This “open” conformation of tetrameric IN could catalyze 3’-processing and half-site integration reactions, however an incorrect spacing (~29 Å) between the two active sites would hamper the concerted integration. <b>(B)</b> A “closed” conformation of HIV-1 IN tetramer. This model was built by Hare <span class="html-italic">et al.</span> using one of the crystal forms of the MVV IN structure [<a href="#" class="html-">47</a>], where the catalytic sites are positioned optimally for concerted integration. It has been proposed that this structure could be stabilized by two viral DNAs [<a href="#" class="html-">47</a>]. However, viral DNAs have not been included in the model. Instead, the relative positioning of two catalytic sites with respect to the target DNA is shown to demonstrate the 5 bps separation consistent with a pair-wise integration. Red arrows point to the target scissile bonds. Individual subunits are colored cyan, green, yellow and orange. Side chains of catalytic residues in green and yellow subunits are depicted in red. For clarity only NTD-CCD fragments are depicted, while the CTDs, which are also present in these models [<a href="#" class="html-">47</a>,<a href="#" class="html-">48</a>], are not shown.<b/></p> Full article ">
791 KiB  
Review
Revisiting Plus-Strand DNA Synthesis in Retroviruses and Long Terminal Repeat Retrotransposons: Dynamics of Enzyme: Substrate Interactions
by Daniele Fabris, John P. Marino and Stuart F. J. Le Grice
Viruses 2009, 1(3), 657-677; https://doi.org/10.3390/v1030657 - 4 Nov 2009
Cited by 3 | Viewed by 13729
Abstract
Although polypurine tract (PPT)-primed initiation of plus-strand DNA synthesis in retroviruses and LTR-containing retrotransposons can be accurately duplicated, the molecular details underlying this concerted series of events remain largely unknown. Importantly, the PPT 3’ terminus must be accommodated by ribonuclease H (RNase H) [...] Read more.
Although polypurine tract (PPT)-primed initiation of plus-strand DNA synthesis in retroviruses and LTR-containing retrotransposons can be accurately duplicated, the molecular details underlying this concerted series of events remain largely unknown. Importantly, the PPT 3’ terminus must be accommodated by ribonuclease H (RNase H) and DNA polymerase catalytic centers situated at either terminus of the cognate reverse transcriptase (RT), and in the case of the HIV-1 enzyme, ~70Å apart. Communication between RT and the RNA/DNA hybrid therefore appears necessary to promote these events. The crystal structure of the HIV-1 RT/PPT complex, while informative, positions the RNase H active site several bases pairs from the PPT/U3 junction, and thus provides limited information on cleavage specificity. To fill the gap between biochemical and crystallographic approaches, we review a multidisciplinary approach combining chemical probing, mass spectrometry, NMR spectroscopy and single molecule spectroscopy. Our studies also indicate that nonnucleoside RT inhibitors affect enzyme orientation, suggesting initiation of plus-strand DNA synthesis as a potential therapeutic target. Full article
(This article belongs to the Special Issue Retroviral Enzymes)
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<p>PPT-primed synthesis of HIV plus-strand DNA. The experimental strategy is outlined in (a), and comprises an RNA/DNA hybrid within which the PPT sequence is embedded. RNA and DNA nucleotides are in lower and upper case, respectively. Inclusion of HIV-1 RT and a full complement of dNTPs (one of which is radiolabeled) is predicted to support cleavage of the plus strand RNA (excluding the PPT) (RNase H-mediated), initiation of DNA synthesis from the PPT 3’ OH (DNA polymerase-mediated) and precise post-priming cleavage of the PPT at the PPT/U3 junction (RNase H-mediated). The experimental outcome is illustrated in (b). NaOH + and – notations indicate whether the final reverse transcription product was subjected to alkaline hydrolysis. C, U, A,G; sequencing reactions to locate the site of plus-strand initiation. Adapted from [<a href="#B2" class="html-bibr">2</a>].</p> Full article ">Figure 2
<p>KMnO<sub>4</sub> footprinting detects anomalous base pairing in the HIV-1 PPT DNA template in the absence of RT. (a), KMnO<sub>4</sub> sensitivity of the minus-strand DNA template hybridized to PPT RNA primers containing a 5- (Lane 3), 10- (Lane 4) or 15-nt DNA extension (Lane 5), creating “staged” +5, +10 and +15 replication complexes. Lane 1, No KMnO<sub>4</sub>; Lane 2, No piperidine. As controls of KMnO<sub>4</sub> sensitivity, +6T/+7T reactivity of the +5 replication complex is eliminated in a +10 complex, while +13T/+14T reactivity is eliminated in the +15 complex. (b), summary of KMnO<sub>4</sub> footprinting data, indicating enhanced reactivity of +1T at the PPT/U3 junction and the distal (a)<sub>4</sub>:(T)<sub>4</sub> tract. See [11] for additional details.</p> Full article ">Figure 3
<p>Pyrimidine isostere insertion into the PPT DNA template alters cleavage specificity. (a), Sequence of the HIV-1 PPT-containing RNA/DNA hybrid. T and C isosteres F and D, respectively, depicted in Panel (b) were added pairwise throughout the T<sub>4</sub> and C<sub>6</sub> tracts of the DNA template. PPT&lt;&gt;U3 denotes the scissile phosphodiester bond. Pairwise isostere insertions are numbered as follows: 1; -1D/-2D; 2, -2D/-3D; 3, -3D/-4D; 4, -4D/-5D; 5, -5D/-6D; 6, -7F/-8F; 7, -8F/-9F; 8, -9F/-10F. (c), Hydrolysis profiles of isostere-substituted PPT RNA/DNA hybrids. Lane notations correspond to the numbering system of Panel (a). Lane C, unmodified DNA. Adapted from [17].</p> Full article ">Figure 4
<p>SHAMS – combining chemical modification and mass spectrometry to investigate PPT architecture. (a) Nano-ESI mass spectrum of the HIV-1 PPT RNA and DNA strands after treatment of the hybrid with a 10-fold excess of NMIA. Under these conditions, the majority of the RNA strand is unmodified, while the DNA strand, lacking a 2’ OH, is unaffected. (b), SORI-CID spectrum of the unmodified RNA substrate in the -5 charge state. For clarity, only the main sequence ions (c and y ions) are labeled using standard mass spectrometry notation. Gray lines through the sequence mark the cleavage positions corresponding to labeled fragments. (c) SORI-CID spectrum of the triply-modified RNA substrate in the -5 charge state. Only modified fragments are labeled for clarity. Due to modification of the RNA 2’ OH, the d-H<sub>2</sub>O series cannot undergo loss of H<sub>2</sub>O and therefore is observed as a c series. The gray arrow indicates the mass shift of 133.05 Da for one NMIA modification from the unmodified to the modified c<sub>1</sub> ion. (●) indicates the number of NMIA modifications observed on the corresponding fragment, for example the c<sub>5</sub>●● ion corresponds to the ‘caaaa’ fragment with two modifications. (d), Schematic of the wild type HIV-1 PPT RNA/DNA hybrid. Gray pentagons indicate positions of NMIA sensitivity. (e) NMIA sensitivity of an HIV-1 PPT RNA/DNA hybrid whose DNA template contains a -8T -&gt; dF substitution. Additional details of the SHAMS approach can be found in [21].</p> Full article ">Figure 5
<p>Examining RT orientational dynamics by single molecule spectroscopy. (a), <span class="html-italic">Upper, </span>HIV-1 RT is site-specifically labeled with Cy3 (green) at its C-terminal RNase H domain. <span class="html-italic">Lower, </span>Cy3-labeled RT interacts with surface-immobilized Cy5-labeled DNA, and fluorescence of individual substrates is followed by total-internal-reflection fluorescence (TIRF) microscopy with alternating laser excitations at 532 and 635nm. Panels (b) – (d), Alternative RT orientations on the PPT. FRET histograms derived from Cy3 RNase H-labeled RT incubated with RNA/DNA hybrids whose PPT RNA primer was (b), extended at its 3’ terminus with two ribonucleotides (PPTr2), (c), lacking an extension (PPT) or (d), extended by two deoxynucleotides (PPTD2). (e) Ternary complex formation promotes enzyme binding to the primer terminus in a polymerization orientation. Histogram of RT bound to PPT:dd2 hybrid (filled, grey) in the presence of 10 μM (purple trace) and 1mM TTP (cyan trace) (f), NNRTI binding induces RT binding to the PPT 3’ terminus in an RNase H orientation. Histograms of RT bound to PPT:d2 substrates in the absence (filled grey trace) presence of 10 μM (red trace) and 100 μM nevirapine (NVP) (orange trace). See [36] for additional details.</p> Full article ">Figure 6
<p>Investigating aminoglycoside binding to the HIV-1 PPT by mass spectrometry. (a) Sequence and masses of the wild type HIV-1 PPT (PPT<sub>WT</sub>), an all-DNA version (PPT<sub>DNA</sub>,), an all RNA version (PPT<sub>RNA</sub>), and a hybrid whose DNA and RNA sequences were interchanged (PPT<sub>SWP</sub>); (b) ESI-FTICR spectrum of an equimolar mixture of PPT variants in the presence of neomycin B (NB). Mass signatures for unliganded duplexes are illustrated in the grey filled portion of the spectrum. In the presence of neomycin B, mass increments for each duplex suggest that PPT<sub>RNA</sub>, PPT<sub>DNA</sub> and PPT<sub>SWP</sub> bind one equivalent of ligand, while two equivalents bind to PPT<sub>WT</sub>. (c) Determination of the neomycin B binding sites on PPT<sub>WT</sub> by tandem mass spectrometry. The ion series observed for the DNA and RNA strands constituting the substrate are reported on the respective sequences to highlight nucleotides prevented from undergoing fragmentation in the presence of ligand [48]. The lines mark the phosphodiester bonds cleaved by gas-phase dissociation. Conversely, the absence of phosphodiester bond cleavage indicates protection by bound neomycin B. See [44] for additional details.</p> Full article ">Figure 7
<p>One-dimensional water flip-back watergate <sup>1</sup>H NMR spectra of the imino region of the PPT<sub>wt</sub> duplex after titration with neomycin B (Panel (a)), or HIV-1 RT (Panel b)). In (a), 1D <sup>1</sup>H NMR spectra were obtained in the absence (<span class="html-italic">upper</span>) and presence of 1.0 equivalents (<span class="html-italic">middle</span>) and 2.0 equivalents (<span class="html-italic">lower</span>) of neomycin B. Imino resonances for +2G, +1T and -1g where chemical shift changes can be tracked are highlighted. Dotted lines and arrows indicate the shift in position of the resonances. (b) 1D <sup>1</sup>H NMR spectra for PPT<sub>wt</sub> in the absence (<span class="html-italic">upper</span>) and presence of 1.0 equivalent (<span class="html-italic">lower</span>) of p66/p51 HIV-1 RT. Imino protons were assigned using 2D NOESY experiments; assignments are listed above each peak. Adapted from [41].</p> Full article ">
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