Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise
2012; Elsevier BV; Volume: 287; Issue: 49 Linguagem: Inglês
10.1074/jbc.r112.389056
ISSN1083-351X
Autores Tópico(s)Biochemical and Molecular Research
ResumoSynthesis of integration-competent, double-stranded DNA from the (+)-RNA strand genome of retroviruses and long terminal repeat-containing retrotransposons reflects a multistep process catalyzed by the virus-encoded reverse transcriptase (RT). In conjunction with RNA- and DNA-templated DNA synthesis, a hydrolytic activity of the same enzyme (RNase H) is required to remove genomic RNA of the RNA/DNA replication intermediate. Together, these combined synthetic and degradative functions ensure correct selection, extension, and removal of the RNA primers of (−)- and (+)-strand DNA synthesis (tRNA and the polypurine tract, respectively). For HIV-1 RT, a quarter century of research has not only illuminated the biochemical properties, structure, and conformational dynamics of this highly versatile enzyme but has also witnessed drug discovery advances from the first Food and Drug Administration-approved anti-RT drug to recent use of RT inhibitors as potential colorectal microbicides. Salient features of HIV-1 RT and extension of these findings into programs of drug discovery are reviewed here. Synthesis of integration-competent, double-stranded DNA from the (+)-RNA strand genome of retroviruses and long terminal repeat-containing retrotransposons reflects a multistep process catalyzed by the virus-encoded reverse transcriptase (RT). In conjunction with RNA- and DNA-templated DNA synthesis, a hydrolytic activity of the same enzyme (RNase H) is required to remove genomic RNA of the RNA/DNA replication intermediate. Together, these combined synthetic and degradative functions ensure correct selection, extension, and removal of the RNA primers of (−)- and (+)-strand DNA synthesis (tRNA and the polypurine tract, respectively). For HIV-1 RT, a quarter century of research has not only illuminated the biochemical properties, structure, and conformational dynamics of this highly versatile enzyme but has also witnessed drug discovery advances from the first Food and Drug Administration-approved anti-RT drug to recent use of RT inhibitors as potential colorectal microbicides. Salient features of HIV-1 RT and extension of these findings into programs of drug discovery are reviewed here. The individual steps of HIV-1 DNA synthesis, catalyzed by the multifunctional reverse transcriptase (RT), 2The abbreviations used are: RTreverse transcriptasePPTpolypurine tractNNRTInon-nucleoside RT inhibitorSMSsingle-molecule spectroscopyNVPnevirapineNRTInucleoside RT inhibitorTFVtenofovirINDOPYindolopyridoneDPVdapivirine. are summarized schematically in Fig. 1. (−)-Strand DNA synthesis, initiated from a cellular tRNA (tRNA3Lys) hybridized to the genome-encoded primer-binding site, continues to the 5′ terminus, creating (−)-strong-stop DNA. RNase H-mediated degradation of the resulting RNA/DNA hybrid promotes relocation of nascent (−)-DNA to the genome 3′ terminus by a strand transfer event that exploits sequence homology between the 5′ and 3′ termini. RNA-templated DNA synthesis continues, accompanied by RNase H-mediated degradation of the RNA genome, the exception to which are two short purine-rich segments (the 3′- and central polypurine tracts (PPTs)) from which (+)-strand DNA-dependent DNA synthesis is initiated. Newly synthesized (−)-strand DNA and 18 nucleotides of the covalently attached tRNA3Lys primer provide the template for 3′-PPT-primed (+)-strand DNA synthesis until the replication complex stalls at a position corresponding to the first modified tRNA base (A57). As a consequence, the C-terminal RNase H domain is positioned at the (−)-DNA/tRNA junction, and degradation of the tRNA "template" promotes a second or (+)-strand transfer event supported by homology between (−)- and (+)-strand DNA primer-binding sites. Although bidirectional DNA synthesis would be sufficient to complete DNA synthesis, HIV utilizes a second, central PPT primer, thereby producing a (+)-strand discontinuity (1.Lavigne M. Roux P. Buc H. Schaeffer F. DNA curvature controls termination of plus strand DNA synthesis at the centre of HIV-1 genome.J. Mol. Biol. 1997; 266: 507-524Crossref PubMed Scopus (36) Google Scholar). Following (+)-strand transfer, 3′-PPT-mediated DNA synthesis continues, displacing ∼100 nucleotides of central PPT-primed (+)-DNA, but abruptly ceases at the central termination sequence, a prominent feature of which is phased A-tracts that induce minor groove compression (1.Lavigne M. Roux P. Buc H. Schaeffer F. 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Although the extreme p51 C terminus has not been resolved crystallographically, its contribution to maintaining RT architecture is supported by observations that reconstituted enzymes with short deletions show increased RNase H inhibitor sensitivity (28.Chung S. Miller J.T. Johnson B.C. Hughes S.H. Le Grice S.F. Mutagenesis of human immunodeficiency virus reverse transcriptase p51 subunit defines residues contributing to vinylogous urea inhibition of ribonuclease H activity.J. Biol. Chem. 2012; 287: 4066-4075Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) and altered thermal stability. 3S. Chung and S. F. Le Grice, unpublished data. The p66 nucleic acid-binding cleft is formed by its finger, palm, and thumb subdomains, and co-crystal structures of HIV-1 RT with duplex DNA and an RNA/DNA hybrid have identified numerous contacts with both strands of the template and primer (20.Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. 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Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance.Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1358) Google Scholar) showed that the template overhang ahead of the polymerase active site was not co-linear with the duplex but was bent away and contacting the p66 fingers, revealing contacts with nucleobases +1, +2, and +3. The palm subdomain of HIV-1 RT houses the DNA polymerase active site (Fig. 2b) characterized by the Asp-110, Asp-185, and Asp-186 catalytic triad, a common feature of nucleic acid-polymerizing enzymes (19.Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Crystal structure at 3.5 Å resolution of HIV-1 reverse transcriptase complexed with an inhibitor.Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1757) Google Scholar, 20.Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughesi S.H. Arnold E. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 Å resolution shows bent DNA.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 6320-6324Crossref PubMed Scopus (1120) Google Scholar). Among polymerase families, palm subdomain architecture is also highly conserved, comprising a four- to six-stranded β-sheet flanked on one side by two α-helices (47.Brautigam C.A. Steitz T.A. Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes.Curr. Opin. Struct. Biol. 1998; 8: 54-63Crossref PubMed Scopus (336) Google Scholar). In nucleic acid-containing crystal structures, catalytic aspartates are close to the 3′ terminus of the primer. Asp-185 and Asp-186 are part of the conserved -Tyr-Met-Asp-Asp- motif, which adopts an unusual β-turn conformation (29.Ding J. Das K. Hsiou Y. Sarafianos S.G. Clark Jr., A.D. Jacobo-Molina A. Tantillo C. Hughes S.H. Arnold E. Structure and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody Fab fragment at 2.8 Å resolution.J. Mol. Biol. 1998; 284: 1095-1111Crossref PubMed Scopus (303) Google Scholar, 35.Hsiou Y. Ding J. Das K. Clark Jr., A.D. Hughes S.H. Arnold E. Structure of unliganded HIV-1 reverse transcriptase at 2.7 Å resolution: implications of conformational changes for polymerization and inhibition mechanisms.Structure. 1996; 4: 853-860Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 48.Esnouf R. Ren J. Ross C. Jones Y. Stammers D. Stuart D. Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors.Nat. Struct. Biol. 1995; 2: 303-308Crossref PubMed Scopus (447) Google Scholar), possibly to promote their positioning for catalysis, whereas the Tyr-183 phenoxy side chain is involved in hydrogen bonding with nucleobases at position −2 (29.Ding J. Das K. Hsiou Y. Sarafianos S.G. Clark Jr., A.D. Jacobo-Molina A. Tantillo C. Hughes S.H. Arnold E. Structure and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody Fab fragment at 2.8 Å resolution.J. Mol. Biol. 1998; 284: 1095-1111Crossref PubMed Scopus (303) Google Scholar). Comparison of the crystal structures of DNA-bound RT with unliganded or non-nucleoside RT inhibitor (NNRTI)-bound enzymes reveals significant conformational differences for the -Tyr-Met-Asp-Asp- motif, implicating a high degree of structural flexibility (29.Ding J. Das K. Hsiou Y. Sarafianos S.G. Clark Jr., A.D. Jacobo-Molina A. Tantillo C. Hughes S.H. Arnold E. Structure and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody Fab fragment at 2.8 Å resolution.J. Mol. Biol. 1998; 284: 1095-1111Crossref PubMed Scopus (303) Google Scholar). The incoming dNTP is tightly coordinated by p66 finger residues Lys-65 and Arg-72, the main chain NH groups of Asp-113 and Ala-114, and two divalent metal ions, whereas its ribose is accommodated by a pocket lined by Asp-113, Tyr-115, and Phe-116 on one side and Glu-151 and Arg-72 on the other. Additional dNTP contacts involve base pairing and base stacking with the template overhang (30.Huang H. Chopra R. Verdine G.L. Harrison S.C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance.Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1358) Google Scholar). The fidelity of dNTP insertion is critically influenced by interactions of the γ-phosphate with Lys-65 (49.Garforth S.J. Kim T.W. Parniak M.A. Kool E.T. Prasad V.R. Site-directed mutagenesis in the fingers subdomain of HIV-1 reverse transcriptase reveals a specific role for the β3-β4 hairpin loop in dNTP selection.J. Mol. Biol. 2007; 365: 38-49Crossref PubMed Scopus (18) Google Scholar), whereas mutagenesis studies have designated Tyr-115 as the "steric gate," suggesting that it aids in discriminating between deoxy- and ribonucleoside triphosphates (50.Martín-Hernández A.M. Domingo E. Menéndez-Arias L. Human immunodeficiency virus type 1 reverse transcriptase: role of Tyr115 in deoxynucleotide binding and misinsertion fidelity of DNA synthesis.EMBO J. 1996; 15: 4434-4442Crossref PubMed Scopus (70) Google Scholar, 51.Cases-Gonzalez C.E. Gutierrez-Rivas M. Menéndez-Arias L. Coupling ribose selection to fidelity of DNA synthesis. The role of Tyr-115 of human immunodeficiency virus type 1 reverse transcriptase.J. Biol. Chem. 2000; 275: 19759-19767Abstract Full Text Ful
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