title: Molecular mechanism of HIV-1 resistance to 3'-azido-2',3'-dideoxyguanosine.
abstract: We reported that 3'-azido-2',3'-dideoxyguanosine (3'-azido-ddG) selected for the L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain of HIV-1 reverse transcriptase (RT). In this study, we have defined the molecular mechanisms of 3'-azido-ddG resistance by performing in-depth biochemical analyses of HIV-1 RT containing mutations L74V, F77L, V106I, L214F, R277K, and K476N (SGS3). The SGS3 HIV-1 RT was from a single-genome-derived full-length RT sequence obtained from 3'-azido-ddG resistant HIV-1 selected in vitro. We also analyzed two additional constructs that either lacked the L74V mutation (SGS3-L74V) or the K476N mutation (SGS3-K476N). Pre-steady-state kinetic experiments revealed that the L74V mutation allows RT to effectively discriminate between the natural nucleotide (dGTP) and 3'-azido-ddG-triphosphate (3'-azido-ddGTP). 3'-azido-ddGTP discrimination was primarily driven by a decrease in 3'-azido-ddGTP binding affinity (Kd) and not by a decreased rate of incorporation (kpol). The L74V mutation was found to severely impair RT's ability to excise the chain-terminating 3'-azido-ddG-monophosphate (3'-azido-ddGMP) moiety. However, the K476N mutation partially restored the enzyme's ability to excise 3'-azido-ddGMP on an RNA/DNA, but not on a DNA/DNA, template/primer by selectively decreasing the frequency of secondary RNase H cleavage events. Collectively, these data provide strong additional evidence that the nucleoside base structure is major determinant of HIV-1 resistance to the 3'-azido-2',3'-dideoxynucleosides.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: jdm69@pitt.edu.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Meteer
authors.affiliation: Center for AIDS Research, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA; Veterans Affairs Medical Center, Decatur, GA, USA. Electronic address: rschina@emory.edu.
authors.forename: Raymond F
authors.initials: RF
authors.lastname: Schinazi
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: jwm1@pitt.edu.
authors.forename: John W
authors.initials: JW
authors.lastname: Mellors
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: nps2@pitt.edu.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Sluis-Cremer
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D015224:Dideoxynucleosides / Q000494:pharmacology*; D024882:Drug Resistance, Viral; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D012641:Selection, Genetic; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D019380:Anti-HIV Agents; D015224:Dideoxynucleosides; D050505:Mutant Proteins; D054302:pol Gene Products, Human Immunodeficiency Virus; C051073:3'-azido-2',3'-dideoxyguanosine; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase
keywords: 3′-Azido-2′,3′-dideoxyguanosine; 3′-Azido-ddG; 3′-Azido-ddGMP; 3′-Azido-ddGTP; 3′-azido-2′,3′-dideoxyguanosine; 3′-azido-ddG-monophosphate; 3′-azido-ddG-triphosphate; 50% inhibitory concentration; AZT; DP; Discrimination; Excision; HIV-1; IC(50); MP; NRTI; NRTIs; RNase H; RT; Resistance; Reverse transcriptase; T/P; TP; WT; diphosphate; monophosphate; nucleoside RT inhibitor; nucleoside reverse transcriptase inhibitors; reverse transcriptase; ribonuclease H; template/primer; triphosphate; wild-type; zidovudine