title: Selection of mutations in the connection and RNase H domains of human immunodeficiency virus type 1 reverse transcriptase that increase resistance to 3'-azido-3'-dideoxythymidine.
abstract: Recent work indicates that mutations in the C-terminal domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) increase 3'-azido-3'-dideoxythymidine (AZT) resistance. Because it is not known whether AZT selects for mutations outside of the polymerase domain of RT, we carried out in vitro experiments in which HIV-1(LAI) or AZT-resistant HIV-1(LAI) (M41L/L210W/T215Y) was passaged in MT-2 cells in increasing concentrations of AZT. The first resistance mutations to appear in HIV-1(LAI) were two polymerase domain thymidine analog mutations (TAMs), D67N and K70R, and two novel mutations, A371V in the connection domain and Q509L in the RNase H domain, that together conferred up to 90-fold AZT resistance. Thereafter, the T215I mutation appeared but was later replaced by T215F, resulting in a large increase in AZT resistance ( approximately 16,000-fold). Mutations in the connection and RNase H domains were not selected starting with AZT-resistant virus (M41L/L210W/T215Y). The roles of A371V and Q509L in AZT resistance were confirmed by site-directed mutagenesis: A371V and Q509L together increased AZT resistance approximately 10- to 50-fold in combination with TAMs (M41L/L210W/T215Y or D67N/K70R/T215F) but had a minimal effect without TAMs (1.7-fold). A371V and Q509L also increased cross-resistance with TAMs to lamivudine and abacavir, but not stavudine or didanosine. These results provide the first evidence that mutations in the connection and RNase H domains of RT can be selected in vitro by AZT and confer greater AZT resistance and cross-resistance to nucleoside RT inhibitors in combination with TAMs in the polymerase domain.
authors.affiliation: University of Pittsburgh School of Medicine, S818 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA.
authors.forename: Jessica H
authors.initials: JH
authors.lastname: Brehm
authors.forename: Dianna
authors.initials: D
authors.lastname: Koontz
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Meteer
authors.forename: Vinay
authors.initials: V
authors.lastname: Pathak
authors.forename: Nicolas
authors.initials: N
authors.lastname: Sluis-Cremer
authors.forename: John W
authors.initials: JW
authors.lastname: Mellors
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology*; D001665:Binding Sites; D002460:Cell Line; D024882:Drug Resistance, Viral; D008040:Genetic Linkage; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D016914:Ribonuclease H / Q000378:metabolism*; D015215:Zidovudine / Q000494:pharmacology*
chemical_list: D019380:Anti-HIV Agents; D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H