Impaired rescue of chain-terminated DNA synthesis associated with the L74V mutation in human immunodeficiency virus type 1 reverse transcriptase.
The L74V and M184V mutations in the reverse transcriptase (RT) gene of human immunodeficiency virus type 1 (HIV-1) are frequently associated with resistance to the nucleoside reverse transcriptase inhibitors abacavir, didanosine, and lamivudine. Yet viruses containing any of these mutations often display hypersusceptibility to zidovudine (ZDV). Two distinct mechanisms have been described to explain HIV-1 drug resistance. One of these involves diminished rates of incorporation of the nucleotide analogue by mutated RT, while the other mechanism involves increased rates of phosphorolytic excision of the drug-terminated primer. To understand the biochemical mechanisms responsible for the hypersensitization of L74V-containing viruses to ZDV, we studied the efficiency of excision of ZDV-monophosphate (ZDV-MP)-terminated primers by recombinant wild-type and mutated HIV-1 RTs in cell-free assays. We observed that the L74V mutation in RT caused reductions in ATP-dependent removal of ZDV-MP from newly synthesized viral DNA. In addition, we determined that the L74V and M184V mutations did not affect the ratio between the populations of RT-DNA/DNA complexes found at pre- and posttranslocational stages; however, they might have affected proper alignment between incorporated chain terminator and pyrophosphate donor, substrate orientation, affinity for ATP, and/or primer-template substrate. Finally, we confirmed previous findings that L74V-containing viruses display diminished replication capacity and that this is associated with reduced levels of synthesis of early reverse-transcribed viral DNA molecules.
Frankel
Fernando A
FA
McGill AIDS Centre, Lady Davis Institute-Jewish General Hospital, 3755 Côte-Ste-Catherine Road, Montreal, Quebec H3T 1E2, Canada.
Marchand
Bruno
B
Turner
Dan
D
Götte
Matthias
M
Wainberg
Mark A
MA
D000255:Adenosine Triphosphate / Q000378:metabolism; D019380:Anti-HIV Agents / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D024921:Drug Resistance, Multiple, Viral; D054303:HIV Reverse Transcriptase / Q000187:drug effects / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D014779:Virus Replication; D015215:Zidovudine / Q000494:pharmacology*
D019380:Anti-HIV Agents; D004279:DNA, Viral; D011756:Diphosphates; D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; D000255:Adenosine Triphosphate; D054303:HIV Reverse Transcriptase