In vitro study of resistance-associated genotypic mutations to nucleoside analogs.
In spite of a rather long period of investigations, the problem of HIV drug resistance remains unsolved, and more that, at present HIV-1 mutants resistant to all known nucleoside inhibitors being used in clinical therapy against the human immunodeficiency syndrome are discovered. In this study we selected HIV-1 mutants resistant to the nucleoside inhibitors of HIV reverse transcriptase (NRTI): 3'-azido-2',3'-dideoxythymidine (AZT), 5'-phosphit 3'-azido-2',3'-dideoxythymidine (ph-AZT), dideoxyinosine (ddI) and didehydrodeoxythymidine (d4T). Selection of resistant mutants was carried out by gradually increasing of drug concentration in the culture medium during propagation of the HIV-1EVK on fresh MT-4 cells. Phenotypic resistance was defined as an increase in ID50 of 160-fold for AZT, 8 for ph-AZT, 10 for ddI, 7 for d4T. In comparison studies it was determined that the viral resistance to these drugs was appeared variously in a similar conditions and duration of selection. The nucleotide sequences of the RT region of the HIV-1 variants were compared with the HIV-1EVK from "0" passage. For some of selected HIV-1 mutants NRTI resistance mutations were detected. Selected AZT resistant variants contained amino acid substitutions in positions D67A and K70R. Our studies was not revealed substitution at position 75 for ph-AZT resistant variants, whereas substitution at position L214F have been observed in both experiments using AZT and ph-AZT. Selected d4T resistant mutants contained amino acid substitutions in positions N54D and P52R. Selected ddI resistant mutants contained only one amino acid substitution in position P143S. Collection of drug-resistant mutants should prove to be a convenient tool for rapid investigations a new antiretroviral agents on cross drug-resistance.
Gashnikova
N
N
State Research Center of Virology and Biotechnology Vector, Institute of Molecular Biology, Koltsovo, Novosibirsk Region, Russia. gapv@online.sinor.ru
Plyasunova
O
O
Kiseleva
Y
Y
Fedyuk
N
N
Pokrovsky
A
A
D016049:Didanosine / Q000737:chemistry / Q000494:pharmacology; D024882:Drug Resistance, Viral / Q000235:genetics*; D014644:Genetic Variation; D005838:Genotype; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009705:Nucleosides / Q000737:chemistry / Q000494:pharmacology*; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D018119:Stavudine / Q000737:chemistry / Q000494:pharmacology; D015215:Zidovudine / Q000737:chemistry / Q000494:pharmacology*
D009705:Nucleosides; D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; D018119:Stavudine; D054303:HIV Reverse Transcriptase; D016049:Didanosine