13th International HIV Drug Resistance Workshop


8–12 June 2004, Tenerife Sur-Costa Adeje, Canary Islands, Spain


FITNESS OF T215Y VERSUS T215F MUTANTS IN HIV-1 RT: COMPARISON OF SPECIFIC THYMIDINE ANALOGUE RESISTANCE MUTATION PATTERNS

Antiviral Therapy 2004; 9:S68

ZX Hu, P Reid, H Hatano, J Lu and DR Kuritzkes
Section of Retroviral Therapeutics, Brigham & Women’s Hospital, Division of AIDS, Harvard Medical School, Boston, Mass., USA

BACKGROUND: Resistance to zidovudine (ZDV) results from sequential accumulation of thymidine analogue resistance mutations (TAMs) at HIV-1 reverse transcriptase (RT) codons 41, 67, 70, 210, 215 and 219. Two mutations are possible at codon 215: T215Y or T215F; both involve double nucleotide mutations (acc to tac or ttc, respectively). The 210W mutation usually is found in combination with 215Y, and rarely occurs together with the alternative mutation at this codon, 215F. To explain this association, we compared relative replication rates, fitness and infectivity of recombinant HIV-1 carrying various combinations of TAMs.

METHODS: TAMs were introduced by site-directed mutagenesis into the cloned PR-RT-coding segment of pol from the ZDV-sensitive (WT) HIV-1 isolate, HXB2. Infectious recombinant viruses carrying hisD or gfp gene fragments as sequence tags in nef were generated by co-transfecting into MT-2 cells the appropriate PR-RT-deleted recombination vector together with the PR-RT gene of interest. Growth kinetics of the resulting virus stocks were tested in MT-2 cells in the absence or presence of ZDV. Relative fitness was determined in growth competition assays using a recombinant marker virus assay developed in our laboratory. Zidovudine susceptibility was determined by a standardized drug susceptibility assay; infectivity titres were measured by the MAGI/CPRG infectivity assay.

RESULTS: Recombinant viruses carrying RT mutations 41L/210W/215Y or 67N/70R/219Q showed somewhat faster replication kinetics as compared to wild-type. By contrast, 215F, 41L/215F 41L/ 210W/215F, 41L/67N/210W/215F mutant replicated poorly in the absence and presence of ZDV. Growth competition assays showed that the 215F, 41L/215F 41L/210W/215F, 41L/67N/210W/215F mutants were substantially less fit than 215Y, 41L/215Y 41L/ 210W/215Y or 41L/67N/210W/215Y. Introduction of the 210W mutation into 41L215Y or 41L215F backgrounds reduced fitness of both viruses, but 41L210W215Y viruses showed greater infectivity in the presence of ZDV compared to 41L210W215F.

CONCLUSION: The relative fitness of these mutants is summarized as follows: 215Y >> 215F, 41L/215Y >> 41L/215F, 41L/210W/215Y >> 41L/210W/215F, 41L/ 67N/210W/215Y >> 41L/67N/210W/215F; 41L/215F >> 41L/210W/215F, 41L/215Y >> 41L/210W/215Y. Results of these experiments show that the combination of 215F together with 41L and 210W is highly unfavourable, and helps explain why this combination is rarely observed in clinical isolates from ZDV-treated patients.

PRESENTING AUTHOR: ZX Hu

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2004-06-08
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