15th International HIV Drug Resistance Workshop 13-17 June 2006, Sitges, Spain

FITNESS COMPARISON OF THYMIDINE ANALOGUE RESISTANCE PATHWAY MUTANTS IN HIV-1 REVERSE TRANSCRIPTASE

Antivir Ther. 2006, 11:S47 (abstract no. 39)

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

BACKGROUND: Resistance to zidovudine (ZDV) results from 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: Y or F. Whereas 215Y occurs alone or with 41L and 210W (TAM-1), 215F rarely occurs with these mutations or by itself; it is usually found with 67N, 70R and 219Q (TAM-2). The 210W mutation most often occurs with 41L and 215Y and rarely occurs with 215F or TAM-2 mutations. We previously demonstrated that the 215F mutant is less fit than T215 wild-type or 215Y (Hu et al., Antiviral Therapy 2004; 9:S68). In the present study, we explored the virological basis for clustering of 210W with other TAM-1 but not TAM-2 mutants, and the clustering of 215F, but not 215Y, with TAM-2 mutants.

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. ZDV susceptibility was determined by a standardized drug susceptibility assay; infectivity titers were measured by the MAGI/CPRG infectivity assay.

RESULTS: Whereas introduction of 210W improved infectivity and relative fitness of an 41L/215Y mutant in the presence of ZDV, introduction of this mutation into a 67N/70R/219Q background resulted in decreased infectivity and relative fitness in the presence or absence of drug. By contrast, introduction of 215F, but not 215Y into the 67N/70R/219Q background increased fitness of the TAM-2 mutant in the presence of ZDV.

CONCLUSIONS: These results, together with our earlier data, help explain the clustering of 210W with TAM-1 mutations and 215F with TAM-2 mutations.

2006-06-13
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