Variations in reverse transcriptase and RNase H domain mutations in human immunodeficiency virus type 1 clinical isolates are associated with divergent phenotypic resistance to zidovudine

Abstract

Mutations in the RNase H domain of human immunodeficiency virus type 1 RT have been reported to cause resistance to zidovudine (ZDV) in vitro. However, very limited data on the in vivo relevance of these mutations in patients exist to date. This study was designed to determine the relationship between mutations in the RNase H domain and viral susceptibility to nucleoside analogues. Viruses harboring complex thymidine analogue mutation (TAM) and nucleoside analogue mutation (NAM) profiles were evaluated for their phenotypic susceptibilities to ZDV, tenofovir (TNF), and the nonapproved nucleoside reverse transcriptase inhibitors (NRTIs) beta-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (Reverset), beta-D-5-fluorodioxolane-cytosine, and apricitabine. As controls, viruses from NRTI-naïve patients were also studied. The pol RT region (codons 21 to 250) of the viruses were sequenced and evaluated for mutations in the RNase H domain (codons 441 to 560) and the connection domain (codons 289 to 400). The results showed that viruses from patients failing multiple NRTI-containing regimens had distinct TAM and NAM profiles that conferred various degrees of resistance to ZDV (0.9- to >300-fold). Sequencing of the RNase H domain identified five positions (positions 460,468, 483, 512, and 519) at which extensive amino acid polymorphisms common in both wild-type viruses and viruses from treated patients were identified. No mutations were observed at positions 539 and 549, which have previously been associated with ZDV resistance. Mutations in the RNase H domain did not appear to correlate with the levels of phenotypic resistance to ZDV. Although some mutations were also observed in the connection domain, the simultaneous presence of the L74V and M184V mutations was the most significant determinant of phenotypic resistance to ZDV in patients infected with viruses with TAMs.

FIG. 1.

Distribution of amino acid mutations and polymorphisms in the RNase H domain of wild-type viruses, viruses with NNRTI resistance-conferring mutations only, and viruses containing complex TAM and NAM profiles. Dots, identical amino acids; regions in gray, regions in which common polymorphisms were observed; +, <20-fold resistance to ZDV; +++, >50-fold resistance to ZDV.

FIG. 2.

Comparative phenotypes of recombinant clones of viruses harboring the L74V and/or M184V mutation for susceptibilities to ATC and ZDV in CBMCs. Bars with dots, fold resistance to ZDV; bars with slashes, fold resistance to ATC; wt, wild type. Recombinant clones containing the L74V or M184V mutation, or both, were tested for their susceptibilities to ATC and ZDV in cell culture assays. The mean EC₅₀s of the mutants were compared to those of wild-type strain pNL4-3, which were 5.87 ± 0.29 and 0.07 ± 0.03 for ATC and ZDV, respectively. The results are shown as means ± standard deviations.