Mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors demonstrate altered rates of RNase H cleavage that correlate with HIV-1 replication fitness in cell culture

Three mutants of human immunodeficiency virus type 1 (HIV-1) reverse transc riptase (V106A, V179D, and Y181C), which occur in clinical isolates and con fer resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), were analyzed for RNA- and DNA-dependent DIVA polymerization and RNase H cl eavage. All mutants demonstrated processivities of polymerization that were indistinguishable from wild-type enzyme under conditions in which deoxynuc leoside triphosphates were not limiting. The V106A reverse transcriptase de monstrated a three- to fourfold slowing of both DNA 3'-end-directed and RNA 5'-end-directed RNase H cleavage relative to both wild-type and V179D enzy mes, similar to what was observed for P236L in a previously published study (P. Gerondelis et al., J, Virol, 73:5803-5813, 1999). In contrast, the Y18 1C reverse transcriptase demonstrated a selective acceleration of the secon dary RNase H cleavage step during both modes of RNase H cleavage. The relat ive replication fitness of these mutants in Ho cells was assessed in parall el infections as well as in growth competition experiments. Of the NNRTI-re sistant mutants, V179D was more fit than Y181C, and both of these mutants w ere more fit than V106A, which demonstrated the greatest reduction in RNase H cleavage. These findings, in combination with results from previous work , suggest that abnormalities in RNase H cleavage are a common characteristi c of HIV-1 mutants resistant to NNRTIs and that combined reductions in the rates of DNA 3'-end- and RNA 5'-end-directed cleavages are associated with significant reductions in the replication fitness of HIV-1.