Low-abundance HIV species and their impact on mutational profiles in patients with virological failure on once-daily abacavir/lamivudine/zidovudine and tenofovir.

BACKGROUND: HIV clonal genotypic analysis (CG) was used to investigate whether a more sensitive analysis method would detect additional low-abundance mutations compared with population genotyping (PG) in antiretroviral-naive patients who experienced virological failure (VF) during treatment with abacavir/lamivudine/zidovudine and tenofovir. METHODS: HIV was analysed by PG and CG (771 baseline and 657 VF clones) from subjects with VF (confirmed HIV RNA > or = 400 copies/mL at 24-48 weeks). RESULTS: Fourteen of 123 subjects (11%) met VF criteria; their median baseline HIV RNA was 5.4 log(10) copies/mL, and 4.0 log(10) copies/mL at VF. By baseline PG, 2/14 had HIV-1 with nucleoside reverse transcriptase inhibitor (NRTI) or non-NRTI mutations. By baseline CG, 9/14 had HIV-1 with NNRTI and/or NRTI mutations; 7/9 had study drug-associated mutations. By PG at VF, 10/14 had selected for resistance mutations [2, K65R; 1, M184V; and 7, thymidine analogue mutations (TAMs) +/- M184V]. By CG at VF, for subjects with TAMs, T215F was more commonly detected (5/14 samples) than T215Y (2/14). For one subject who selected K65R at VF, both K65R-containing clones and TAM-containing clones (both T215A and T215F) were observed independently but not conjunctively in the same clone in a post-VF sample. CONCLUSIONS: The majority of subjects with VF had major and minor mutations detected at VF; CG detected additional low-abundance variants at baseline and VF that could have influenced mutation selection pathways. Both PG and CG data suggest TAMs, not K65R selection, are the preferred resistance route, biased towards 215F selection. No HIV clone contained both K65R and T215F/Y mutations, suggesting in vivo antagonism between the two mutations. The once-daily zidovudine usage and high baseline viraemia may also have contributed to rapid selection of HIV with multiple mutations in VFs.

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 transcriptase (V106A, V179D, and Y181C), which occur in clinical isolates and confer resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), were analyzed for RNA- and DNA-dependent DNA polymerization and RNase H cleavage. All mutants demonstrated processivities of polymerization that were indistinguishable from wild-type enzyme under conditions in which deoxynucleoside triphosphates were not limiting. The V106A reverse transcriptase demonstrated 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 enzymes, 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 Y181C reverse transcriptase demonstrated a selective acceleration of the secondary RNase H cleavage step during both modes of RNase H cleavage. The relative replication fitness of these mutants in H9 cells was assessed in parallel infections as well as in growth competition experiments. Of the NNRTI-resistant mutants, V179D was more fit than Y181C, and both of these mutants were 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 characteristic 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.

The P236L delavirdine-resistant human immunodeficiency virus type 1 mutant is replication defective and demonstrates alterations in both RNA 5'-end- and DNA 3'-end-directed RNase H activities.

The nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) delavirdine (DLV) selects in vitro for the human immunodeficiency virus type 1 (HIV-1) RT mutation P236L, which confers high-level resistance to DLV but not other NNRTIs. Unexpectedly, P236L has developed infrequently in HIV-1 isolates obtained from patients receiving DLV; K103N is the predominant resistance mutation observed in that setting. We characterized the replication fitness of viruses derived from pNL4-3 containing P236L or K103N in both H9 and primary human peripheral blood mononuclear cell cultures infected in parallel with the two mutants. In the absence of DLV, p24 production by wild-type virus occurred more rapidly and to higher levels than with either mutant; P236L consistently demonstrated a two- to threefold decrease in p24 relative to K103N. At low levels of DLV, growth of wild-type virus was severely inhibited, and K103N replicated two- to threefold more efficiently than P236L. At high concentrations of DLV, P236L replication and K103N replication were both inhibited. Recombinant RTs containing K103N or P236L were analyzed for DNA polymerization on heteropolymeric RNA templates and RNase H degradation of RNA-DNA hybrids. Neither mutant demonstrated defects in polymerization. K103N demonstrated normal RNA 5'-end-directed RNase H cleavage and slowed DNA 3'-end-directed RNase H cleavage compared to wild-type RT. P236L demonstrated slowing of both DNA 3'-end- and RNA 5'-end-directed RNase H cleavage, consistent with its reduced replication efficiency relative to K103N. These data suggest that NNRTI resistance mutations can lead to reductions in the efficiency of RNase H cleavage, which may contribute to a reduction in the replication fitness of HIV-1.

HIV-1 drug susceptibilities and reverse transcriptase mutations in patients receiving combination therapy with didanosine and delavirdine.

Previous studies have shown that the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase mutation Y181C, which confers high-level resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), develops rarely during therapy with NNRTIs plus zidovudine. To determine whether didanosine (ddI) is also effective in preventing the emergence of Y181C, we analyzed delavirdine (DLV) susceptibilties and reverse transcriptase sequences of isolates obtained from patients enrolled in a pharmacokinetic study of DLV and ddI. Nine NNRTI-naive patients were evaluated. Seven received DLV/ddI and two received DLV/ddI/zidovudine. Median durations of prior zidovudine and ddI were 26 and 15 months, respectively. Isolates from eight of nine patients had a mutation(s) associated with nucleoside resistance at entry. After treatment with DLV and ddI alone, isolates from five of seven patients developed Y181C, four in combination with K103N. Thus, in this group of nucleoside-experienced patients, combination therapy with DLV/ddI did not prevent the emergence of Y181C.

Development of zidovudine resistance mutations in patients receiving prolonged didanosine monotherapy.

Human immunodeficiency virus type 1 (HIV-1) isolates from 2 patients who received didanosine (ddI) monotherapy for > 2 years were analyzed for reverse transcriptase (RT) mutations by sequencing of proviral DNA from peripheral blood mononuclear cell cultures. One patient was otherwise antiretroviral-naive; the other had received zidovudine for 5 months before beginning ddI therapy. Isolates obtained from both patients before initiation of ddI monotherapy were free of HIV-1 RT mutations associated with zidovudine or ddI resistance. However, after prolonged ddI monotherapy, mutations associated with zidovudine resistance (M41L, D67N, K70R, and/or T215Y) were detected in HIV-1 isolates from both patients. There was no evidence that surreptitious use of zidovudine or technical artifact caused these findings. This observation suggests that prolonged ddI monotherapy may decrease the efficacy of subsequent zidovudine therapy in some patients.