Zidovudine (ZDV) and nevirapine (NVP) target the HIV-1 reverse transcriptase (RT) by distinct mechanisms of action and belong to the nucleoside RT inhibitor (NRTI) and nonnucleoside RT inhibitor (NNRTI) classes, respectively. The combination of ZDV and NVP has been recommended for antiretroviral therapy to delay the emergence of drug-resistant strains and the onset of disease progression. Potency of the ZDV and NVP combination can be ascribed to synergistic inhibition of HIV-1 replication and the selection of mutations in the RT that are distinct to each drug class and that demonstrate antagonism.1,2 These include the thymidine analogue mutations (TAMs) and NNRTI mutations located in the N-terminal RT region from codons 1–240.
N348I, located in the connection subdomain of the HIV-1 RT, has been associated with ZDV and NVP treatment.3 N348I confers decreased susceptibility to ZDV, NVP, and efavirenz and potentiates RT inhibitor resistance when combined with TAMs and key NNRTI resistance mutations in clade B and non–subtype B strains.3–6 N348I is highly prevalent in RT inhibitor-treated compared with drug-naive individuals and normally appears before TAMs,3 providing a genetic pathway for the selection of TAMs and mutations that are known to antagonize ZDV resistance.7 Although increases in viral load similar to that seen with each of the individual TAMs have been observed, the role of N348I in virological failure remains unclear.3
Synergistic inhibition of HIV-1 by NVP and ZDV is mediated by NNRTIs blocking basal phosphorolysis in wild-type (WT) RT, and the removal of ZDV-terminated primers by RT containing TAMs, leading to ZDV resensitization.8,9 Apart from TAMs, N348I also confers decreased susceptibility to ZDV; however, resistance to ZDV is mediated by a ribonuclease H (RNase H)–dependent mechanism.3,10–12 N348I decreases formation of RNase H secondary cleavage products favoring the RT to bind to the template/primer (T/P) in a polymerase-dependent mode (with the polymerase active site at the 3′ end of the primer) leading to increased excision of ZDV-monophosphate (ZDV-MP). Conversely, NNRTIs promote binding of RT in an RNase H competent mode (with the RNase H active site positioned to cleave the RNA/DNA strand), thereby enhancing RNase H cleavage leading to inhibition of ZDV-MP excision on a RNA/DNA duplex and increased susceptibility to ZDV.10,13 Given the opposing actions of N348I and NNRTIs on RNase H cleavage and ZDV resistance, we investigated whether N348I counteracts synergistic inhibition of HIV-1 conferred by ZDV and NVP.
Drugs and Reagents
RT inhibitors ZDV and NVP were obtained from the AIDS Research and Reference Reagent Program. 3′-azido-3′ deoxythymidine triphosphate (ZDV-TP) was purchased from TriLink Biotechnologies. All other nucleotides were purchased from GE Healthcare.
Plasmids, Mutagenesis, and Virus Production
The NL4.3-derived HIV-1 molecular clone, pDRNL, was mutated by site-directed mutagenesis as previously described3 to generate HIVN348I, harboring N348I. All HIV-1 constructs were verified by nucleotide sequencing. HIV-1 was generated by transfection of 293T cells, concentrated by ultracentrifugation and the viral titer determined in TZM-bl cells by staining for β-galactosidase activity as previously described.3
Expression, Purification, and Characterization of Recombinant HIV-1 RT
Recombinant HIV-1LAI RT containing N348I (RTN348I) was expressed in Escherichia coli, purified by metal chelate affinity chromatography and characterized as published.3
Drug Susceptibility Assays
Assays were performed in TZM-bl cells as previously described3 except with the following modifications. Assays were performed in the presence of ZDV, NVP, or combinations of ZDV and NVP at fixed ratios. Cells were infected with ∼250 blue foci-forming units of virus and at 48 hours postinfection, luciferase activity in cell lysates was measured as described14 and the 50% effective concentration (EC50) determined as published.3
Evaluation of synergistic drug interactions was determined using the CalcuSyn software (Biosoft, Cambridge, United Kingdom) according to instructions for fixed ratio combinations. The analysis is based on the median effect principle. Combination index (CI) values at effective doses of 50% (ED50), 75% (ED75), and 90% (ED90) were calculated from 5 (for HIVWT) and 3 (for HIVN348I) independent assays with r ≥ 0.93.
WT (RTWT) and N348I RT (RTN348I) susceptibility to NVP was examined using an RNA/DNA T/P (sequence in Fig. 1A) as described previously.7 Primer rescue assays were performed using a 5′-32P-radiolabelled 26-nucleotide DNA primer (pr26) chain terminated with ZDV-MP (pr26 + ZDV) annealed to a 35-nucleotide RNA (T-RNA) template (Fig. 1A) as reported previously.7 RNase H cleavage of a 5′-32P-labelled 35-nucleotide RNA template annealed to a 26-nucleotide ZDV-MP–terminated primer was performed as described.3 The effect of NVP on RNase H cleavage was performed in the presence of 1 μM of drug.
Statistical significance of differences between drug EC50 values, fold-resistance, or CI values was determined by using the Wilcoxon rank sum test.
To determine whether N348I decreases susceptibility to ZDV and NVP combinations, we evaluated the capacity of HIVN348I to replicate in the presence of fixed ratios of ZDV and NVP compared with WT (HIVWT) (Table 1). ZDV and NVP combinations at fixed ratios of 1:1, 1:10, and 10:1 resulted in significant increases in the EC50 values for HIVN348I compared with HIVWT of 5- (P < 0.001, n = 7), 6- (P < 0.01, n = 6), and 3-fold (P < 0.01, n = 6), respectively (Table 1). Consistent with previous studies, HIVN348I demonstrated 2- (P < 0.05, n = 17) and 7-fold (P < 0.003, n = 13) decreased susceptibility to ZDV and NVP, respectively compared to HIVWT.3
We next investigated whether decreased susceptibility to ZDV and NVP combinations was because of reversal of their synergistic activity against HIV-1. As previously reported,1 the combination of ZDV and NVP tested at a 1:1 fixed ratio was strongly synergistic for HIVWT inhibition at the ED50 (CI = mean ± SEM; 0.3 ± 0.1), ED75 (CI = 0.3 ± 0.1), and ED90 (CI = 0.3 ± 0.04) levels. In contrast, N348I dramatically reversed the synergistic activity of ZDV and NVP (at a 1:1 fixed ratio) with slight antagonism observed at the ED50 (CI = 1.1 ± 0.1), additivity at the ED75 (CI = 1.0 ± 0.2), and moderate synergism at the ED90 (CI = 0.7 ± 0.2) levels. Taken together, these data demonstrate that in addition to conferring decreased susceptibility to each drug alone, N348I confers decreased susceptibility to the combination of ZDV and NVP and reverses the strong synergism normally seen with this drug combination against WT virus.
Our previous studies demonstrate that N348I confers more efficient ZDV-MP excision and DNA polymerization of recombinant HIV-1 RT in the presence of ZDV-TP and ATP on an RNA but not a DNA template.3 We investigated whether N348I improves DNA polymerization efficiency of recombinant RT in the presence of NVP under steady-state conditions. RTWT or RTN348I was incubated for 5 minutes at 37°C in the presence of 20 nM of T/P, 1 μM of dNTP, and variable concentrations of NVP. The short incubation time ensured that the reaction was in the linear phase and that there was no substrate depletion. RTN348I was more efficient in DNA polymerization in the presence of NVP (50% inhibitory concentration ± standard deviation, 3.5 ± 0.5 μM, n = 3) compared with RTWT (0.8 ± 0.1 μM)(data not shown). Next, we determined the ZDV-MP excision activity of RTN348I compared with RTWT. RTN348I showed greater ATP-mediated excision of ZDV-MP and rescue of DNA polymerization compared with RTWT in the absence and the presence of 1, 2.5, 5, and 10 μM of NVP (Figs. 1B, C). Notably, the excision rate of RTN348I in the presence of 2.5 μM of NVP was similar to the RTWT in the absence of drug (Fig. 1C). These data show that N348I enables RT to unblock a ZDV-MP–terminated primer and resume DNA polymerization even in the presence of high concentrations of NVP.
We next evaluated the ability of N348I to counteract increased RNase H cleavage activity mediated by NVP on a 5′-32P-labelled RNA/DNA duplex with a ZDV-MP–terminated primer. NVP increased the efficiency of RNase H cleavage by RTWT resulting in an accumulation of a 19-nucleotide single-stranded RNA template corresponding to the 10-nucleotide excision incompetent RNA/DNA duplex (Figs. 1D, E). Cleavage to the 10-nucleotide duplex was more efficient with RTN348I when the reaction was performed in the presence of NVP with the efficiency of cleavage similar to RTWT in the absence of NVP. These data show that N348I counteracts the accelerated RNase H cleavage by HIV-1 RT in both the absence and the presence of NVP.
ZDV and NVP synergistically inhibit HIV-1, and this is mediated by the ability of NVP to block ZDV-MP excision on DNA/DNA and RNA/DNA template primers.1,9,10 Here, we show that N348I abrogates synergistic inhibition of HIV-1 by ZDV and NVP. N348I conferred decreased susceptibility to ZDV:NVP combinations at different drug ratios compared with WT. In addition, recombinant RT containing N348I also showed greater efficiency in the rescue of DNA polymerization from a ZDV-MP–terminated primer in the presence of NVP compared with WT. The mechanism by which N348I reverses the synergistic inhibition of HIV-1 by ZDV and NVP can be ascribed to decreased RNase H cleavage activity of RTN348I allowing greater preservation of the RNA/DNA duplex relative to RTWT subjected to similar conditions (ie, RTN348I + NVP compared with RTWT + NVP). Notably, the rate of excision of RTN348I in the presence of NVP was equivalent to RTWT in the absence of drug (Fig. 1E). Preservation of the RNA/DNA duplex to >12 nucleotides provides more time for ZDV-MP excision to occur consistent with previous reports.10,13
N348I is observed early in therapy before the selection of TAMs and is highly associated with NVP and ZDV therapy.3 N348I is associated with the NNRTI mutations K103N, Y181C/I, and G190A/S and the lamivudine mutations M184V/I.3,15 These mutations are known to suppress ZDV resistance mediated by TAMs by directly antagonizing the ATP-mediated ZDV-MP excision reaction.2,7 In contrast, N348I compensates for the antagonism of ZDV resistance conferred by Y181C and M184V on RNA/DNA template/primers by an indirect RNase H-dependent mechanism. This involves the reduction of secondary RNase H cleavage products allowing more time for the excision reaction to take place. Therefore, N348I overcomes the suppression of ZDV resistance mediated by Y181C and NVP by similar RNase H-dependent mechanisms, resulting in decreased susceptibility to the combination of ZDV and NVP.
The World Health Organization recommends that first-line antiretroviral therapy in resource-constrained settings comprises a NNRTI with 2 NRTIs, one of which should be ZDV or tenofovir and that stavudine should be phased out because of its toxicities.16 The roll out of tenofovir could be hampered because of its cost.17 Accordingly, treatment with lamivudine, stavudine, and NVP, that are widely used in Africa,18 are likely to be maintained or could be replaced with a combination that includes ZDV, NVP, and lamivudine. Of note, both of these treatments have been associated with the selection of N348I.3,19
We propose that N348I, due to its ability to counteract the synergistic inhibition of HIV by ZDV and NVP, would allow for the selection of mutations that confer higher levels of resistance, for example, Y181C and TAMs. In contrast, Y181C, which confers NVP resistance and hypersusceptibility to ZDV, in the absence of N348I, would make it relatively more difficult to select TAMs. The role of N348I in providing a genetic pathway for the selection of TAMs, and the Y181C and M184V mutations that antagonize TAMs7 may have implications in the efficacy of recommended first-line therapies in resource-constrained settings.6
S. H. Yap, B. D. Herman, J. Radzio, N. Sluis-Cremer, and G. Tachedjian conceived and designed the experiments; S. H. Yap, B. D. Herman, and J. Radzio performed the experiments; S. H. Yap, B. D. Herman, J. Radzio, N. Sluis-Cremer, and G. Tachedjian analyzed the data; G. Tachedjian performed statistical analyses; and S. H. Yap, N. Sluis-Cremer, and G. Tachedjian wrote the paper. The authors thank Yanille Scott for experimental assistance. The authors thank the NIH AIDS Research & Reference Reagent Program for providing ZDV, NVP, and the TZM-bl cell line that was contributed by Dr John C. Kappes, Dr Xiaoyun We, and Tranzyme Inc.
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