Connection Domain Mutations N348I and A360V in HIV-1 Reverse Transcriptase Enhance Resistance to 3′-Azido-3′-deoxythymidine through Both RNase H-dependent and -independent Mechanisms

Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2008; 283(32):22222-32. DOI: 10.1074/jbc.M803521200
Source: PubMed


Thymidine analogue-associated mutations (TAMs) in reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) cause resistance to 3'-azido-3'-deoxythymidine (AZT) through excision of the incorporated monophosphate. Mutations in the connection domain of HIV-1 RT can augment AZT resistance. It has been suggested that these mutations compromise RNase H cleavage, providing more time for AZT excision to occur. However, the underlying mechanism remains elusive. Here, we focused on connection mutations N348I and A360V that are frequently observed in clinical samples of treatment-experienced patients. We show that both N348I and A360V, in combination with TAMs, decrease the efficiency of RNase H cleavage and increase excision of AZT in the presence of the pyrophosphate donor ATP. The TAMs/N348I/A360V mutant accumulates transiently formed, shorter hybrids that can rebind to RT before the template is irreversibly degraded. These hybrids dissociate selectively from the RNase H-competent complex, whereas binding in the polymerase-competent mode is either not affected with N348I or modestly improved with A360V. Both connection domain mutations can compensate for TAM-mediated deficits in processive DNA synthesis, and experiments with RNase H negative mutant enzymes confirm an RNase H-independent contribution to increased levels of resistance to AZT. Moreover, the combination of diminished RNase H cleavage and increased processivity renders the use of both PP(i) and ATP advantageous, whereas classic TAMs solely enhance the ATP-dependent reaction. Taken together, our findings demonstrate that distinct, complementary mechanisms can contribute to higher levels of excision of AZT, which in turn can amplify resistance to this drug.

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    • "positively associated with treatment experience in a 345 subtype-B-infected patient cohort (Dau et al., 2010). Associations of these and other CN mutations with drug exposure and TAMs has also been confirmed through other subtype-B-infected patient cohorts and in vitro studies (Cane et al., 2007; Dau et al., 2010; Ehteshami et al., 2008; Gupta et al., 2010; Hachiya et al., 2008; Lengruber et al., 2011; Michels et al., 2010; Price et al., 2010; von Wyl et al., 2010a; von Wyl et al., 2010b; Waters et al., 2009; Yap et al., 2007) Recently, CN mutation A360V was shown to be selected in subtype-B-infected patients receiving AZT monotherapy (Brehm et al., 2012b). "
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    ABSTRACT: Mutations in the connection subdomain (CN) and RNase H domain (RH) of HIV-1 reverse transcriptase (RT) from subtype B-infected patients enhance nucleoside and nonnucleoside RT inhibitor (NRTI and NNRTI) resistance by affecting the balance between polymerization and RNase H activity. To determine whether CN mutations in subtype C influence drug sensitivity, single genome sequencing was performed on Brazilian subtype C-infected patients failing RTI therapy. CN mutations identified were similar to subtype B, including A376S, A400T, Q334D, G335D, N348I, and A371V, and increased AZT resistance in the presence of thymidine analog mutations. CN mutations also enhanced NNRTI resistance in the presence of classical NNRTI mutations: etravirine resistance was enhanced 6- to 11-fold in the presence of L100I/K103N/Y181C. These results indicate that selection of CN mutations in treatment-experienced patients also occurs in subtype-C-infected patients and are likely to provide valuable information in predicting clinical RTI resistance.
    Virology 10/2012; 435(2). DOI:10.1016/j.virol.2012.09.021 · 3.32 Impact Factor
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    • "The mechanism by which R284K confers a selective advantage in the context of TAM1 mutations is different from others previously described for mutations in the thumb-connection subdomains and in the RNase H domain. Thus, mutations such as N348I, A360V and Q509L increased chain-terminated primer rescue with RNA/DNA complexes, but not with DNA/DNA template-primers [29,30,32-34,57,58]. In combination with TAMs, N348I and A360V decreased the efficiency of RNase H cleavage and increased excision of AZT in the presence of ATP [34]. "
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    ABSTRACT: Background Thymidine analogue resistance mutations (TAMs) selected under treatment with nucleoside analogues generate two distinct genotypic profiles in the HIV-1 reverse transcriptase (RT): (i) TAM1: M41L, L210W and T215Y, and (ii) TAM2: D67N, K70R and K219E/Q, and sometimes T215F. Secondary mutations, including thumb subdomain polymorphisms (e.g. R284K) have been identified in association with TAMs. We have identified mutational clusters associated with virological failure during salvage therapy with tenofovir/emtricitabine-based regimens. In this context, we have studied the role of R284K as a secondary mutation associated with mutations of the TAM1 complex. Results The cross-sectional study carried out with >200 HIV-1 genotypes showed that virological failure to tenofovir/emtricitabine was strongly associated with the presence of M184V (P < 10-10) and TAMs (P < 10-3), while K65R was relatively uncommon in previously-treated patients failing antiretroviral therapy. Clusters of mutations were identified, and among them, the TAM1 complex showed the highest correlation coefficients. Covariation of TAM1 mutations and V118I, V179I, M184V and R284K was observed. Virological studies showed that the combination of R284K with TAM1 mutations confers a fitness advantage in the presence of zidovudine or tenofovir. Studies with recombinant HIV-1 RTs showed that when associated with TAM1 mutations, R284K had a minimal impact on zidovudine or tenofovir inhibition, and in their ability to excise the inhibitors from blocked DNA primers. However, the mutant RT M41L/L210W/T215Y/R284K showed an increased catalytic rate for nucleotide incorporation and a higher RNase H activity in comparison with WT and mutant M41L/L210W/T215Y RTs. These effects were consistent with its enhanced chain-terminated primer rescue on DNA/DNA template-primers, but not on RNA/DNA complexes, and can explain the higher fitness of HIV-1 having TAM1/R284K mutations. Conclusions Our study shows the association of R284K and TAM1 mutations in individuals failing therapy with tenofovir/emtricitabine, and unveils a novel mechanism by which secondary mutations are selected in the context of drug-resistance mutations.
    Retrovirology 08/2012; 9(1):68. DOI:10.1186/1742-4690-9-68 · 4.19 Impact Factor
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    • "TAMs in HIV-1 RT confer AZT resistance by enabling the enzyme to excise the chain-terminating AZT-MP moiety from the 3'-end of the DNA primer using ATP as a phosphate donor [15]. Previous biochemical studies demonstrated that N348I in HIV-1 RT indirectly increases AZT resistance by decreasing the frequency of secondary RNase H cleavages that significantly reduce the RNA/DNA duplex length of the T/P and diminish the efficiency of AZT-MP excision [6,8]. As such, we first assessed the AZT-MP excision activity of the WT and N348 mutant enzymes on a well-defined RNA/DNA T/P substrate that is routinely used in our laboratory [6,7,16,17]. "
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    ABSTRACT: N348I in HIV-1 reverse transcriptase (RT) confers resistance to zidovudine (AZT) and nevirapine. Biochemical studies demonstrated that N348I indirectly increases AZT resistance by decreasing the frequency of secondary ribonuclease H (RNase H) cleavages that reduce the RNA/DNA duplex length of the template/primer (T/P) and diminish the efficiency of AZT-monophosphate (MP) excision. By contrast, there is some discrepancy in the literature in regard to the mechanisms associated with nevirapine resistance: one study suggested that it is due to decreased inhibitor binding while others suggest that it may be related to the decreased RNase H cleavage phenotype. From a structural perspective, N348 in both subunits of RT resides distal to the enzyme's active sites, to the T/P binding tract and to the nevirapine-binding pocket. As such, the structural mechanisms associated with the resistance phenotypes are not known. Using a novel modelled structure of RT in complex with an RNA/DNA T/P, we identified a putative interaction between the β14-β15 loop in the p51 subunit of RT and the RNA template. Substitution of the asparagine at codon 348 in the p51 subunit with either isoleucine or leucine abrogated the observed protein-RNA interaction, thus, providing a possible explanation for the decreased RNase H phenotype. By contrast, alanine or glutamine substitutions exerted no effect. To validate this model, we introduced the N348I, N348L, N348A and N348Q mutations into RT and purified enzymes that contained subunit-specific mutations. N348I and N348L significantly decreased the frequency of secondary RNase H cleavages and increased the enzyme's ability to excise AZT-MP. As predicted by the modelling, this phenotype was due to the mutation in the p51 subunit of RT. By contrast, the N348A and N348Q RTs exhibited RNase H cleavage profiles and AZT-MP excision activities similar to the wild-type enzyme. All N348 mutant RTs exhibited decreased nevirapine susceptibility, although the N348I and N348L mutations conferred higher fold resistance values compared to N348A and N348Q. Nevirapine resistance was also largely due to the mutation present in the p51 subunit of RT. This study demonstrates that N348I-mediated AZT and nevirapine resistance is due to the mutation in the p51 subunit of RT.
    Retrovirology 08/2011; 8(1):69. DOI:10.1186/1742-4690-8-69 · 4.19 Impact Factor
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