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: 4.6). 07/2008; 283(32):22222-32. DOI: 10.1074/jbc.M803521200
Source: PubMed

ABSTRACT 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 · 3.28 Impact Factor
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    • "Recently, it has been shown that the N348I mutation decreases the efficiency of RNase H cleavage and increases excision of AZT from AZTterminated primer/templates, in the presence of the pyrophosphate donor ATP (Delviks- Frankenberry et al., 2008; Ehteshami et al., 2008; Yap et al., 2007). The decreased degradation of the RNA template by the diminished RNase H cleavage has been proposed to provide additional time for RT to excise AZT-MP and hence result in the observed increased AZT resistance(Delviks-Frankenberry et al., 2008; Ehteshami et al., 2008).. "
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    ABSTRACT: Some mutations in the connection subdomain of the polymerase domain and in the RNase H domain of HIV-1 reverse transcriptase (RT) have been shown to contribute to resistance to RT inhibitors. However, the clinical relevance of such mutations is not well understood. To address this point we determined the prevalence of such mutations in a cohort of antiretroviral treatment-naïve patients (n=123) and assessed whether these substitutions are associated with drug resistance in vitro and in vivo. We report here significant differences in the prevalence of substitutions among subtype B, and non-subtype B HIV isolates. Specifically, the E312Q, G333E, G335D, V365I, A371V and A376S substitutions were present in 2-6% of subtype B, whereas the G335D and A371V substitutions were commonly observed in 69% and 75% of non-B HIV-1 isolates. We observed a significant decline in the viral loads of patients that were infected with HIV-1 carrying these substitutions and were subsequently treated with triple drug regimens, even in the case where zidovudine (AZT) was included in such regimens. We show here that, generally, such single substitutions at the connection subdomain or RNase H domain have no influence on drug susceptibility in vitro by themselves. Instead, they generally enhance AZT resistance in the presence of excision-enhancing mutations (EEMs, also known as thymidine analogue-associated mutations, TAMs). However, N348I, A376S and Q509L did confer varying amounts of nevirapine resistance by themselves, even in the absence of EEMs. Our studies indicate that several connection subdomain and RNase H domain substitutions typically act as pre-therapy polymorphisms.
    Antiviral research 07/2009; 82(3):115-21. DOI:10.1016/j.antiviral.2009.02.189 · 3.94 Impact Factor
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    ABSTRACT: HIV-1 reverse transcriptase (RT) polymerase domain is known as target of nonnucleoside (NNRTIs) and nucleoside (NRTIs) reverse transcriptase inhibitors. However, resistance mutations in the polymerase domain lead repeatedly to therapeutic failure. The Connection (CN) and RNAse H domains of HIV-1 RT have recently gained more interest. They also might serve as independent new drug targets. Recently, in several in vitro studies several mutations in the CN and RNAse H domains were suggested to interfer with Polymerase resistance mutations (TAMs or finger domain mutations). In our in vivo approach, a clear differentiation between different HIV-1 subtypes was performed. Sequences of 57 HIV-1 subtype B infected patients were analysed. Their mutation status in the Connection and RNAse H domains was compared to a subtype-specific reference sequence. The sequences were studied for mutations. A potential correlation to Polymerase domain resistance mutation was analysed. Highly conserved amino acids and a number of natural polymorphisms were found for most of the studied positions. Subtype specific amino acid patterns were found. However, for positions 333, 359, 371, 390 and 558 a significant correlation to Polymerase domain resistance mutations was found. In conclusion, five positions were detected that might be involved in resistance mechanisms. The creation of a subtype specific reference sequence was necessary in order to distinguish between drug related mutations, subtype specific conservation or natural polymorphism. Extended sequence analysis to these regions are not recommended due to the small number of mutations found. Subtype speficication is highly recommended in order to gain resilient data.
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