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Mechanisms involved in the selection of HIV-1 reverse transcriptase thumb subdomain polymorphisms associated with nucleoside analogue therapy failure.

ABSTRACT: Previous studies showed an increased prevalence of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) thumb subdomain polymorphisms Pro272, Arg277, and Thr286 in patients failing therapy with nucleoside analogue combinations. Interestingly, wild-type HIV-1(BH10) RT contains Pro272, Arg277, and Thr286. Here, we demonstrate that in the presence of zidovudine, HIV-1(BH10) RT mutations P272A/R277K/T286A produce a significant reduction of the viral replication capacity in peripheral blood mononuclear cells in both the absence and presence of M41L/T215Y. In studies carried out with recombinant enzymes, we show that RT thumb subdomain mutations decrease primer-unblocking activity on RNA/DNA complexes, but not on DNA/DNA template-primers. These effects were observed with primers terminated with thymidine analogues (i.e., zidovudine and stavudine) and carbovir (the relevant derivative of abacavir) and were more pronounced when mutations were introduced in the wild-type HIV-1(BH10) RT sequence context. RT thumb subdomain mutations increased by 2-fold the apparent dissociation equilibrium constant (K(d)) for RNA/DNA without affecting the K(d) for DNA/DNA substrates. RNase H assays carried out with RNA/DNA complexes did not reveal an increase in the reaction rate or in secondary cleavage events that could account for the decreased excision activity. The interaction of Arg277 with the phosphate backbone of the RNA template in HIV-1 RT bound to RNA/DNA and the location of Thr286 close to the RNA strand are consistent with thumb polymorphisms playing a role in decreasing nucleoside RT inhibitor excision activity on RNA/DNA template-primers by affecting interactions with the template-primer duplex without involvement of the RNase H activity of the enzyme.

SUBMITTER: Betancor G

PROVIDER: S-EPMC2976120 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Mechanisms involved in the selection of HIV-1 reverse transcriptase thumb subdomain polymorphisms associated with nucleoside analogue therapy failure.

Betancor Gilberto G Puertas Maria C MC Nevot María M Garriga César C Martínez Miguel A MA Martinez-Picado Javier J Menéndez-Arias Luis L

Antimicrobial agents and chemotherapy 20100823 11

Previous studies showed an increased prevalence of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) thumb subdomain polymorphisms Pro272, Arg277, and Thr286 in patients failing therapy with nucleoside analogue combinations. Interestingly, wild-type HIV-1(BH10) RT contains Pro272, Arg277, and Thr286. Here, we demonstrate that in the presence of zidovudine, HIV-1(BH10) RT mutations P272A/R277K/T286A produce a significant reduction of the viral replication capacity in peripher ...[more]

PMID: 20733040

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Project description:Nucleoside reverse-transcriptase inhibitor (NRTI) therapy for human immunodeficiency virus (HIV) infection has been associated with mitochondrial DNA (mtDNA) polymerase-gamma inhibition and subsequent mtDNA depletion. Effects on mtDNA mutation, although suggested by critical involvement of polymerase-gamma in DNA-repair reactions, are unknown. In the present study, we assessed the nature and frequency of mitochondrial genome sequence differences in peripheral-blood samples taken prior to NRTI therapy and after 6-77 mo of treatment in 16 NRTI-treated patients. Samples from 10 HIV-infected, treatment-naive control individuals were taken at similar time intervals. Single-stranded conformation polymorphism (SSCP) and DNA-sequencing analysis techniques were used to detect mitochondrial genome sequence variants between paired longitudinal samples, and heteroplasmic populations were quantified after cloning and repeat SSCP/sequencing. Of 16 individuals treated with NRTIs, 5 exhibited altered SSCP profiles associated with the development of novel heteroplasmic DNA sequence changes, whereas no SSCP pattern change within these regions was observed in the control individuals. Heteroplasmic sequence changes were distributed across four regions of the genome: the noncoding region to 12S ribosomal RNA, reduced-nicotinamide-adenine-dinucleotide dehydrogenase 1, and cytochrome oxidase subunits I and III. Of the total of 26 patients who were examined in the present study, 4 of 5 patients with detectable mtDNA sequence changes since commencement of therapy developed evidence of peripheral fat wasting (lipoatrophy) between sample intervals (P=.031). One patient, without detectable sequence changes on NRTI therapy, also developed lipoatrophy. Levels of mtDNA copies/cell in blood samples were determined by quantitative PCR for 11 of the 16 NRTI-exposed patients; 7 of these 11 patients showed reduced levels of mtDNA in blood after therapy, including all 3 patients tested with evidence of mtDNA sequence changes on therapy. These data indicate that NRTI therapy provides conditions permissive for the development of peripheral-blood mtDNA mutations in vivo.

Project description:Infection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H domains of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, therefore, we analyzed the polymerase, connection, and RNase H domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation, V111I, in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNase H domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-to-115 loop region, which may affect deoxynucleoside triphosphate (dNTP) binding and polymerase activity.Mutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues but increases the fitness of viruses with drug resistance mutations K65R and Q151M.

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Dataset Information

Mechanisms involved in the selection of HIV-1 reverse transcriptase thumb subdomain polymorphisms associated with nucleoside analogue therapy failure.

Publications

Mechanisms involved in the selection of HIV-1 reverse transcriptase thumb subdomain polymorphisms associated with nucleoside analogue therapy failure.

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