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Thymidine analogue excision and discrimination modulated by mutational complexes including single amino acid deletions of Asp-67 or Thr-69 in HIV-1 reverse transcriptase.

ABSTRACT: Single amino acid deletions in the ?3-?4 hairpin loop of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been identified in heavily treated patients. The deletion of Asp-67 together with mutations T69G and K70R (?67 complex) are usually associated with thymidine analog resistance mutations (TAMs) (e.g. M41L, T215Y, etc.) while the deletion of Thr-69 (?69) is rarely found in isolates containing TAMs. Here, we show that the complex ?67/T69G/K70R enhances ATP-dependent phosphorolytic activity on primers terminated with 3'-azido-3'-deoxythymidine (AZT) or 2',3'-didehydro-2',3'-dideoxythymidine (d4T) both in the presence or absence of TAMs (i.e. M41L/T215Y), while ?69 (or the complex S68G/?69/K70G) antagonize the effects of TAMs in ATP-mediated excision. These effects are consistent with AZT susceptibility data obtained with recombinant HIV-1 bearing the relevant RTs. Molecular dynamics studies based on models of wild-type HIV-1 RT and mutant ?69, ?67/T69G/K70R, and D67N/K70R RTs support a relevant role for Lys/Arg-70 in the excision reaction. In ?69 RT, the side chain of Lys-70 locates away from the putative pyrophosphate binding site. Therefore, its participation in interactions required for the excision reaction is unlikely. Our theoretical studies also suggest a role for Lys-219 in thymidine analog excision/discrimination. However, pre-steady-state kinetics revealed only minor differences in selectivity of AZT-triphosphate versus dTTP between deletion-containing RTs and their homologous enzymes having the K219E mutation. K219E reduced both ATP- and pyrophosphate-mediated excision of primers terminated with thymidine analogues, only when introduced in RTs bearing ?69 or S68G/?69/K70G, providing further biochemical evidence that explains the lack of association of ?69 and TAMs in HIV-1 isolates.

SUBMITTER: Kisic M

PROVIDER: S-EPMC3121480 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Thymidine analogue excision and discrimination modulated by mutational complexes including single amino acid deletions of Asp-67 or Thr-69 in HIV-1 reverse transcriptase.

Kisic Mónica M Matamoros Tania T Nevot María M Mendieta Jesús J Mendieta Jesús J Martinez-Picado Javier J Martínez Miguel A MA Menéndez-Arias Luis L

The Journal of biological chemistry 20110419 23

Single amino acid deletions in the β3-β4 hairpin loop of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been identified in heavily treated patients. The deletion of Asp-67 together with mutations T69G and K70R (Δ67 complex) are usually associated with thymidine analog resistance mutations (TAMs) (e.g. M41L, T215Y, etc.) while the deletion of Thr-69 (Δ69) is rarely found in isolates containing TAMs. Here, we show that the complex Δ67/T69G/K70R enhances ATP-dependent p ...[more]

PMID: 21504903

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Project description: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.

Dataset Information

Thymidine analogue excision and discrimination modulated by mutational complexes including single amino acid deletions of Asp-67 or Thr-69 in HIV-1 reverse transcriptase.

Publications

Thymidine analogue excision and discrimination modulated by mutational complexes including single amino acid deletions of Asp-67 or Thr-69 in HIV-1 reverse transcriptase.

Similar Datasets

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