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Clinical, virological and biochemical evidence supporting the association of HIV-1 reverse transcriptase polymorphism R284K and thymidine analogue resistance mutations M41L, L210W and T215Y in patients failing tenofovir/emtricitabine therapy

Gilberto Betancor1, César Garriga2, Maria C Puertas3, María Nevot3, Lourdes Anta4, José L Blanco5, M Jesús Pérez-Elías6, Carmen de Mendoza4, Miguel A Martínez3, Javier Martinez-Picado37, Luis Menéndez-Arias1* and for the Resistance Platform of the Spanish AIDS Research Network (ResRIS)

Author Affiliations

1 Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, Madrid, Spain

2 Centro Nacional de Epidemiología, Instituto de Salud Carlos III, Madrid, Spain

3 AIDS Research Institute IrsiCaixa, Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Universidad Autònoma de Barcelona, Badalona, Spain

4 Hospital Carlos III, Madrid, Spain

5 Hospital Clìnic, Barcelona, Spain

6 Hospital Ramón y Cajal (Instituto Ramón y Cajal de Investigación Sanitaria), Madrid, Spain

7 Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain

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Retrovirology 2012, 9:68  doi:10.1186/1742-4690-9-68

Published: 13 August 2012

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.