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Mechanisms Involved in the Selection of HIV-1 Reverse Transcriptase Thumb Subdomain Polymorphisms Associated with Nucleoside Analogue Therapy Failure

Authors :
Gilberto Betancor
Javier Martinez-Picado
Luis Menéndez-Arias
Miguel Angel Martinez
Cesar Garriga
Maria Nevot
Maria C. Puertas
Ministerio de Ciencia e Innovación (España)
Fundación para la Investigación y la Prevención del Sida en España
Instituto de Salud Carlos III
Fundación Ramón Areces
European Commission
Source :
Digital.CSIC. Repositorio Institucional del CSIC, instname, Antimicrobial Agents and Chemotherapy; Vol 54
Publication Year :
2010
Publisher :
American Society for Microbiology, 2010.

Abstract

13 páginas, 8 figuras, 2 tablas.<br />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-1BH10 RT contains Pro272, Arg277, and Thr286. Here, we demonstrate that in the presence of zidovudine, HIV-1BH10 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-1BH10 RT sequence context. RT thumb subdomain mutations increased by 2-fold the apparent dissociation equilibrium constant (Kd) for RNA/DNA without affecting the Kd 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.<br />Funding for this work was provided by grants from the Spanish Ministry of Science and Innovation (BIO2007/60319), Fundacio´n para la Investigacio´n y Prevencio´n del SIDA en Espan˜a (FIPSE) (grant 36771/08), Fondo de Investigacio´n Sanitaria (through the “Red Tema ´tica de Investigacio´n Cooperativa en SIDA” RD06/0006), and an institutional grant of Fundacio´n Ramo´n Areces. Work at the Fundacio´ irsiCaixa was supported by the European Community’s Seventh Framework Programme (FP7/2007-2013) under the “Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)” project grant agreement 223131 and the Spanish Ministry of Science and Innovation through grant PI07/0098 (to M.A.M.).

Details

ISSN :
10986596 and 00664804
Volume :
54
Database :
OpenAIRE
Journal :
Antimicrobial Agents and Chemotherapy
Accession number :
edsair.doi.dedup.....9cd0a3e9605ed4fa10c8f886af7d3832
Full Text :
https://doi.org/10.1128/aac.00716-10