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Mechanisms Involved in the Selection of HIV-1 Reverse Transcriptase Thumb Subdomain Polymorphisms Associated with Nucleoside Analogue Therapy Failure
- 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.).
- Subjects :
- HIV Infections
Biology
Virus Replication
Antiviral Agents
Cell Line
law.invention
03 medical and health sciences
chemistry.chemical_compound
law
Drug Resistance, Viral
medicine
Humans
Pharmacology (medical)
RNase H
Cells, Cultured
030304 developmental biology
Pharmacology
0303 health sciences
Polymorphism, Genetic
Nucleoside analogue
030306 microbiology
RNA-Directed DNA Polymerase
RNA
Nucleotidyltransferase
Virology
Molecular biology
Dideoxynucleosides
HIV Reverse Transcriptase
Reverse transcriptase
3. Good health
Stavudine
Infectious Diseases
chemistry
Recombinant DNA
biology.protein
Reverse Transcriptase Inhibitors
Zidovudine
DNA
medicine.drug
Subjects
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