title: Molecular basis of the association of H208Y and thymidine analogue resistance mutations M41L, L210W and T215Y in the HIV-1 reverse transcriptase of treated patients.
abstract: Thymidine analogue resistance mutations (TAMs) in HIV-1 reverse transcriptase (RT) associate in two clusters: (i) TAM1 (M41L, L210W and T215Y) and TAM2 (D67N, K70R, K219E/Q, and sometimes T215F). The amino acid substitution H208Y shows increased prevalence in patients treated with nucleoside analogues and is frequently associated with TAM1 mutations. We studied the molecular mechanism favoring the selection of H208Y in the presence of zidovudine, tenofovir and other nucleoside RT inhibitors (NRTIs). NRTI susceptibility was not affected by the addition of H208Y in phenotypic assays carried out in MT-4 cells using recombinant HIV-1 containing wild-type (subtype B, BH10), H208Y, M41L/L210W/T215Y or M41L/H208Y/L210W/T215Y RTs. However, enzymatic studies carried out with purified RTs revealed that in the presence of M41L/L210W/T215Y, H208Y increases the RT's ability to unblock and extend primers terminated with zidovudine, tenofovir and in a lesser extent, stavudine. These effects were observed with DNA/DNA complexes (but not with RNA/DNA) and resulted from the higher ATP-dependent excision activity of the M41L/H208Y/L210W/T215Y RT compared with the M41L/L210W/T215Y mutant. The increased rescue efficiency of the M41L/H208Y/L210W/T215Y RT was observed in the presence of ATP but not with GTP or ITP. Molecular dynamics studies predict an alteration of the stacking interactions between Tyr(215) and the adenine ring of ATP due to long-distance effects caused by tighter packaging of Tyr(208) and Trp(212). These studies provide a mechanistic explanation for the association of TAM-1 and H208Y mutations in viral isolates from patients treated with NRTIs.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid), Madrid, Spain.
authors.forename: Gilberto
authors.initials: G
authors.lastname: Betancor
authors.affiliation: Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Badalona, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Nevot
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid), Madrid, Spain; Biomol-Informatics, Parque Científico de Madrid, Madrid, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Mendieta
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid), Madrid, Spain.
authors.forename: Paulino
authors.initials: P
authors.lastname: Gómez-Puertas
authors.affiliation: Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Badalona, Spain.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Martínez
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid), Madrid, Spain. Electronic address: lmenendez@cbm.csic.es.
authors.forename: Luis
authors.initials: L
authors.lastname: Menéndez-Arias
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000627:therapeutic use; D019380:Anti-HIV Agents / Q000627:therapeutic use*; D002460:Cell Line; D024882:Drug Resistance, Viral; D015658:HIV Infections / Q000188:drug therapy* / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests; D020125:Mutation, Missense; D063065:Organophosphonates / Q000627:therapeutic use; D048348:Reverse Transcription / Q000187:drug effects; D000068698:Tenofovir; D015215:Zidovudine / Q000627:therapeutic use
chemical_list: D019380:Anti-HIV Agents; D063065:Organophosphonates; D015215:Zidovudine; D000068698:Tenofovir; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D000225:Adenine
keywords: Drug resistance; HIV; Reverse transcriptase; Tenofovir; Thymidine analogues; Zidovudine