title: Molecular determinants of multi-nucleoside analogue resistance in HIV-1 reverse transcriptases containing a dipeptide insertion in the fingers subdomain: effect of mutations D67N and T215Y on removal of thymidine nucleotide analogues from blocked DNA primers.
abstract: Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3 '-azido-3 '-deoxythymidine (AZT) and other nucleoside analogues. Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y. The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers. In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity. The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance. Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion. The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT. However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme. Removal reactions can be inhibited by the next complementary dNTP. Both Tyr-215 and the dipeptide insertion affect RT-DNA.DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet. Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.
authors.affiliation: Centro de Biologìa Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientìficas-Universidad Autónoma de Madrid, 28049 Madrid, Spain.
authors.forename: Tania
authors.initials: T
authors.lastname: Matamoros
authors.forename: Sandra
authors.initials: S
authors.lastname: Franco
authors.forename: Blanca M
authors.initials: BM
authors.lastname: Vázquez-Alvarez
authors.forename: Antonio
authors.initials: A
authors.lastname: Mas
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Martínez
authors.forename: Luis
authors.initials: L
authors.lastname: Menéndez-Arias
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D024882:Drug Resistance, Viral; D017353:Gene Deletion; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D018119:Stavudine / Q000494:pharmacology; D013936:Thymidine / Q000737:chemistry; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D015215:Zidovudine / Q000494:pharmacology
chemical_list: D003062:Codon; D017931:DNA Primers; D010455:Peptides; D018894:Reverse Transcriptase Inhibitors; D014443:Tyrosine; D015215:Zidovudine; D000255:Adenosine Triphosphate; D004247:DNA; D018119:Stavudine; D054303:HIV Reverse Transcriptase; D013936:Thymidine