title: Hypersusceptibility to substrate analogs conferred by mutations in human immunodeficiency virus type 1 reverse transcriptase.
abstract: Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs (A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an important determinant of substrate specificity. To examine the functional role of this region, we performed "random scanning mutagenesis" of 11 motif B residues and screened replication-competent mutants for altered substrate analog sensitivity in culture. Single amino acid replacements throughout the targeted region conferred resistance to lamivudine and/or hypersusceptibility to zidovudine (AZT). Substitutions at residue Q151 increased the sensitivity of HIV-1 to multiple nucleoside analogs, and a subset of these Q151 variants was also hypersusceptible to the pyrophosphate analog phosphonoformic acid (PFA). Other AZT-hypersusceptible mutants were resistant to PFA and are therefore phenotypically similar to PFA-resistant variants selected in vitro and in infected patients. Collectively, these data show that specific amino acid replacements in motif B confer broad-spectrum hypersusceptibility to substrate analog inhibitors. Our results suggest that motif B influences RT-deoxynucleoside triphosphate interactions at multiple steps in the catalytic cycle of polymerization.
authors.affiliation: Department of Pathology, University of Washington, K-084 HSB, Box 357705, 1959 NE Pacific St., Seattle, 98195, USA. smithra@u.washington.edu
authors.forename: Robert A
authors.initials: RA
authors.lastname: Smith
authors.forename: Donovan J
authors.initials: DJ
authors.lastname: Anderson
authors.forename: Bradley D
authors.initials: BD
authors.lastname: Preston
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D002384:Catalysis; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics; D017245:Foscarnet / Q000031:analogs & derivatives / Q000494:pharmacology; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000235:genetics*; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D016296:Mutagenesis; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics*; D015215:Zidovudine / Q000494:pharmacology
chemical_list: D018894:Reverse Transcriptase Inhibitors; D017245:Foscarnet; D015215:Zidovudine; D054303:HIV Reverse Transcriptase