title: A role for dNTP binding of human immunodeficiency virus type 1 reverse transcriptase in viral mutagenesis.
abstract: HIV-1 reverse transcriptase (RT) is a highly error prone DNA polymerase. We assessed whether the ability of RT to bind nucleotide substrates affects viral mutagenesis. Structural modeling predicts that the V148 and Q151 residues influence the interaction between RT and the incoming dNTP. When we introduce either a V148I or Q151N mutation, RT fidelity increases 8.7- or 13-fold, respectively, as measured by the M13 lacZalpha forward mutation assay. Interestingly, pre-steady state kinetic studies demonstrated that these mutations do not alter polymerase fidelity during the first step of mutation synthesis, misincorporation. Rather, the V148I and Q151N mutations alter RT fidelity by weakening the ability of the polymerase to complete mismatch extension, the second step of mutation synthesis. While both these mutations minimally affect the binding of RT (K(D)) to a mismatched template-primer complex (T/P), these mutant RTs are significantly impaired in their ability to bind (K(d)) and chemically incorporate (k(pol)) nucleotide substrate onto a mismatched T/P. These differences in binding and catalysis translate into 24- and 15.9-fold increase in mismatch extension fidelity for the V148I and Q151N RT mutants, respectively. Finally, we employed a cell-based pseudotyped HIV-1 mutation assay to determine whether changes in these dNTP binding residues alter RT fidelity in vivo. We found that the V148I and Q151N mutant viruses had 3.8- and 5.7-fold higher fidelities than wild-type viruses, respectively, indicating that the molecular interaction between HIV-1 RT and the dNTP substrate contributes to viral mutagenesis.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 672, Rochester, New York 14642, USA.
authors.forename: Kellie K
authors.initials: KK
authors.lastname: Weiss
authors.forename: Renxiang
authors.initials: R
authors.lastname: Chen
authors.forename: Mark
authors.initials: M
authors.lastname: Skasko
authors.forename: Holly M
authors.initials: HM
authors.lastname: Reynolds
authors.forename: Kwi
authors.initials: K
authors.lastname: Lee
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
authors.forename: Louis M
authors.initials: LM
authors.lastname: Mansky
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D017104:Bacteriophage M13 / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism*; D005787:Gene Frequency; D005973:Glutamine / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics*; D007532:Isoleucine / Q000235:genetics; D007700:Kinetics; D007763:Lac Operon; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011485:Protein Binding / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013698:Templates, Genetic; D014633:Valine / Q000235:genetics
chemical_list: D017931:DNA Primers; D003854:Deoxyribonucleotides; D007532:Isoleucine; D005973:Glutamine; D001216:Asparagine; D054303:HIV Reverse Transcriptase; D014633:Valine