Molecular architecture of the mutagenic active site of human immunodeficiency virus type 1 reverse transcriptase: roles of the beta 8-alpha E loop in fidelity, processivity, and substrate interactions.

Important note: Information in this article was accurate in 2000. The state of the art may have changed since the publication date.

Biochemistry. 2000 Sep 5;39(35):10684-94. Unique Identifier : AIDSLINE MED/20435243
Weiss KK; Isaacs SJ; Tran NH; Adman ET; Kim B; Department of Microbiology and Immunology, University of; Rochester Medical Center, 601 Elmwood Avenue, Box 672, Rochester,; New York 14642, USA.

Abstract: Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a putative source of the genomic hypermutation that promotes rapid evolution of HIV-1. To understand the molecular strategies that create a highly mutagenic DNA polymerase active site in HIV-1 RT, we investigated the roles of four residues in the beta 8-alpha E loop. Gln151, which interacts with the sugar of the incoming dNTP, and Lys154, which interacts with the template, yielded site-directed mutants with increased fidelity, suggesting that these residues are directly involved in the mutagenic architecture of the active site. Mutations at Gln151 and Lys154 also reduced processivity. Q151N RT showed enhanced ability to discriminate between TTP and AZT triphosphate, consistent with the observation that the Q151M mutation confers AZT resistance in vivo. Mutations at Gly152 greatly decreased RT activity; molecular modeling suggests that Gly152 is critical for the proper geometric alignment that permits base-pairing of the incoming dNTP with the template. Mutations at Trp153 reduced the expression level, and presumably the stability, of RT proteins in bacteria. These observations support the conclusion that interactions of active site residues in the beta 8-alpha E loop with incoming dNTPs and the template are determinants of the accuracy, processivity, and substrate selectivity of HIV-1 RT.

Keywords: JOURNAL ARTICLE Amino Acid Substitution/GENETICS Anti-HIV Agents/PHARMACOLOGY Base Pair Mismatch Binding Sites/GENETICS DNA Mutational Analysis DNA Primers/CHEMISTRY/GENETICS Glutamine/GENETICS Glycine/GENETICS Human HIV-1 Reverse Transcriptase/ANTAGONISTS & INHIB/*CHEMISTRY/ *GENETICS/METABOLISM Lac Operon Lysine/GENETICS *Mutagenesis, Site-Directed Protein Processing, Post-Translational/*GENETICS Protein Structure, Secondary/GENETICS RNA/CHEMISTRY/GENETICS Substrate Specificity/GENETICS Support, U.S. Gov't, P.H.S. Templates Thymine Nucleotides/PHARMACOLOGY Tryptophan/GENETICS Zidovudine/*ANALOGS & DERIVATIVES/PHARMACOLOGY

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