Insertions into the β3-β4 hairpin loop of HIV-1 reverse transcriptase reveal a role for fingers subdomain in processive polymerization

Yvonne Kew, Laurence R. Olsen, Anthony J. Japour, Vinayaka R. Prasad

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Abstract

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) displays a characteristic poor processivity during DNA polymerization. Structural elements of RT that determine processivity are poorly understood. The three-dimensional structure of HIV-1 RT, which assumes a hand-like structure, shows that the fingers, palm, and thumb subdomains form the template-binding cleft and may be involved in determining the degree of processivity. To assess the influence of fingers subdomain of HIV-1 RT in polymerase processivity, two insertions were engineered in the β3-β4 hairpin of HIV-I(NL4-3) RT. The recombinant mutant RTs, named FE20 and FE103, displayed wild type or near wild type levels of RNA-dependent DNA polymerase activity on all templates tested and wild type or near wild type-like sensitivities to dideoxy-NTPs. When polymerase activities were measured under conditions that allow a single cycle of DNA polymerization, both of the mutants displayed 25-30% greater processivity than wild type enzyme. Homology modeling the dimensional structures of wild type HIV-I(NL4-3) RT and its finger insertion mutants revealed that the extended loop between the β3 and β4 strands protrudes into the cleft, reducing the distance between the fingers and thumb subdomains to ~12 A. Analysis of the models for the mutants suggests an extensive interaction between the protein and template- primer, which may reduce the degree of super- structure in the template- primer. Our data suggest that the β3-β4 hairpin of fingers subdomain is an important determinant of processive polymerization by HIV-1 RT.

Original languageEnglish (US)
Pages (from-to)7529-7537
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number13
DOIs
Publication statusPublished - Mar 27 1998

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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