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

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 HTV-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 beta 3-beta 4 hairpin of HIV-1(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 three-dimensional structures of wild type HIV-1(NL4-3), RT and its finger insertion mutants revealed that the extended loop between the beta 3 and beta 4 strands protrudes into the cleft, reducing the distance between the fingers and thumb subdomains to similar to 12 Angstrom. Analysis of the models for the mutants suggests an extensive interaction between the protein and template-primer, which may reduce the degree of superstructure in the template-primer, Our data suggest that the beta 3-beta 4 hairpin of fingers subdomain is an important determinant of processive polymerization by HIV-1 RT.