Effects of Mutations F61A and A62V in the Fingers Subdomain of HIV-1 Reverse Transcriptase on the Translocational Equilibrium

Abstract

Changes of the translocational status of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) can affect susceptibility to antiretroviral drugs. The pyrophosphate analogue phosphonoformic acid (PFA) binds specifically to and traps the pretranslocated complex of HIV-1 RT, while nucleotide-competing RT inhibitors trap the posttranslocated conformation. Here, we attempted to assess the potential role of residues in the fingers subdomain as determinants of polymerase translocation. The fingers can exist in open and closed conformations; however, the relationship between such conformational changes and the translocation status of HIV-1 RT remains elusive. We focused on substitution F61A and the neighboring A62V that is frequently associated with drug-resistance-conferring mutations. The proximity of these residues to the nucleic acid substrate suggested a possible role in translocation for these amino acid changes. We employed site-specific footprinting, binding assays, and DNA-synthesis inhibition experiments to study F61A and A62V, alone and against a background of known drug-resistance mutations. We demonstrate that F61A causes a strong bias to the posttranslocational state, while A62V shows a subtle bias toward pretranslocation regardless of the mutational background. Increases in the population of pretranslocated complexes were accompanied by increases in PFA activity, while F61A is literally resistant to PFA. Our data shed light on equilibria between pre- and posttranslocated complexes with the fingers subdomain in its open or closed conformations. We propose that a binary, pretranslocated complex in a closed conformation is stabilized with A62V and destabilized with F61A.