Mutations in the primer grip region of HIV reverse transcriptase can increase replication fidelity

Mutations in the primer grip region of human immunodeficiency virus reverse transcriptase (HIV-RT) affect its replication fidelity. The primer grip re gion (residues 227-235) correctly positions the 3'-ends of primers. Point m utations were created by alanine substitution at positions 224-235, Error f requencies were measured by extension of a dG:dA primer-template mismatch. Mutants E224A, P225A, P226A, L228A, and E233A were approximately equal to t he wild type in their ability to extend the mismatch. Mutants F227A, W229A, M230A, G231A, and Y232A extended 40, 66, 54, 72, and 76% less efficiently past a dG:dA mismatch compared with the wild type. We also examined the mis insertion rates of dG, dC, or dA across from a DNA template dA using RT mut ants F227A and W229A Mutant W229A exhibited high fidelity and did not produ ce a dC:dA or dC:dA mismatch. Interestingly, mutant F227A displayed high fi delity for dG:dA and dC:dA mismatches but low fidelity for dA:dA misinserti ons, This indicates that F227A discriminates against particular base substi tutions. However, a primer extension assay with three dNTPs showed that F22 7A generally displays higher fidelity than the wild type RT. Clearly, prime r grip mutations can improve or worsen either the overall or base-specific fidelity of HIV-RT, We hypothesize that wild type RT has evolved to a fidel ity that allows genetic variation without compromising yield of viable viru ses.