Substitutions of Phe⁶¹ located in the vicinity of template 5′-overhang influence polymerase fidelity and nucleoside analog sensitivity of HIV-1 reverse transcriptase

Human immunodeficiency virus type 1 reverse transcriptase (RT) is an error-prone DNA polymerase. Structural determinants of its fidelity are incompletely understood. RT/template primer contacts have been shown to influence its fidelity and sensitivity to nucleoside analog inhibitors. The Phe⁶¹ residue, located within the β3 sheet of the finger subdomain, is highly conserved among retroviral RTs. The crystal structure of a ternary complex revealed that Phe⁶¹ contacts the first and second bases of the 5′-template overhang. To determine whether such contacts influence the dNTP-binding pocket, we performed a limited vertical scanning mutagenesis (Phe → Ala, Leu, Trp, or Tyr) at Phe⁶¹. The F61A mutant displayed the highest increase in fidelity, followed by the F61L and F61W variants, which had intermediate phenotypes. F61Y RT had a minimal effect. The increase in fidelity of the F61A mutant was corroborated by a 12-fold decrease in its forward mutation rate. The Phe⁶¹ mutant RTs also displayed large reductions in sensitivity to 2′,3′-dideoxythymidine triphosphate and 2′,3′-dideoxy,2′3′-didehydrothymidine triphosphate. Mutants displaying the largest increase in fidelity (F61A and F61L) were also the most resistant. These results suggest that contacts between the finger subdomain of human immunodeficiency virus type 1 RT and the template 5′-overhang are important determinants of the geometry of the dNTP-binding pocket.