Lys66 residue as a determinant of high mismatch extension and misinsertion rates of HIV-1 reverse transcriptase

A major factor contributing to the high mutation rate of HIV-1 reverse transcriptase (RT) is its high propensity for misincorporation. Misincorporation requires both deoxyribonucleotide triphosphate (dNTP) misinsertion and the subsequent extension of the mismatched terminus thus formed. We hypothesized that Lys66 is a determinant of mismatch extension based on its position near the primer terminus. This hypothesis was tested by steady-state kinetic studies using wild-type HIV-1 RT and four Lys66 substitution mutants: Lys66Arg, Lys66Ala, Lys66Asn and Lys66Thr. The mismatch extension efficiency was reduced for all mutants, with Lys66Ala, Lys66Asn and Lys66Thr showing a four- to six-fold reduction compared with wild-type HIV-1 RT. Surprisingly, the nonconservative substitutions also led to large decreases in misinsertion efficiency, ranging from as low as three-fold to values much higher than 23-fold. Thus, the Lys66Arg mutant was akin to wild-type HIV-1 RT, whereas all nonconservative mutants displayed significantly decreased efficiency for both events. Our results suggest that Lys66, much like Lys65, is a determinant of both dNTP misinsertion and mismatch extension efficiency. While Lys65 is known to contact the γ-phosphate of incoming dNTP, the Lys66 side chain is in the vicinity of the primer terminus. However, our results suggest that both residues have a similar influence on dNTP misinsertion and mispair extension efficiencies of HIV-1 RT. When we tested the mutants for susceptibility to selected nucleoside analog and non-nucleoside analog drugs, similarly to Lys65Arg, the Lys66Ala and Lys66Asn mutants displayed mild resistance to the nucleoside analog drug 3'-azido-3'-deoxythymidine-5'-triphosphate (AZTTP).