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Lys66 residue as a determinant of high mismatch extension and misinsertion rates of HIV ‐1 reverse transcriptase
Author(s) -
Lwatula Chisanga,
Garforth Scott J.,
Prasad Vinayaka R.
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2012.08807.x
Subject(s) - reverse transcriptase , mutant , wild type , primer extension , nucleotide , nucleoside , chemistry , nucleoside analogue , microbiology and biotechnology , stereochemistry , biology , biochemistry , rna , gene
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 L ys66 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 L ys66 substitution mutants: L ys66Arg, L ys66Ala, L ys66Asn and L ys66 T hr. The mismatch extension efficiency was reduced for all mutants, with L ys66Ala, L ys66Asn and L ys66Thr 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 L ys66 A rg mutant was akin to wild‐type HIV ‐1 RT , whereas all nonconservative mutants displayed significantly decreased efficiency for both events. Our results suggest that L ys66, much like L ys65, is a determinant of both dNTP misinsertion and mismatch extension efficiency. While L ys65 is known to contact the γ‐phosphate of incoming d NTP , the L ys66 side chain is in the vicinity of the primer terminus. However, our results suggest that both residues have a similar influence on d NTP 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 L ys65 A rg, the L ys66 A la and L ys66 A sn mutants displayed mild resistance to the nucleoside analog drug 3′‐azido‐3′‐deoxythymidine‐5′‐triphosphate ( AZTTP ). Database EC 2.7.7.49 , EC 2.7.7.7