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

ABSTRACT: 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 ?-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).

SUBMITTER: Lwatula C

PROVIDER: S-EPMC3472125 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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

Lwatula Chisanga C Garforth Scott J SJ Prasad Vinayaka R VR

The FEBS journal 20120927 21

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 ...[more]

PMID: 22925131

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Project description:N348I in HIV-1 reverse transcriptase (RT) confers resistance to zidovudine (AZT) and nevirapine. Biochemical studies demonstrated that N348I indirectly increases AZT resistance by decreasing the frequency of secondary ribonuclease H (RNase H) cleavages that reduce the RNA/DNA duplex length of the template/primer (T/P) and diminish the efficiency of AZT-monophosphate (MP) excision. By contrast, there is some discrepancy in the literature in regard to the mechanisms associated with nevirapine resistance: one study suggested that it is due to decreased inhibitor binding while others suggest that it may be related to the decreased RNase H cleavage phenotype. From a structural perspective, N348 in both subunits of RT resides distal to the enzyme's active sites, to the T/P binding tract and to the nevirapine-binding pocket. As such, the structural mechanisms associated with the resistance phenotypes are not known.Using a novel modelled structure of RT in complex with an RNA/DNA T/P, we identified a putative interaction between the ?14-?15 loop in the p51 subunit of RT and the RNA template. Substitution of the asparagine at codon 348 in the p51 subunit with either isoleucine or leucine abrogated the observed protein-RNA interaction, thus, providing a possible explanation for the decreased RNase H phenotype. By contrast, alanine or glutamine substitutions exerted no effect. To validate this model, we introduced the N348I, N348L, N348A and N348Q mutations into RT and purified enzymes that contained subunit-specific mutations. N348I and N348L significantly decreased the frequency of secondary RNase H cleavages and increased the enzyme's ability to excise AZT-MP. As predicted by the modelling, this phenotype was due to the mutation in the p51 subunit of RT. By contrast, the N348A and N348Q RTs exhibited RNase H cleavage profiles and AZT-MP excision activities similar to the wild-type enzyme. All N348 mutant RTs exhibited decreased nevirapine susceptibility, although the N348I and N348L mutations conferred higher fold resistance values compared to N348A and N348Q. Nevirapine resistance was also largely due to the mutation present in the p51 subunit of RT.This study demonstrates that N348I-mediated AZT and nevirapine resistance is due to the mutation in the p51 subunit of RT.

Dataset Information

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

Publications

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

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