Subunit-specific mutational analysis of residue N348 in HIV-1 reverse transcriptase

Jessica Radzio,Nicolas Sluis-Cremer

doi:10.1186/1742-4690-8-69

Jessica Radzio, Nicolas Sluis-Cremer

Open Access

https://doi.org/10.1186/1742-4690-8-69

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Journal: Retrovirology	Publication Date: Aug 22, 2011
Citations: 29	License type: CC BY 2.0

Affiliation: University of Pittsburgh

Abstract

BackgroundN348I 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.ResultsUsing a novel modelled structure of RT in complex with an RNA/DNA T/P, we identified a putative interaction between the β14-β15 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.ConclusionsThis study demonstrates that N348I-mediated AZT and nevirapine resistance is due to the mutation in the p51 subunit of RT.

Highlights

N348I in HIV-1 reverse transcriptase (RT) confers resistance to zidovudine (AZT) and nevirapine
There is some discrepancy in the literature in regard to the mechanisms associated with nevirapine resistance: one study has suggested it is due to decreased inhibitor binding [9], while other studies suggest that it may be due to the decreased ribonuclease H (RNase H) cleavage phenotype of the N348I HIV-1 RT [10,11]
When the N348I mutation is introduced into the p51 subunit in this structure by molecular modelling (Figure 1D), the position of the b14-b15 loop is shifted such that P345 and F346 no longer contact the RNA template

Summary

Introduction

N348I in HIV-1 reverse transcriptase (RT) confers resistance to zidovudine (AZT) and nevirapine. In the available crystal structures of HIV-1 RT, residue N348 in both subunits of the enzyme is located distal to the DNA polymerase and RNase H active sites, to the T/P substrate, to residues that comprise the nucleic acid binding tract and to the NNRTIbinding pocket [Figure 1A, B]. It is not evident how N348I in HIV-1 RT impacts the RNase H cleavage of the enzyme or decreases drug susceptibility.

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Conclusion