The N348I Mutation at the Connection Subdomain of HIV-1 Reverse Transcriptase Decreases Binding to Nevirapine

Matthew M Schuckmann,Bruno Marchand,Atsuko Hachiya,Eiichi N Kodama,Karen A Kirby,Kamalendra Singh,Stefan G Sarafianos

doi:10.1074/jbc.m110.153783

Matthew M Schuckmann, Bruno Marchand + Show 5 more

Open Access

PDF Available

https://doi.org/10.1074/jbc.m110.153783

Copy DOI

Export

Save

Cite

Abstract
Highlights/Summary
Full-Text PDF
Similar Papers

Abstract

Listen

The N348I mutation at the connection subdomain of HIV-1 reverse transcriptase (RT) confers clinically significant resistance to both nucleoside and non-nucleoside RT inhibitors (NNRTIs) by mechanisms that are not well understood. We used transient kinetics to characterize the enzymatic properties of N348I RT and determine the biochemical mechanism of resistance to the NNRTI nevirapine (NVP). We demonstrate that changes distant from the NNRTI binding pocket decrease inhibitor binding (increase K(d)(-NVP)) by primarily decreasing the association rate of the inhibitor (k(on-NVP)). We characterized RTs mutated in either p66 (p66(N348I)/p51(WT)), p51 (p66(WT)/p51(N348I)), or both subunits (p66(N348I)/p51(N348I)). Mutation in either subunit caused NVP resistance during RNA-dependent and DNA-dependent DNA polymerization. Mutation in p66 alone (p66(N348I)/p51(WT)) caused NVP resistance without significantly affecting RNase H activity, whereas mutation in p51 caused NVP resistance and impaired RNase H, demonstrating that NVP resistance may occur independently from defects in RNase H function. Mutation in either subunit improved affinity for nucleic acid and enhanced processivity of DNA synthesis. Surprisingly, mutation in either subunit decreased catalytic rates (k(pol)) of p66(N348I)/p51(N348I), p66(N348I)/p51(WT), and p66(WT)/p51(N348I) without significantly affecting affinity for deoxynucleotide substrate (K(d)(-dNTP)). Hence, in addition to providing structural integrity for the heterodimer, p51 is critical for fine tuning catalytic turnover, RNase H processing, and drug resistance. In conclusion, connection subdomain mutation N348I decreases catalytic efficiency and causes in vitro resistance to NVP by decreasing inhibitor binding.

Highlights

We demonstrate that changes distant from the non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) binding pocket decrease inhibitor binding by primarily decreasing the association rate of the inhibitor
nucleoside RT inhibitors (NRTIs) mimic deoxynucleotide triphosphate substrates required for DNA synthesis
Once integrated into the nascent viral DNA, NRTIs act as chain terminators because they lack a 3Ј-OH group required for formation of a phosphodiester bond with the incoming nucleotide (1, 2)

Summary

Introduction

To determine optimal rates of dNTP incorporation (kpol) and dNTP binding to the enzyme-DNA complex (Kd-dATP), observed burst rates (kobs) were fit to the following hyperbolic equation. Nevirapine Binding Assays—Solutions of RT (30 nM active sites), Td31/Cy3-Pd18 (30 nM), and EDTA (5 mM) in RT buffer were incubated with various concentrations of NVP for 10 min at room temperature before initiating reactions with MgCl2 (0.5 mM) and saturating dATP (100 ␮M). Effect of N348I on Enzyme-Template/Primer Complex Formation—We determined the effect of N348I on DNA/DNA binding affinity using a pre-steady state kinetics assay where we titrated the amount of nucleic acid that can be bound to, and extended by, RT (Fig. 1).

Results

Conclusion