K70Q Adds High-Level Tenofovir Resistance to “Q151M Complex” HIV Reverse Transcriptase through the Enhanced Discrimination Mechanism

Abstract

HIV-1 carrying the “Q151M complex” reverse transcriptase (RT) mutations (A62V/V75I/F77L/F116Y/Q151M, or Q151Mc) is resistant to many FDA-approved nucleoside RT inhibitors (NRTIs), but has been considered susceptible to tenofovir disoproxil fumarate (TFV-DF or TDF). We have isolated from a TFV-DF-treated HIV patient a Q151Mc-containing clinical isolate with high phenotypic resistance to TFV-DF. Analysis of the genotypic and phenotypic testing over the course of this patient's therapy lead us to hypothesize that TFV-DF resistance emerged upon appearance of the previously unreported K70Q mutation in the Q151Mc background. Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF. However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF. Biochemical experiments established that the increased resistance to tenofovir is not the result of enhanced excision, as K70Q/Q151Mc RT exhibited diminished, rather than enhanced ATP-based primer unblocking activity. Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain. Molecular dynamics simulations suggest that changes in the hydrogen bonding pattern in the polymerase active site of K70Q/Q151Mc RT may contribute to the observed changes in binding and incorporation of TFV-DP. The novel pattern of TFV-resistance may help adjust therapeutic strategies for NRTI-experienced patients with multi-drug resistant (MDR) mutations.

Standardized Comparison of the Relative Impacts of HIV-1 Reverse Transcriptase (RT) Mutations on Nucleoside RT Inhibitor Susceptibility

“…Several of these mutations occur at known NRTI resistance positions: K65N, D67G/E/S/T, K70Q/N/G/S/T, L74I, V75M/A/S, and K219N/R/W/ D/H (2,4,11,25,30,37). Others are at novel positions in the 5= polymerase coding domain: E40F, K43E/Q/N, E44D/A, V118I, E203K, H208Y, D218E, K223Q/E, and L228H/R (9, 12, 33) (13,30).…”

Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

“…Construct pET-28a-MRT, encoding full-length (FL) wild-type MoMLV RT, was provided by Mukund Modak (New Jersey Medical School, Newark, NJ). HIV-1 RT was cloned into a pETDuet-1 vector and purified as previously described (4,32,56,75). Plasmid pCSR231, encoding protein p15-Ec, a catalytically active chimeric HIV-1 RNase H domain fragment containing an ␣-helical substrate binding loop derived from E. coli RNase H1 (41,78,83), was a generous gift from Daria Hazuda (Merck, West Point, PA).…”

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) Inhibits HIV-1 Reverse Transcriptase with Multiple Mechanisms

“…The optimal rate of polymerization (k pol ) was determined using the rapid quench-flow method as described before (19,26,27,29). Specifically, a solution containing 50 nM RT and 50 nM T d31 /P d18 or T d26 /P d18ϩ5 in RT buffer was rapidly mixed with a solution of 5 mM MgCl 2 and 1-50 M dATP or EFdA-TP for reaction times ranging between 0.005 and 2 s followed by quenching with 150 mM EDTA.…”