Silent mutations at codons 65 and 66 in reverse transcriptase alleviate indel formation and restore fitness in subtype B HIV-1 containing D67N and K70R drug resistance mutations.

Sushama Telwatte,Katie Moore,Gilda Tachedjian,Mary Tachedjian,Nicolas Sluis-Cremer,Paul Agius,P Richard Harrigan,Anna C Hearps,Adam Johnson,Secondo Sonza,Catherine F Latham

doi:10.1093/nar/gkv128

Abstract

Resistance to combined antiretroviral therapy (cART) in HIV-1-infected individuals is typically due to nonsynonymous mutations that change the protein sequence; however, the selection of synonymous or ‘silent’ mutations in the HIV-1 genome with cART has been reported. These silent K65K and K66K mutations in the HIV-1 reverse transcriptase (RT) occur in over 35% of drug-experienced individuals and are highly associated with the thymidine analog mutations D67N and K70R, which confer decreased susceptibility to most nucleoside and nucleotide RT inhibitors. However, the basis for selection of these silent mutations under selective drug pressure is unknown. Using Illumina next-generation sequencing, we demonstrate that the D67N/K70R substitutions in HIV-1 RT increase indel frequency by 100-fold at RT codons 65–67, consequently impairing viral fitness. Introduction of either K65K or K66K into HIV-1 containing D67N/K70R reversed the error-prone DNA synthesis at codons 65–67 in RT and improved viral replication fitness, but did not impact RT inhibitor drug susceptibility. These data provide new mechanistic insights into the role of silent mutations selected during antiretroviral therapy and have broader implications for the relevance of silent mutations in the evolution and fitness of RNA viruses.

Highlights

Nucleoside and nucleotide reverse transcriptase (RT) inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) are essential components of combined antiretroviral therapy to control human immunodeficiency virus (HIV) infection [1]
Consistent with the ability of silent mutations to reverse RT pausing observed in biochemical assays due to the thymidine analog mutations (TAMs) D67N/K70R [19], we demonstrate that K65K and K66K decrease the frequency of deleterious insertion and deletion mutations introduced in the HIV type 1 (HIV-1) genome during virus replication that could alleviate the fitness defect due to TAMs
TAMs including D67N and K70R are significantly associated with K65K or K66K, suggesting that these silent mutations contribute to strategies employed by the virus to escape from the inhibitory effects of antiretroviral drugs in vivo [19]

Summary

Introduction

Nucleoside and nucleotide reverse transcriptase (RT) inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) are essential components of combined antiretroviral therapy (cART) to control human immunodeficiency virus (HIV) infection [1]. Given that we did not observe differences in RTn efficiency due to silent mutations in a single-cycle infection (Figure 3) and that homopolymeric nucleotide regions are associated with increased rates of synthesis errors [38,39], we considered the possibility that the change in nucleotide sequence impacts on the fidelity of RTn. we examined whether the TAMs D67N/K70R increase errors introduced by HIV-1 RT during intracellular RTn that are alleviated by silent mutations K65K and K66K. Mutations were considered above background if their frequency was >0.06% This stringent threshold is ∼40 times greater than the error rate for HIV-1 RT determined using a HIV-1 vector containing the LacZ ␣ reporter gene [40]

Methods

Results

Conclusion