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

Nucleic Acids Research
Sushama TelwatteGilda Tachedjian

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 o...Continue Reading

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Methods Mentioned

BETA
transfection
ELISA
PCR
transfections
amplicon sequencing

Software Mentioned

Create Sequencing QC Report
GraphPad
CLC Genomics Workbench
GraphPad Prism
Stata

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