Role of silent mutations K65K and K66K in subtype B HIV-1 reverse transcriptase selected during drug therapy

Abstract

Resistance to combined antiretroviral therapy (cART) in HIV-1 infected individuals is typically due to non-synonymous 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 (TAMs) D67N and K70R, which confer decreased susceptibility to most HIV-1 nucleoside/nucleotide RT inhibitor drugs. However, the basis for the selection of these silent mutations under drug pressure and their role in HIV-1 replication are unknown. Introduction of either K65K or K66K into HIV-1 containing D67N/K70R improved viral replication fitness but did not impact RT inhibitor drug susceptibility. Using Illumina next- generation sequencing, it is demonstrated that D67N/K70R substitutions in HIV-1 RT increase indel frequency by 100-fold at RT codons 65-67, consequently impairing viral fitness. The fitness advantage conferred by K65K/K66K is likely due to their ability to alleviate decreased RT efficiency on homopolymeric stretches of nucleotides and reverse the introduction of indels due to the emergence of TAMs. A retrospective population analysis of the British Columbia cohort revealed that these silent mutations were increasing in prevalence in drug-naive individuals. Additionally, K65K and K66K emerge in antiretroviral drug treated patients in the absence of TAMs in over 13% of the cohort analysed, suggesting there may be a role for these silent mutations independent of TAMs. In this regard, preliminary data suggest that neither K65K nor K66K are likely to be deleterious in the context of wild type virus. A parallel investigation of the role of homopolymeric stretches of nucleotides in the HIV-1 genome in regions distinct from the RT coding region revealed that an extended stretch of iterated sequence can result in virus fitness defects relative to wild type. These studies suggest that the emergence of a silent mutation to disrupt this homopolymeric stretch could restore viral fitness. Taken together, this study has demonstrated that the silent mutations can act as compensatory mutations to alleviate defects conferred by D67N and K70R. These data provide new mechanistic insights into the role of silent mutations selected during cART and have broader implications for the relevance of silent mutations in the evolution and fitness of RNA viruses.