OBJECTIVE: Synonymous substitutions K65K or K66K in HIV-1 reverse transcriptase (RT) alleviate fitness and fidelity defects in HIV-1 molecular clones harboring thymidine analog mutations (TAMs); however their potential for transmission and persistence is unknown. Here we investigated the temporal appearance of K65K/K66K relative to TAMs in a HIV-1 cohort, their prevalence over time, and their impact on viral fitness in the context of patient-derived RT sequences. METHODS: Retrospective analyses of the temporal appearance and longitudinal prevalence of synonymous substitutions and drug resistance mutations were performed using the British Columbia Centre for Excellence in HIV/AIDS Drug Treatment Program (DTP) database. Plasma-derived HIV-1 from the DTP was used to generate infectious molecular clones. Growth competition assays were performed to determine viral fitness. RESULTS: The prevalence of K65K/K66K in drug-naive individuals tripled from 11% in 1997 to 37% in 2014 (P < 0.0001, n = 5221), with K66K mainly accounting for the increase. These mutations emerged in drug-treated individuals without TAMs in 14% of the cohort and conferred a fitness advantage in the context of patient-derived multidrug-resistant (MDR) virus in the absence of drug. CONCLUSIONS: The appearance of K65K/K66K in drug-treated individuals was largely independent of TAMs, suggesting alternative factors are likely associated with their emergence. The increasing K65K/K66K prevalence to over a third of treatment-naive individuals in the mostly subtype B DTP cohort and their ability to confer a fitness advantage to MDR virus might explain the transmission and persistence of virus harbouring K65K/K66K in untreated individuals, and highlights their role in adaptive HIV-1 evolution.
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GT was supported by a National Health and Medical Research Council of Australia
(NHMRC) grant  and NHMRC Senior Research Fellowship . ST
was supported by a Monash Postgraduate Publication Award and an Australian
Postgraduate Award through Monash University. N.SC was supported by: United
States National Institutes of Health [R01AI081571]. We acknowledge the
contribution to this work of the Victorian Operational Infrastructure Support Program
received by the Burnet Institute.