Investigating the fitness benefit of reverse transcriptase (RT) mutation A62V whenco-occurring with M184V and K65R inHIV-1 subtype C

[摘要] ENGLISH ABSTRACT: Background and AimsTenofovir disoproxil fumarate (TDF) and lamivudine (3TC) or emtricitabine (FTC) combined with efavirenz is the predominant first-line antiretroviral regimen in the Southern African region. Resistance to TDF and 3TC/FTC is largely through the occurrence of the drug resistance mutations (DRMs) K65R and M184V, respectively. Preliminary data from a large laboratory-based dataset of HIV drug resistance that showed a high prevalence of these mutations in patients who received the TDF regimen also revealed a significant co-occurrence of A62V with M184V and K65R. The aim of this study was to investigate the functional interaction and effect on viral fitness that A62V has when it co-occurs with M184V and K65R reverse transcriptase mutations in HIV-1 Subtype C.Materials and MethodsUsing Infusion™ cloning and site-directed mutagenesis techniques, eight full-length genome infectious clones containing the HIV-1 subtype C polymerase gene were synthesised having all combinations of DRMs - A62V, M184V and K65R, either being present or absent. The mutations in these constructs were verified by sequencing. The constructs were transfected into 293T cells for virion production and harvested virus was infected in the TZM-bl cell line in head to head growth competition experiments and assayed for growth kinetics using an allele-specific quantitative real-time polymerase chain reaction (PCR) assay.ResultsThe growth competition experiment between two viruses (A62V+K65R+M184V vs K65R+M184V) evaluated by taking the mean of 3 biological replicates in the assay in the absence of antiretroviral drugs, revealed that A62V mutation has no significant impact on fitness (Wilcoxon signed rank test p-value = 0.56). The overall coefficient of variation (CV) in the experiment was 12.8% indicating the high reproducibility of the growth competition assay using real-time PCR measurement of relative growth.Conclusion and recommendationsA62V mutation has no effect on fitness when it co-occurs with M184V and K65R. The co-occurrence with M184V and K65R remains unexplained and might be due to an effect on TDF resistance in combination with K65R.This requires investigation in future studies as TDF regimens are part of 1st line therapy in many Sub-Saharan countries in the treatment of HIV-1. Finally, the cloning and mutagenesis techniques used coupled with a very sensitive and reproducible real-time quantitative PCR assay provide an efficient system for detection of mutation fitness interactions and can be used in any future work to study HIV mutation fitness interactions.

[发布机构] Stellenbosch University