Investigating the fitness benefit of reverse transcriptase (RT) mutation A62V when co-occurring with M184V and K65R in HIV-1 subtype C

Njenda, Duncan Tazvinzwa (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Background and Aims Tenofovir 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 Methods Using 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. Results The 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 recommendations A62V 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.

AFRIKAANSE OPSOMMING: Agtergrond en doel Tenofovir disoproksil fumaraat (TDF) en lamivudien (3TC) of emitricitabine (FTC) gekombineer met efavirenz is die oorheersende eerste lyn antiretrovirale regimen in die Suider-Afrikaanse streek. Weerstand teen TDF en 3TC / FTC is grootliks deur die voorkoms van die middel-- weerstandigheid-mutasies (DRM's) K65R en M184V, onderskeidelik. Voorlopige data van 'n groot laboratorium- gebaseerde datastel van MIV middel-weerstand het bewys dat daar 'n hoë voorkoms van hierdie mutasies is in pasiënte wat die tenofovir regimen ontvang en het ook 'n beduidende mede-voorkoms van A62V met M184V en K65R onthul. Die doel van hierdie studie was om die funksionele interaksie en invloed op virale fiksheid te ondersoek wat A62V het wanneer dit voorkom saam met M184V en K65R trutranskriptase mutasies in MIV-1 Subtype C. Materiale en metodes Infusion ™ kloning en mutagenese tegnieke is gebruik om 8 vollengte MIV rekombinante klone te verkry met alle kombinasies van DRM's, A62V, M184V en K65R, hetsy teenwoordig of afwesig en die mutasies in hierdie konstrukte is bevestig deur nukleinsuurbasisbaarvolgordebepaling. Die konstrukte was getransfekteer in 293T selle vir virion produksie. Virus supernatant is geoes en die TZM-bl sellyn is geinfekteer en gebruik in kop-aan-kop groei kompetisie eksperimente;virusproduksie is getoets met behulp van 'n alleel-spesifieke in-huis kwantitatiewe reële-tyd polimerase kettingreaksie (PKR) om die groei kinetika te ondersoek. Resultate Die groei kompetisie eksperiment tussen twee virusse (A62V + K65R + M184V vs K65R + M184V) wat geëvalueer was deur die neem van die gemiddelde van 3 biologiese herhalings in die teenwoordigheid van anti-retrovirale middels, het getoon dat A62V mutasie het geen beduidende impak op fiksheid (“Wilcoxon singned rank” toets p-waarde = 0.56). Die algehele koëffisiënt van variasie (KV) in die eksperiment was 12,8% wat aandui op die hoë herhaalbaarheid van die groei-kompetisie eksperiment met reële tyd PKR kwantifisering van relatiewe groeikoers. Gevolgtrekking en aanbevelings A62V mutasie het geen effek op fiksheid wanneer dit voorkom met M184V en K65R nie. Die gesamentlike voorkoms van A62V met M184V en K65R bly onverklaarbaar, maar dit mag weens verhoogde weerstandigheid teen TDF wees wanneer A62V met K65R voorkom. Om dit te verklaar benodig verdere studies aangesien TDF- gebaseerde kombinasieterapie in baie Sub-Saharan lande gebruik word vir 1ste-lyn behandeling van MIV-1.Ten slotte, die kloning en mutageniese tegnieke, wat gebruik is, tesame met 'n baie sensitiewe en herhaalbare reële-tyd kwantitatiewe PKR toets verleen 'n doeltreffende benaderingom fiksheids interaksies te ondersoek.

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