Investigating the structural impact of hiv-1 integrase natural occurring polymorphisms and novel mutations identified among group m subtypes circulating in sub-Saharan Africa

Thesis (DScMedSc)--Stellenbosch University, 2020.Introduction HIV/AIDS remains a major health concern worldwide, with sub-Saharan Africa (SSA) carrying the largest burden.HIV is characterised by extremely high genetic diversity, with all the major groups and subtypes circulating in SSA. Combination antiretroviral therapy (cART) have substantially reduced HIV related deaths, but this is counteracted by the development of HIVdrug resistance, caused by certain drug resistance-associated mutations (RAMS). Integrase (IN) strand transferase inhibitors (INSTIs), the newest class of antiretroviral drugs,has a high genetic barrier and can be used in individuals that previously exhibited resistance to other classes of drugs. The World Health Organisation (WHO) approved the use of Dolutegravir (DTG) as part of first-line cART. Methods This is a descriptive experimental design study, which aimed to identify IN natural occurring polymorphisms (NOP) among different HIV-1 group M subtypes and Drug resistance mutations within the HIV-1 pol gene fragment of INSTI naïve patients from South Africa (SA) and Cameroon (CR), using the Stanford University genotypic resistance interpretation algorithm. Structural computational methods that included; homology modelling, molecular docking, molecular dynamics simulations and interaction analysis was performed to understand the structural impact of mutations from diverse HIV-1 subtypes on DTG drug binding. ResultsWe observed low-level RAMs against INSTIs in SA (2.2%) and CR sequences (5.4%). Through Fisher’sexact test we noted that the two NOPs occurred: VI72I and R269K, with p-values ≤0. 05, were statistically enriched. The impact of having these mutations are yet to be fully understood. Through molecular modelling and stability predictions, we observed a destabilizing effect of the known G140S mutant on the HIV-1C IN protein structure and simulation analysis showed that it affected structural stability and flexibility of the protein structure. Interactions analysis of different drug binding conformations to different HIV-1 IN subtypes reported differences in the number of binding interactions to different HIV-1 IN subtypes, but we did not observe any significant differences in binding affinity for each INSTIs. This implies no significant alteration to the binding site in the wild type IN, which may not prevent INSTIs drug binding. In addition, all accessory mutations that resulted in a change in the number of interactions encompassing residues were found within the stable alpha-helix secondary structure element and not in close proximity to the drug active site.ConclusionThe study data indicate that RAMS against INSTIs remain low both in SA and in CR.Subtype C in SA and CRF02_AG in CR continues to be the driving force ofthe epidemic. We further reported on the impact of various NOPs on drug susceptibility. The analyses suggested that NOPs does not have an impact on IN protein structure and stability,and does not affect drug binding in the WT IN, but the known mutation G140S affect DTG binding. The study support recommendations made by the WHO to use DTG as part of salvage therapy in patients with RAM’s and accessory mutations. Data obtained from thisstudy can help to tailor effective treatment strategies in the African population, where diverse HIV subtypes circulate.InleidingMIV/vigs bly wêreldwyd ’n ernstige gesondheidskwessie, en Afrika suid van die Saharadra die swaarste las. MIV word deur uiters hoë genetiese diversiteit gekenmerk, waarvan al die vernaamste groepe en subtipes in Afrika suid van die Sahara in omloop is. Kombinasie-antiretrovirale terapie (kART) het ’n aansienlike vermindering in MIV-verwante sterftes tot gevolg, hoewel dít teengewerk word deur die ontwikkeling van MIV-middelweerstandigheid vanweë sekere middelweerstandigheidsverwante mutasies (oftewel RAM’s). Integrasestringtransferase-inhibitors (INSTI’s), die jongste klas antiretrovirale middels, het ’n hoë genetiese skans en kan gebruik word by individue wat voorheen weerstandigheid teen ander klasse middels getoon het. Die Wêreldgesondheidsorganisasie (WGO) het die gebruik van dolutegravir (DTG) as deel van eerstelinie-kART goedgekeur. MetodesHierdie studie gebruik ’n beskrywende proefondervindelike ontwerp om natuurlike integrase-(IN-) polimorfismes (NOP’s) in verskillende MIV-1-groep-M-subtipes en middelweerstandige mutasies in die MIV-1-pol-geenfragment van INSTI-naïewe pasiënte van Suid-Afrika (SA) en Kameroen (KR) te identifiseer. Dit word met behulp van die Universiteit van Stanford se algoritme vir genotipiese weerstandigheidsvertolking gedoen. Strukturele berekeningsmetodes soos homologiemodellering, molekulêre koppeling, molekulêre dinamikasimulasies en interaksieontleding is uitgevoer om die strukturele impak van mutasies uit diverse MIV-1-subtipes op DTG-middelbinding te verstaan. ResultateLaevlak-RAM’s teen INSTI’s is in reekse van SA (2,2%) én KR (5,4%) opgemerk. Fisher se eksakte toets het twee NOP’s opgespoor –VI72I en R269K –met p-waardes van ≤0,05, wat statisties verryk was. Die impak van hierdie mutasies is nog nie ten volle duidelik nie. Deur molekulêre modellering en stabiliteitsvoorspellings het ons bepaal dat die bekende G140S-mutant ’n destabiliseringsuitwerking het op die MIV-1C-IN-proteïenstruktuur. Simulasieontleding het getoon dat dít die strukturele stabiliteit en buigbaarheid van die proteïenstruktuur beïnvloed. Interaksieontleding van middelbindingskonformasies met MIV-1-IN-subtipes het verskille in die getal bindingsinteraksies met verskillende subtipes opgelewer, maar geen beduidende verskille in bindingsaffiniteit is vir enige van die INSTI’s opgemerk nie. Dít impliseer dat daar geen beduidende aanpassing is in die bindingsetel by die wilde-tipe IN wat INSTI-middelbinding kan verhoed nie. Daarbenewens is alle bykomstige mutasies wat ’n verandering in die getal interaksies in residu’s veroorsaak het in die stabiele alfaheliks-sekondêre struktuurelement aangetref, en nie naby die aktiewe setel van die middel nie.GevolgtrekkingDie studiedata toon dat RAM’s teen INSTI’s steeds laag is in sowel SA as KR. Subtipe C in SA en CRF02_AG in KR bly die dryfkrag agter dieepidemie. Daarbenewens is daar oor die impak van verskillende NOP’s op middelvatbaarheid verslag gedoen. Die ontledings toon dat NOP’s nie ’n impak op IN-proteïenstruktuur en -stabiliteit het nie, en ook nie middelbinding in die WT-IN beïnvloed nie. Nogtans het die bekende mutasie G140S wél ’n invloed op DTG-binding. Die studie ondersteun die WGO se aanbeveling dat DTG as deel van reddingsbehandeling by pasiënte met RAM’s en bykomstige mutasies gebruik word. Die data uit hierdie studie kan doeltreffende behandelingstrategieë help ontwikkel vir die bevolking van Afrika, waar diverse MIV-suptipes in omloop is.Doctora