Madlala, Paradise Zamokuhle.Mkhize, Zakithi Zinhle.2024-05-282024-05-2820232023https://hdl.handle.net/10413/23002Doctoral Degree. University of KwaZulu-Natal, Durban.The HIV-1 Transactivator of transcription (Tat) enhances the ability of the viral promoter 5’ long terminal repeat (LTR) to drive viral gene transcription and is important for HIV-1 pathogenesis. Tat binds to the transactivator RNA (TAR) element of the 5’LTR and subsequently recruits the host positive transcription elongation factor b (P-TEFb) for efficient viral gene transcription. Inter- and intra-subtype Tat genetic variation that translates to functional differences has been reported. Specifically, HIV-1 subtype C (HIV-1C) exhibiting Alanine at position 21 of the Tat protein (TatA21) was reported to be associated with reduced LTR transcriptional activity compared to Tat exhibiting Proline at position 21 mutation (TatP21). However, the effect of Tat variation on its ability to recruit P-TEFb is unknown. Therefore, this study seek to determine the effect of HIV-1 subtype C TatA21 mutant on the ability of Tat to recruit P-TEFb to 5’ LTR to enhance viral gene transcription. To this effect, site-directed mutagenesis (SDM) was performed on the Plasmid pcDNA3.1(+) HIV-1C BL43/02 TatA21 to introduce TatP21 alone or together with other mutations using designed primers and the Q5 DNA polymerase kit. The effect of Tat mutations was measured using Tat transactivation assay where the luciferase activity was the measured output in TZM-bl cell lines and the impact of TatA21 was further assessed on ability of the LTR to drive GFP and Gag expression in Jurkat and A72 cells respectively. Next, protein modelling was performed using Hdock software, followed by RNA immunoprecipitation (RNA IP) was performed using stably expressing TatA21 and TatP21 in Jurkat cells. Lastly, co-immunoprecipitation of TatA21 and associated with significantly reduced LTR transcription activity compared to TatP21 (p = 0.0004). TatA21 resulted in had significantly lower GFP expression Jurkat cells (p = 0.0439) and lower Gag expression in A72 cells compared to TatP21. Although TatA21 reduced the LTR transcription activity compared to TatP21, protein modelling using Hdock software revealed that TatA21 and TatP21 protein structures were the same. Consistently, molecular docking showed that TatA21 had a lower binding affinity than TatP21. The RNA IP showed that TatA21 had significantly reduced affinity to bind to TAR compared to TatP21 (p = 0.0151). Moreover, TatA21 and TatP21 formed a complex with cycT1 and CDK9. Taken together, our data shows that HIV-1C TatA21 significantly reduced its transactivation activity but does not affect its ability to recruit P-TEFb. Interestingly, TatP21 is able to bind TAR more efficiently than TatA21 thus revealing a possible mechanism but which the reduced functionality of SDMs and patient derived TatA21 variants was observed. The effect of TatA21 and TatP21 on the propensity of HIV-1 latency development or reversal. To this effect, a recombinant viral vector exhibiting either TatA21 (C731CTatA21C) or TatP21 (C731CTatP21C) were generated. The C731CTatA21C or C731CTatP21C were separately co-transfected together with VSV-G and R8.91 into Jurkat cells for virus production. This virus was then used to infect Jurkat cells for 3 days. Followed by cell sorting of GFP- cells, which represented either truly negative or latently infected cells was then performed. We were able to successfully generate C731CTatA21C virus and characterized it to a 1.2% reactivation. However, the generation of C731CTatP21C recombinant viral vector was unsuccessful and thus could not be used for comparison. Future studies should involve the characterization of TatP21 in the propensity of latency development and/ or reactivation.enHIV.Mutation.Thesishttps://doi.org/10.29086/10413/23002