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bioRxiv · 10.1101/2025.01.23.634640

HIV-1 subtype C LTR Sp1IIIT5A mutant enhances transcription activity and Sp1 binding affinity

Msthali, N.·Kehinde, I.·Khan, R.·Soliman, M. E.·Ndung'u, T.·Madlala, P.
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Abstract

BackgroundGenetic variation within HIV-1 subtype C (HIV-1C) long terminal repeat (LTR) transmitted/founder viruses influences transcription activation and clinical disease outcomes. The role of specific mutations such as thymine-to-adenine (T5A) mutation at position five of the Specificity protein 1 (Sp1) III motif (Sp1IIIT5A) remains underexplored. This study investigates the impact of Sp1IIIT5A on HIV-1C LTR transcription activity and Sp1 binding affinity. MethodsThe Sp1IIIT5A mutant and consensus HIV-1C LTR sequences were cloned into the pGL3 Luciferase Basic reporter vector, sequenced, and transfected into SVG and Jurkat cell lines, independently. Transcription activity and Sp1 expression were assessed via luciferase assays and Western blot. Structural models of Sp1IIIT5A, consensus LTRs and Sp1 were generated, and docking scores calculated using HDOCK, HADDOCK, and pyDockDNA. Molecular dynamics simulations analyzed stability and interactions of Sp1IIIT5A LTR-Sp1 complexes. Results and DiscussionThe Sp1III5A mutant significantly increased basal (SVG: p<0.0001; Jurkat: p=0.0052) and Tat-mediated (SVG and Jurkat: p<0.0001) HIV-1C LTR transcription activity in both cell lines, with stronger effects in SVG cells. Sp1 expression levels remained similar across cell lines (p=0.0814). Sp1III5A exhibited higher binding affinity (-332.7, -174.6, and -279.2 kcal/mol) than the canonical sequence (-311.4, -157.0, and -247.3 kcal/mol). ConclusionThe Sp1IIIT5A mutation significantly enhances HIV-1C LTR transcription activity and Sp1 binding affinity, indicating its potential tole in modulating HIV-1C transcription and pathogenesis. Further investigation is needed to elucidate its impact on HIV-1C latency. ImportanceIn this study we show that the thymine-to-adenine (T5A) mutation at position five of the Sp1 III motif (Sp1IIIT5A) within the HIV-1 subtype C (HIV-1C) long terminal repeat (LTR) increases viral transcription. This mutation enhances the interaction between HIV-1C and the cellular transcription factor Sp1, promoting the viral strains ability to replicate. Our findings provide insight into why certain HIV-1C strains behave differently, potentially leading to heterogenous rates of disease progression. Understanding the Sp1IIIT5A mutation could lead to improved strategies for controlling HIV-1C and developing cure strategies to clear the infection or result in virus remission.

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Msthali, N., Kehinde, I., Khan, R., Soliman, M. E., Ndung'u, T., Madlala, P.. 2025-01-24. HIV-1 subtype C LTR Sp1IIIT5A mutant enhances transcription activity and Sp1 binding affinity. https://doi.org/10.1101/2025.01.23.634640

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