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

Abstract

Background: Human immunodeficiency virus type 1 subtype C (HIV-1C) long terminal repeat (LTR) variants play a crucial role in influencing transcriptional activation and disease outcomes. However, it is unclear how much effect Sp1IIIT5A has on HIV-1C LTR transcription activity. To address this gap, the current study investigates the impact of a specific single mutation, T5A, which has been reported to affect the HIV-1 subtype B (HIV-1B), within the Specific Protein 1 (Sp1) III motif on HIV-1C LTR transcription activity and Sp1 binding affinity. Methods: HIV-1C LTR consensus and LTR mutant exhibiting a single T5A mutation into the Sp1III motif were (LTR Sp1IIIT5A) independently cloned into the pGL3 Luciferase basic reporter vector and sequenced to confirm the presence of the mutation. Consensus LTR-pGL3 and LTR Sp1IIIT5A-pGL3 recombinants were transfected into SVG and Jurkat cell lines to determine their transcription activity. The Sp1 transcription factor expression levels in these cell lines, were quantified by western blotting assay. Additionally, we modeled the crystal structures of the HIV-1C LTR and Sp1 protein, and performed docking calculations using various web servers, including HDOCK, HADDOCK, and pyDockDNA. Lastly, molecular dynamics simulations were carried out to evaluate the stability and binding affinity of Sp1 to LTR consensus and LTR Sp1IIIT5A. Results and Discussion: Our data show that LTR exhibiting Sp1IIIT5A mutation was successfully cloned into pGL3 basic reporter vector. The Sp1III5A mutation significantly enhances basal transcription activity compared to the canonical Sp1III motif in SVG cells (p<0.00001) and Jurkat cells (p=0.00521). This suggest that LTR Sp1III5A mutation is associated with enhanced transcription activity. Similarly, the Sp1IIIT5A mutation exhibited significantly increased Tat-mediated HIV-1C LTR transcription activity in SVG cells (p<0.00001) and Jurkat cells (p<0.00001). This further reinforces the notion that Sp1III5A enhances HIV-1C LTR transcription activity. Notably, transcription activity exhibited by LTR Sp1IIIT5A was more pronounced in SVG then in Jurkat cell line. Furthermore, the expression levels of the Sp1 transcription factor were comparable (p=0.08140) between SVG and Jurkat cell lines, suggesting that differences in transcriptional activity observed are not attributable to variations in Sp1 expression levels. Moreover, the binding affinity analysis revealed that the Sp1IIIT5A mutation exhibits stronger interactions with the Sp1 transcription factor than its canonical counterpart, with interaction values of -332.7, -174.6, and -279.2 kcal/mol compared to -311.4, -157.0, and -247.3 kcal/mol for the canonical sequence. These suggest that the Sp1IIIT5A mutation not only potentially enhances transcriptional efficiency but also stabilizes the binding of Sp1 transcription factor. Conclusion: The HIV-1 LTR Sp1IIIT5A mutation enhances transcription activity and binding affinity of Sp1 compared to the Sp1III canonical sequence in both SVG and Jurkat cell lines. Future studies should investigate the effect of Sp1IIIT5A mutation on the HIV-1 subtype C latency profile.