Biology subjects

Madlala, P.

Publications and source records attributed to Madlala, P..

4 recordsLinked to original sources

bioRxiv2025

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

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.

microbiology↗

bioRxiv2024

Development of a latency model based on HIV-1 subtype C to study how long terminal repeat genetic variation impacts viral persistence and latency reversal

Most people living with HIV (PLWH) reside in sub-Saharan Africa. South Africa is the epicentre where 98% of HIV-1 infections are subtype C. However, partially due to unavailability of non-subtype B latency models, most studies of HIV-1 latency and cure have focused on HIV-1 subtype B (HIV-1B) which predominates Europe and USA. Moreover, the effect of inter- and intra-subtype genetic variation of the viral promoter, long terminal repeat (LTR), from PLWH on latency reversal is unknown. We constructed a retroviral vector expressing green fluorescent protein and HIV-1 subtype C (HIV-1C) consensus Tat protein under the control of either HIV-1C consensus or PLWH-derived transmitted/founder (T/F) LTR, produced respective LTR pseudotyped viruses, infected Jurkat E6 and primary CD4+ T cells in vitro, enriched for latently infected cells, and treated these cells with different latency reversing agents. We show that the HIV-1C LTR exhibited lower reactivation compared to HIV-1B. Furthermore, HIV-1C T/F LTR pseudotyped proviral variants with four NF-{kappa}B motifs exhibited lower reactivation compared to those with three NF-{kappa}B motifs. Our data indicate that inter- and intra-subtype HIV-1 LTR genetic variation in combination with host variation modulates latency reversal. Author summaryAntiretroviral therapy (ART) suppresses HIV-1 replication, but it is not curative due to a persistent latent reservoir established early in infection. Although HIV-1 subtype C (HIV-1C) is responsible for about 50% of global and 98% of southern Africa infections, it is underrepresented in HIV-1 cure studies. The unavailability of non-subtype B latency models has led to most studies on HIV-1 latency and cure focusing on HIV-1 subtype B (HIV-1B) which predominates in western countries. The viral promoter, long terminal repeat (LTR) drives viral gene transcription and is important for the HIV-1 life cycle. In this study we undertook to develop a latency model based on HIV-1 subtype C to investigate the effect of inter- and intra- LTR genetic variation on viral persistence and latency reversal. Our data show that the HIV-1C LTR exhibited lower reactivation compared to HIV-1B. Furthermore, HIV-1C T/F LTR pseudotyped proviral variants with four NF-{kappa}B motifs exhibited lower reactivation compared to those with three NF-{kappa}B motifs. Taken together, our data suggest that inter- and intra-subtype HIV-1 LTR genetic variation in combination with host variation modulates latency reversal.

molecular biology↗

bioRxiv2023

SQuHIVLa: A novel assay for Specific Quantification of inducible HIV-1 reservoir by LAMP

Strategies toward HIV-1 cure aim to clear, inactivate, reduce or immunologically control the virus from a pool of latently infected cells such that combination antiretroviral therapy (cART) can be safely interrupted. In order to assess the impact of any putative curative interventions on the size and inducibility of the latent HIV-1 reservoir, robust and scalable assays are needed to precisely quantify the frequency of infected cells containing inducible replication competent HIV-1. Here, we present Specific Quantification of Inducible HIV-1 by LAMP (SQuHIVLa), a novel assay that leverages the high sensitivity and specificity of RT-LAMP, performed in a single reaction, to detect and quantify cells expressing Tat/Rev msRNA upon activation. Our LAMP primer/probe design exclusively detects subtype-specific HIV-1 Tat/Rev msRNA and exhibits high sensitivity, specificity, and reproducibility. Using SQuHIVLa we quantified the inducible viral reservoir in CD4+ T cells from a diverse group of people living with HIV-1 subtypes B and C on cART. SQuHIVLa presents a high throughput, scalable and specific HIV-1 reservoir quantification tool that is amenable to resource limited settings.

microbiology↗

bioRxiv2023

Genetic variation of the HIV-1 subtype C transmitted/founder viruses long terminal repeat elements and the impact on transcription activation potential and clinical disease outcomes

A genetic bottleneck is a hallmark of HIV-1 transmission such that only very few viral strains, termed transmitted/founder (T/F) variants establish infection in a newly infected host. Phenotypic characteristics of these variants may determine the subsequent course of disease. The HIV-1 5 long terminal repeat (LTR) promoter drives viral gene transcription and is genetically identical to the 3 LTR. We hypothesized that HIV-1 subtype C (HIV-1C) T/F virus LTR genetic variation is a determinant of transcriptional activation potential and clinical disease outcome. The 3LTR was amplified from plasma samples of 41 study participants acutely infected with HIV-1C (Fiebig stages I and V/VI). Paired longitudinal samples were also available at one year post-infection for 31 of the 41 participants. 3 LTR amplicons were cloned into a pGL3-basic luciferase expression vector, and transfected alone or together with Transactivator of transcription (tat) into Jurkat cells in the absence or presence of cell activators (TNF-, PMA, Prostratin and SAHA). Inter-patient T/F LTR sequence diversity was 5.7% with subsequent intrahost viral evolution observed in 48.4% of the participants analyzed at 12 months post-infection. T/F LTR variants exhibited differential basal transcriptional activity, with significantly higher Tat-mediated transcriptional activity compared to basal (p<0.001). Basal and Tat-mediated T/F LTR transcriptional activity showed significant positive correlation with contemporaneous viral loads and negative correlation with CD4 T cell counts (p<0.05) during acute infection respectively. Furthermore, Tat-mediated T/F LTR transcriptional activity significanly correlated positively with viral load set point and viral load; and negatively with CD4 T cell counts at one year post infection (all p<0.05). Lastly, PMA, Prostratin, TNF- and SAHA cell stimulation resulted in enhanced yet heterologous transcriptional activation of different T/F LTR variants. Our data suggest that T/F LTR variants may influence viral transcriptional activity, disease outcomes and sensitivity to cell activation, with potentional implications for therapeutic interventions. Author summaryThere is heterogeneity in the rates of clinical disease progression in antiretroval therapy-naive people living with HIV (PLWH). In heterosexual HIV-1 transmission, only a single or very few viral strains, called transmitted/founder (T/F) viruses establish infection in a newly infected host. The long terminal repeat (LTR) is the viral promoter that drives viral gene transcription and is important for the HIV-1 life cycle. In this study we investigated the impact of HIV-1 subtype C T/F virus LTR genetic variation on transcriptional activity, clinical disease outcomes and response to cell activation. Our data show inter-patient T/F LTR genetic variation and limited intrahost evolution by 12 months post infection. T/F LTR variants exhibit differential basal LTR transcriptional activity, which is significanly increased in the presence of the Transactivator of transcription (Tat) protein. Furthermore, we show that T/F LTR transcription activity significanly correlates positively with viral load and viral load set point but negatively with CD4 T cell count. Lastly, we show that T/F LTR variants exhibit differential responses to cell activators PMA, TNF-, Prostratin and SAHA. Taken together our data suggest that T/F viruses LTR genetic variation and functional heterogeneity are important determinants of clinical outcomes and virus reactivation potential.

molecular biology↗