Maikoo, S. | bioRxiv Science

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

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↗