HIV-1 latency: the role of G-quadruplexes within the HIV-1 proviral genome

The Human Immunodeficiency virus (HIV) is the etiological agent of the Acquired immunodeficiency syndrome (AIDS). Although antiretroviral therapy reduces the viral load to undetectable levels, treatment interruption leads to viral rebound because of virus persistence in latently infected cells. How HIV-1 establishes latency is not fully understood yet. It is well known that the long terminal repeat (LTR) promoter controls the expression of all viral genes. Given the reported presence of G-quadruplex (G4) folding sequences in the LTR promoter of the HIV-1 provirus and their involvement in transcriptional activation in vitro, we here investigated the role of LTR G4s and their interactors in HIV-1 latency and reactivation. We examined the reactivation of several latently infected cell lines upon treatment with latency-reversing agents (LRAs) and analyse transcriptionally active and silenced cell populations. Our findings provide the first direct evidence of G4 folding in the chromatin context of latently infected cells and demonstrate the interaction of nucleolin with the LTR promoter in the presence of G4 structures. Additionally, we show that in transcriptionally active cells, the HIV-1 promoter binds Sp1 and lacks G4 structures, underscoring the G4 role in maintaining latency. Our results also reveal that the site of proviral integration does not affect G4 folding but influences the percentage of reactivated cells and GFP expression. Given nucleolin key role in modulating HIV latency and reactivation, we examined its localization across different transcriptional states, finding no significant difference between transcriptionally active and silenced cells. Interestingly, nucleolin silencing was associated with LTR reactivation. To extend these findings, we analysed patient-derived peripheral blood mononuclear cells (PBMCs) and confirmed our results in individuals with both high and low viremia, providing further insight into the mechanism of HIV-1 latency. These findings highlight G4s as potential therapeutic targets for preventing viral reactivation, which could significantly improve patient outcomes. We also explored the use of CRISPR/Cas9 to disrupt G4 folding, using point mutations in G-tracts to prevent G4 formation, and plan to investigate whether G4s drive its association with transcriptional silencing in future work.