Al Burtamani, Nawal; (2025) The effect of HIV-1 integration site selection on the establishment of viral latency. Doctoral thesis (Ph.D), UCL (University College London).

Text PhD Thesis- Nawal Al Burtamani_final.pdf - Submitted Version Access restricted to UCL open access staff until 1 July 2026. Download (11MB)

Abstract

HIV-1 latency remains a major challenge in achieving a cure, as the virus persists in a transcriptionally silent state within long-lived cellular reservoirs, evading immune clearance and antiretroviral therapy. The genomic integration site of HIV-1 is a key determinant of latency establishment, maintenance, and reactivation potential. This study investigated how integration site selection influences HIV-1 latency in a Jurkat CD4+ T cell model using WT and N74D mutant HIV-1, which differ in their integration preferences. To explore differential responses to drugs targeting specific PTMs such as DNA methylation, histone methylation and histone acetylation/deacetylation, we treated latently infected cells with various epigenetic inhibitors targeting histone deacetylation, DNA methylation, and histone methylation. SAHA (a pan-HDAC inhibitor) and BIX-01294 (a G9a histone methyltransferase inhibitor) effectively reactivated both WT and N74D HIV-1, suggesting that histone acetylation and H3K9 methylation play a role in maintaining latency. In contrast, DNA methyltransferase inhibitors Aza-C and Azad-C, as well as SUV39H1 inhibitor Chaetocin, failed to induce significant reactivation, indicating that DNA methylation and H3K9me3 deposition by SUV39H1 may not be primary mechanisms of latency maintenance in this model. Notably, M344 (an HDAC6-selective inhibitor) and DZNep (a polycomb inhibitor targeting H3K27me3) reactivated WT HIV-1 more effectively than N74D, reinforcing the idea that integration into transcriptionally active regions makes WT proviruses more susceptible to chromatin-based latency reversal strategies. The study also highlights the role of HDAC6 and the HUSH complex in reinforcing HIV-1 repression. Knockdown of HDAC6 and HUSH components Periphilin and TASOR led to significant viral reactivation, underscoring the complex interplay between integration site selection and epigenetic silencing mechanisms. Through mapping of UISs obtained using LM-PCR, we demonstrate that WT HIV-1 preferentially integrates into transcriptionally active regions, particularly within A1/A2 chromatin sub-compartments, in introns of transcribed genes, near enhancers and TSS. In contrast, N74D HIV-1 predominantly integrates into repressive chromatin domains such as LADs and the B3 sub-compartment, which are enriched in heterochromatin and associated with deeper latency states. We further observed that latent WT proviruses were shifted toward more repressive chromatin marked by H3K27me3, and they were far from enhancers and TSS. Conversely, N74D proviruses remained stably embedded within heterochromatin-rich regions, exhibiting a more intrinsically repressed state. Proximity to CTCF-binding sites and TADs also influenced HIV-1 transcriptional activity, with WT proviruses being initially positioned near these regulatory elements but moving away as latency deepened. These findings provide novel insights into how integration site selection dictates latency establishment and reactivation potential. This research will provide a new understanding for HIV-1 latency and potentially reveal new mechanisms to regulate gene expression in CD4 T cells.

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