Transcriptional Interference Antagonizes Proviral Gene Expression to Promote HIV Latency

Abstract

SUMMARY Eradication of the latent HIV reservoir remains a major barrier to curing AIDS. However, the mechanisms that direct viral persistence in the host are not well understood. Studying a model system of post-integration latency, we found that proviral integration into the actively transcribed host genes led to transcriptional interference caused by the elongating RNA polymerase II transcribing through the viral promoter. As a result, physical exclusion of the pre-initiation complex formation on the 5’LTR promoted the silencing of HIV transcription. Furthermore, we demonstrated that this block could be counteracted through inhibiting the upstream transcription or cooperatively activating transcription initiation and elongation from the 5’LTR. Importantly, using a novel PCR-based method, we detected significant levels of host transcription through the 5’LTR in HIV-infected primary CD4+ T cells. Collectively, our findings suggest that transcriptional interference contributes greatly to HIV latency and has to be considered when attempting to purge the latent reservoir.

Bromodomain and Extra-terminal (BET) Bromodomain Inhibition Activate Transcription via Transient Release of Positive Transcription Elongation Factor b (P-TEFb) from 7SK Small Nuclear Ribonucleoprotein

“…Both compounds released P-TEFb from the 7SK snRNP and potently induced HIV gene expression when the viral LTR was not occluded by transcriptional interference (integration into active genes) (15)(16)(17). In this study, the bromodomain and extra-terminal (BET) bromodomain inhibitor JQ1 (18,19) did the same and acted cooperatively with HMBA and SAHA.…”

“…Glycerol Gradients-Glycerol gradients (10 -30%) were established by pipetting 2 ml of each glycerol fraction (10,15,20,25, and 30%, v/v) in medium-salt buffer A into centrifugation tubes (Beckman 331372). Gradients were formed by standing for 6 h at 4°C.…”

“…Previously, we studied the effects of hexamethylene bisacetamide (HMBA) and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) on HIV transcription in transformed and primary cells, with special emphasis on proviral latency and reactivation (15)(16)(17). Both compounds released P-TEFb from the 7SK snRNP and potently induced HIV gene expression when the viral LTR was not occluded by transcriptional interference (integration into active genes) (15)(16)(17).…”

Viral epigenomes in human tumorigenesis

“…A new mechanism regulating viral gene expression has emerged, explaining the lack of associations between the gene silencing and histone modifications or DNA methylation found in many cases. This is the so-called 'transcriptional interference' by which the constant presence of the host transcriptional machinery in the active region in which the virus is integrated interferes with transcription initiation from the viral promoter (Duverger et al, 2009;Han et al, 2008;Lenasi et al, 2008).…”

Exploring HIV latency using transcription profiling

“…Combined with RT-ddPCR, which enables absolute cDNA quantification 24 , this approach provides a considerable advantage over previous work that mostly focuses on one mechanism of latency at a time and has typically utilised in vitro models of latency, which may not recapitulate what happens in vivo. [18][19][20]22,23,[26][27][28] . Unlike previously employed strategies, specific blocks to transcription and the magnitude of their impact on the prevailing levels of HIV RNA can be simultaneously assessed by determining the expression of these processive transcripts.…”