Volume 10 Supplement 1
The reciprocal antagonistic interplay between HIV-1 Tat and SIN3A specifies the epigenetic switch between de-repression versus maintenance of HIV-1 gene silencing
© Kulkarni et al; licensee BioMed Central Ltd. 2013
Published: 19 September 2013
Post-integration latency is a critical barrier to achieving a complete cure for HIV. Epigenetic micro-environment alterations at the integrated HIV-1 LTR, coupled with modulations of the HIV-1 Tat feedback circuit, influence the onset and maintenance of latency. In this context, we have previously identified the epigenetic core pressor SIN3/HDAC complex as part of the in-vitro HIV-1 Tat nuclear interactome. Here we delineate the dynamics of their antagonistic functional relationship in the control of HIV-1 latency.
Well-studied T-cellular models of HIV-1 latency viz. J-LAT A1 & A72, with latent integrated LTR-Tat-IRES-GFP-LTR and LTR-GFP-LTR cassettes respectively, and ACH-2, with latent full-length integrated HIV-1 provirus in their genome, were employed. We used ChIP-qPCR to study protein occupancy at the HIV-1 LTR; and RT-qPCR and p24 ELISA/GFP FACS to concurrently monitor the reactivation of HIV-1 gene expression. SIN3A knock-down was performed with shRNA targeting sin3a or non-target control using Amaxa nucleofection, and was validated by WB and RT-PCR analysis. Immunofluorescence microscopy was performed in Hela cells co-transfected with Tat and Sin3a.
The latent HIV-1 LTR displays a constitutive enrichment of the SIN3/HDAC complex.
ChIP-qPCR revealed that the SIN3/HDAC key components SIN3A, SAP18, Sap3O and HDAC-1 were enriched in ACH-2, J-LAT A1 and A72 cells. Importantly, a key event in HIV-1 reactivation from latency is the displacement of the SIN3/HDAC complex from the HIV-1 LTR, as TNFα (NF-KB activator), 5-AZA (DNA methylation inhibitor) or SAHA (HDAC inhibitor) treatments disrupted SIN3/HDAC association with the HIV-1 LTR in latently infected cells.
SIN3A is critical for the maintenance of HIV-1 post-integration latency. SIN3A depletion in J-LAT A1 and A72 resulted in HIV-1 reactivation from latency, which was associated with the depletion of SIN3A and concurrent recruitment of POLII and/or HIV-1 Tat, at the HIV-1 LTR. Next we combined SIN3A knock-down with various reactivation treatments and observed that SIN3A depletion potentiated proviral reactivation by TNFα, PMA/ionomycin (Protein Kinase C activator), 5-AZA and SAHA in J-LAT A1 cells. Thus when associated with the HIV-1 LTR, SIN3A participates in the silencing of the provirus.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.