Figure 8. Subcellular localization of p65. Immunofluorescence analysis of the p65 protein in J-Lat clones A7 cells mock treated or treated with M344, or TNF or TSA for 30 minutes or 2 hours. Subcellular localization of p65 was determined via indirect immunofluorescence employing rabbit polyclonal anti-p65 and goat anti-rabbit antibody coupled to Alexa-555. DAPI staining was used to determine the region of nuclei and to assess gross cell morphology. doi:10.1371/journal.pone.0048832.g008
Following HIV-1 binding and entry, the viral genome has to be reverse transcribed into DNA, transported into the nucleus and integrated into cellular genomic DNA and packaged into chromatin [74]. Verdin and his colleagues showed that the chromatin structure of the HIV LTR contained two well-ordered nucleosomes called Nuc-0 and Nuc-1 [75]. Nuc-0 is found just upstream of the enhancer (2415 to 2255), and Nuc-1 is near the viral RNA start site. In order to determine if the reactivation of latent HIV-1 induced by M344 in latently infected cells was due to acetylation of histone at HIV-1 LTR, ChIP assays were performed. Our results showed that M344 increased acetylation of histone H3 (7.4-fold) and histone H4 (16.7-fold) at the nuc-1 region of HIV-1 LTR, which is associated with HIV transcription in A7 cells. This is consistent with a number of studies reporting that the reactivation of HIV transcription requires histone acetylation and remodeling of the critical Nuc-1 by SWI/SNF [47,76?8]. These observations suggest that the acetylation level of histone at the nuc-1 region of the HIV-1 LTR is a key element regulating HIV-1 transcription. Several studies provide evidence that the presence of histone deacetylases (HDACs) at the HIV LTR is strongly correlated with transcriptional repression leading to latency. Margolis and his colleagues demonstrated that the transcription factor YY1 can act as a repressor of HIV transcription by recruiting HDAC-1 to the provirus [19]. Later studies demonstrated that NF-kB p50 homodimers, CBF-1, AP-4, CTIP2, Sp1, and c-Myc could also recruit HDAC1 [21,22,79?2]. HDAC2 and 3 can also occupy a site at the HIV LTR and may play a role in the repression of LTR expression [80,83]. More recently, Sluis-Cremer lab has also demonstrates that HDAC3 resides at the HIV-1 LTR in J89GFP cells and that potent inhibition of HDAC3 may be important for reactivation of latent HIV-1 [73]. It should also be noted that HDAC6 inhibition alone has little effect on HIV LTR expression,
Figure 9. M344 induces RelA recruitment to the latent HIV-1 LTR. J-Lat clones A7 were stimulated with M344 (200 nM) or TNFa(10 ng/ml) for 4 hours, respectively. Chromatin immunoprecipitation assays were performed using anti-p65 or anti-p50 antibodies or rabbit preimmune IgG and probed for the HIV LTR DNA sequences spanning the kB enhancer or for nonspecific control b-actin. Each ChIP experiment was repeated three times to confirm reproducibility of results and real-time quantitation of the fold change relative to untreated control is shown
