(Lower) ChIPed samples were analyzed by PCR using primers specific for the cyclin A2 coding region as a negative control
(Lower) ChIPed samples were analyzed by PCR using primers specific for the cyclin A2 coding region as a negative control. less pgRNA. Altogether our results further support the existence of a complex network of epigenetic events that influence cccDNA function and Dehydroepiandrosterone HBV replication and identify an epigenetic mechanism (i.e., to prevent cccDNA deacetylation) by which HBx controls HBV replication. Keywords:histone acetylation, HATs, HDACs Hepatitis B virus (HBV) infection is a major health problem, with 400 million people chronically infected worldwide who are at high risk of developing liver cirrhosis and hepatocellular carcinoma (HCC) (1). The epidemiological evidence linking HBV infection to HCC is very strong, and despite the mechanisms underlying HBV-associated carcinogenesis remain to be fully defined, a growing number of studies support a direct role of HBV in the process (25). The HBV-encoded regulatory protein hepatitis B virus X protein (HBx) is thought to contribute to HBV oncogenicity (5,6). HBx transforms SV40-immortalized murine hepatocytes, induces cell cycle progression within the regenerating liver, causes liver cancer in some transgenic mice, and acts as a cofactor to accelerate cancer development in other mouse models (611). HBx is a 154-amino acid protein with an N-terminal negative regulatory domain and C-terminal transactivation or coactivation domain that has been detected both in the cytoplasm and in the nuclei of infected hepatocytes (6,12,13). Studies in transfected cells have shown that HBx expression affects several cellular functions such as cytoplasmic calcium regulation, cell signaling, transcription, cell proliferation, DNA repair, and apoptosis (11,1316). To perform its multiple functions, HBx interacts with many cellular partners including the tumor suppressor p53, the UV-stimulated E3-ubiquitin ligase DDB1, the nuclear export protein CRM1, a number of nuclear proteins involved in the regulation of transcription such as the RPB5 subunit of RNA polymerase II, TFIIB, TFIIH, the TATA binding protein (TBP), the basic domain-leucine zipper (bZIP) family transcription factors ATF2, CHOP, and cAMP-response element (CRE)-binding protein (CREB) (6,14). Recently, it has been shown that HBx interacts and cooperate with the CREB-binding protein (CBP)/p300 to modify chromatin dynamics of target genes and to synergistically enhance CREB activity (17). The lack Dehydroepiandrosterone of homology of the X ORF to host protein and its high conservation to other mammalian hepadnaviruses genomes strongly suggest that HBx play a role in viral life cycle (6). Although initial studies suggested that HBx was not required for virus replication in cell culture (18), experiments with the highly related woodchuck hepatitis virus (WHV) system indicate that the WHV X protein (WHx) is required for virus replication in vivo (1922). Studies performed using a plasmid-based replication assays that use greater-than-unit-length HBV genomes transfected in HCC cell lines or injected via the mouse tail vein under hydrodynamic conditions have repeatedly confirmed that HBx Colec11 potentiate HBV replication (2327). The HBV replicative intermediate cccDNA (covalently closed circular HBV DNA), which serves as a template for the transcription of all viral transcripts including the pregenomic RNA (pgRNA), is organized into a minichromosome in the nuclei of infected hepatocytes by histone e non-histone proteins (28). The HBV core protein is a structural component of the HBV minichromosome, and its binding results in a reduction of the nucleosomal spacing of the HBV nucleoprotein complexes (29). Recently, we have developed a ChIP-based quantitative technique to study the recruitment in vivo of cellular and viral proteins onto the HBV minichromosome (30). Chromatin immunoprecipitation (ChIP) techniques allow the identification in vivo of the DNA binding sites of virtually any chromosome component (31). The principle relies upon the fixation of protein-DNA and protein-protein interactions in vivo by the cross-linking agent formaldehyde, and the immunoprecipitation of the cross-linked protein of interest with specific antibody directed against immunoprecipitated DNA is used as a template in real-time PCR. The HBV cccDNA ChIP assay combines a cccDNA ChIP step with a sensitive and specific real-time PCR protocol for cccDNA quantification (30). Using an Dehydroepiandrosterone anti-acetylated-H3 or -H4 cccDNA ChIP assay, we found that HBV replication is regulated, both in HBV replicating Huh7 cells and in the liver of HBV chronically infected patients by the acetylation status of H3/H4 histones bound to the viral cccDNA (30). Here we investigated the recruitment of HBx onto the HBV minichromosome and its ability to regulate the epigenetic control of cccDNA function in HBV replicating cells. == Results == == HBx.