Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function

Abstract

HBV cccDNA, the template for transcription of all viral mRNAs, accumulates in the nucleus of infected cells as a stable episome organized into minichromosomes by histones and non-histone viral and cellular proteins. Using a cccDNA-specific chromatin immunoprecipitation (ChIP)-based quantitative assay, we have previously shown that transcription of the HBV minichromosome is regulated by epigenetic changes of cccDNA-bound histones and that modulation of the acetylation status of cccDNA-bound H3/H4 histones impacts on HBV replication. We now show that the cellular histone acetyltransferases CBP, p300, and PCAF/GCN5, and the histone deacetylases HDAC1 and hSirt1 are all recruited in vivo onto the cccDNA. We also found that the HBx regulatory protein produced in HBV replicating cells is recruited onto the cccDNA minichromosome, and the kinetics of HBx recruitment on the cccDNA parallels the HBV replication. As expected, an HBV mutant that does not express HBx is impaired in its replication, and exogenously expressed HBx transcomplements the replication defects. p300 recruitment is severely impaired, and cccDNA-bound histones are rapidly hypoacetylated in cells replicating the HBx mutant, whereas the recruitment of the histone deacetylases hSirt1 and HDAC1 is increased and occurs at earlier times. Finally, HBx mutant cccDNA transcribes significantly less pgRNA. Altogether our results further support the existence of a complex network of epigenetic events that influence cccDNA function and HBV replication and identify an epigenetic mechanism (i.e., to prevent cccDNA deacetylation) by which HBx controls HBV replication.histone acetylation ͉ HATs ͉ HDACs H epatitis B virus (HBV) infection is a major health problem, with Ϸ400 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 (2-5). 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 (6-11). 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, 13-16). To perform its multiple functions, HBx interacts with many cellular partners including the tumor suppressor p53,...

Hepatitis B virus X protein modulates remodelling of minichromosomes related to hepatitis B virus replication in HepG2 cells

“…After centrifugation for 1 min at 12,000 rpm at 4˚C, the supernatants were treated with 100 µg/ml DNase I (Promega) for 30 min at 37˚C and then 0.5 mg/ml proteinase K at 50˚C for 2 h. Viral DNA released from lysed cores were extracted with phenol/chloroform, precipitated with ethanol, and dissolved in Tris-EDTA. Nuclear HBV cccDNA was extracted as described (15,21). Simply, transfected cells were lysed in cell lysis buffer [50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.2% NP-40, and 150 mM NaCl].…”

HBx affects CUL4–DDB1 function in both positive and negative manners

“…HBx carries out many-faceted functions by interacting with a number of cellular proteins in the cytoplasm and nucleus of host cells [10]. Notably, binding of HBx to damage-specific DNA binding protein 1 (DDB1) is essential for HBx to activate HBV transcription [11].…”

Estudos moleculares sobre o vírus da hepatite B

“…O HBxAg é essencial para a iniciação da transcrição do DNA e manutenção do DNA circular covalentemente fechado (cccDNA) que se acumula no núcleo dos hepatócitos infectados: Este antígeno estimula a acetilação de histonas associadas com cccDNA e é necessário para a transcrição no contexto de infecções por HBV (92). HBxAg promove a expressão dos genes virais recrutando a histona acetilases CBP / p300 e PCAF / GCN5 ao cccDNA (93). Adicionalmente, um estudo recente mostrou que HBx promove a …”