HBV X protein mutations affect HBV transcription and association of histone-modifying enzymes with covalently closed circular DNA

Abstract

The hepatitis B X protein (HBx) plays a role in the epigenetic regulation of hepatitis B virus (HBV) replication. This study investigated the effects of HBx mutations on HBV transcription and the recruitment of HBx, histone acetyl-transferase P300 and histone deacetylase 1 (HDAC1) to circularized HBV DNA (which resembles covalently closed circular DNA [cccDNA]). Compared with wild type, majority of mutants had lower levels of intracellular HBV RNA (44-77% reduction) and secretory HBsAg (25-81% reduction), and 12 mutants had a reduction in intracellular encapsidated HBV DNA (33-64% reduction). Eight mutants with >70% reduction in HBV RNA and/or HBsAg were selected for chromatin immunoprecipitation analysis. Four HBx mutants with mutations in amino acid residues 55-60 and 121-126 had a lower degree of HBx-cccDNA association than wild type HBx (mean % input: 0.02-0.64% vs. 3.08% in wild type). A reduced association between cccDNA and P300 (mean % input: 0.69-1.81% vs. 3.48% in wild type) and an augmented association with HDAC1 (mean % input: 4.01-14.0% vs. 1.53% in wild type) were detected. HBx amino acid residues 55-60 and 121-126 may play an important role in HBV transcription regulation, via their impeded interaction with cccDNA and altered recruitment of histone modifying enzymes to cccDNA.

Tumor suppressive role of mitochondrial sirtuin 4 in induction of G2/M cell cycle arrest and apoptosis in hepatitis B virus-related hepatocellular carcinoma

“…The vectors (pcDNA3.1 and pCR-Blunt II-TOPO) were purchased from Thermo Fisher Scientific. The HBx expression plasmid (pHBx) was constructed as previously reported 46 . SIRT4 plasmid provided by Mammalian Gene Collection (MGC:130046) was obtained from PlasmID Repository at Harvard Medical School.…”

Occult hepatitis B infection and hepatocellular carcinoma: Epidemiology, virology, hepatocarcinogenesis and clinical significance

“…54 A number of cccDNA-associated proteins, including the hepatitis B core and X proteins (HBc and HBx, respectively), transcription factors such as cAMP response element binding protein (CREB), signal transcription factors 1 and 2, chromatin modification proteins such as histone deacetylase 1, p300/CREB-binding protein, protein arginine methyltransferase 1 and 5, and sirtuin 1, have been demonstrated to regulate the transcriptional activity of HBV. 52,[54][55][56][57][58][59][60][61] In theory, these cellular and viral epigenetic factors can facilitate a robust control of viral replication, leading to very low HBV DNA and undetectable HBsAg in patients with OBI. 62 However, to date, direct evidence of the involvement of these epigenetic factors in OBI has yet to be reported.…”

“…68 However, partly due to the scarcity of adequate animal or in vitro models for OBI study, current data have, by necessity, been based on inferences generated from experiments designed to control HBV replication using in vitro systems approximating CHB infection. [54][55][56][57][58][59][60][61] Association of OBI and HCC Woodchuck model of OBI The woodchuck model of OBI is an excellent experimental system to study the pathogenicity of occult hepadnaviral persistence and its role in the development of HCC. 69 Investigators reported that woodchucks infected with low doses of WHV developed asymptomatic, seronegative, molecularly evident persistent occult hepatitis with a low viral load.…”

Strategies to Inhibit Hepatitis B Virus Gene Transcription

“…In the case of HBx-minus HBV mutants, histones are hypoacetylated, while the recruitment of histone deacetylases histone deacetylase 1 (HDAC1) and sirtuin-1 (SIRT1) are profoundly increased [61]. Furthermore, mapping analysis determined that C-terminally mutated HBx recruits less acetyltransferase p300 and more HDAC1 onto cccDNA templates [83]. HBx not only initiates but also maintains transcription from cccDNA.…”