Hepatitis C virus Core overcomes all-trans retinoic acid-induced apoptosis in human hepatoma cells by inhibiting p14 expression via DNA methylation.

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to induce p14 expression via promoter hypomethylation to activate the p14-MDM2-p53 pathway, which leads to activation of the p53-dependent apoptotic pathway and subsequent induction of apoptosis in human hepatoma cells. In the present study, we found that hepatitis C virus (HCV) Core derived from ectopic expression or HCV infection overcomes ATRA-induced apoptosis in p53-positive hepatoma cells. For this effect, HCV Core upregulated both protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b and thereby repressed p14 expression via promoter hypermethylation, resulting in inactivation of the pathway leading to p53 accumulation in the presence of ATRA. As a result, HCV Core prevented ATRA from activating several apoptosis-related molecules, including Bax, p53 upregulated modulator of apoptosis, caspase-9, caspase-3, and poly (ADP-ribose) polymerase. In addition, complementation of p14 in the Core-expressing cells by either ectopic expression or treatment with 5-Aza-2'dC almost completely abolished the potential of HCV Core to suppress ATRA-induced apoptosis. Based on these observations, we conclude that HCV Core executes its oncogenic potential by suppressing the p53-dependent apoptosis induced by ATRA in human hepatoma cells.

Hepatitis C virus core activates proteasomal activator 28γ expression via upregulation of p53 levels to control virus propagation.

The proteasomal activator 28γ (PA28γ), frequently overexpressed in hepatocellular carcinoma, is believed to play several important roles in hepatitis C virus (HCV) replication and viral pathogenesis. However, the underlying mechanism for PA28γ overexpression in hepatocellular carcinoma and its role during HCV replication are still unclear. In the present study, we found that HCV core derived from either ectopic expression or HCV infection upregulates PA28γ levels in p53-positive human hepatocytes. For this effect, HCV core sequentially activated ataxia telangiectasia mutated and checkpoint kinase 2 via phosphorylation at Ser-1981 and Thr-68 residues, respectively, resulting in stabilization of p53 via phosphorylation at Ser-15 and Ser-20 residues and subsequent transcriptional activation of PA28γ expression. The elevated PA28γ in turn downregulated HCV core levels by either inducing its ubiquitination-dependent proteasomal degradation via upregulation of E6AP levels in the presence of p53 or activating an ubiquitin-independent proteasomal degradation pathway in the absence of p53, which ultimately led to a decrease in HCV propagation. HCV core modulates its own protein level via a negative feedback loop involving p53 and PA28γ to control HCV replication in p53-positive hepatocytes, which may help HCV evade immune responses and establish chronic infection.

Hepatitis C virus core protein inhibits E6AP expression via DNA methylation to escape from ubiquitin-dependent proteasomal degradation.

The E6-associated protein (E6AP) is a ubiquitin ligase that mediates ubiquitination and proteasomal degradation of hepatitis C virus (HCV) core protein. Given the role of HCV core protein as a major component of the viral nucleocapsid, as well as a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis, HCV has likely evolved a strategy to counteract the host anti-viral defense mechanism of E6AP and maximize its potential to produce infectious virus particles. In the present study, we found that HCV core protein derived from either ectopic expression or HCV infection inhibits E6AP expression via promoter hypermethylation in human hepatocytes. As a result, the potential of E6AP to ubiquitinate and degrade HCV core protein through the ubiquitin-proteasome system was severely impaired, which in turn led to stimulation of virus propagation. The effects of HCV core protein were almost completely abolished when the E6AP level was restored by ectopic expression of E6AP, treatment with a universal DNA methyltransferase (DNMT) inhibitor, 5-Aza-2'dC, or knock-down of DNMT1. In conclusion, HCV core protein inhibits E6AP expression via DNA methylation to protect itself from ubiquitin-dependent proteasomal degradation and stimulate virus propagation, providing a potential target for the development of anti-viral drugs against HCV.

Hepatitis B virus X protein induces epithelial-mesenchymal transition by repressing E-cadherin expression via upregulation of E12/E47.

Previous reports have demonstrated that hepatitis B virus (HBV) X protein (HBx) represses E-cadherin expression to induce epithelial-mesenchymal transition (EMT), an essential component of cancer progression to more aggressive phenotypes characterized by tumour invasion, migration and metastasis; however, the underlying mechanism for this phenomenon is still unclear. In this study, we found that ectopic expression of HBx in human hepatocytes using overexpression and 1.2-mer WT HBV replicon systems upregulated levels of the transcriptional repressors E12 and E47, resulting in inactivation of the E-cadherin promoter, containing three E-box motifs, and subsequent repression of its expression. E12/E47 knockdown using a specific small interfering RNA almost completely abolished the potential of HBx to repress E-cadherin expression. HBx inhibited the ubiquitin-dependent proteasomal degradation of E12/E47 without affecting their expression at the transcriptional level. Upregulation of E12/E47 by HBx ultimately led to EMT in human hepatocytes, as demonstrated by morphological changes, altered protein levels of EMT markers, including E-cadherin, plakoglobin, fibronectin, vimentin and N-cadherin, and increased capacity for cell detachment and migration.

All-trans retinoic acid induces p53-depenent apoptosis in human hepatocytes by activating p14 expression via promoter hypomethylation.

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, has been extensively studied for the prevention and treatment of cancer; however, the underlying mechanism of its anti-cancer potential is still unclear. Here we found that ATRA induces apoptosis in p53-positive HepG2 cells, but not in p53-negative Hep3B cells. For this effect, ATRA activated p14 expression via promoter hypomethylation, resulting in ubiquitin-dependent degradation of mouse double minute 2 (MDM2) and subsequent stabilization of p53. The potential of ATRA to stabilize p53 was almost completely abolished by knock-down of p14 in HepG2 cells and was not observed in p14-negative A549 cells. Upregulation of p14 also abolished the self-regulatory potential of p53 to repress p14 expression via DNA methylation and transcriptionally activate MDM2 expression. The accumulated p53 then activated several apoptosis-related molecules, including Bax, PUMA, caspase-9, Bid, caspase-8, caspase-3, and PARP. Ectopic expression of DNA methyltransferase 1 almost completely abolished the potential of ATRA to activate the p14-MDM2-p53 pathway and induce p53-dependent apoptosis. Therefore, we conclude that ATRA induces p14 promoter hypomethylation to trigger apoptosis.