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.

Epstein-Barr Virus latent membrane protein 1 overcomes all-trans retinoic acid-induced apoptosis by inhibiting retinoic acid receptor-ß2 expression.

Nasopharyngeal carcinoma is closely associated with infection with Epstein-Barr Virus (EBV); however, the mechanism is still unclear. Here, we report that the EBV oncoprotein, latent membrane protein 1 (LMP1), suppresses apoptotic cell death provoked by all-trans retinoic acid (ATRA) in NPC cells. For this purpose, LMP1 downregulated levels of Bak whilst it upregulated levels of Bcl2, lowering the ratio of Bak to Bcl2. In addition, LMP1 suppressed ATRA-mediated activation of Caspase 9, Caspase 3, and PARP but not Caspase 8 in Ad-AH cells, suggesting that LMP1 acts by blocking the activation of intrinsic apoptosis pathway by ATRA. These effects were almost completely abolished when levels of retinoic acid receptor-ß(2) (RAR-ß(2)) in the LMP1-expressing cells were recovered by either exogenous gene expression or treatment with a universal DNMT inhibitor, 5-Aza-2'dC, indicating that LMP1 executes its antiapoptotic effects by downregulating levels of RAR-ß(2) via DNA methylation.

Hepatitis B virus X protein overcomes all-trans retinoic acid-induced cellular senescence by downregulating levels of p16 and p21 via DNA methylation.

Despite current molecular evidence suggesting that hepatitis B virus (HBV) X protein (HBx) plays an important role during HBV-mediated hepatocarcinogenesis, the detailed mechanism is still controversial. Here, it was shown that HBx overcomes cellular senescence provoked by all-trans retinoic acid (ATRA) in HepG2 cells, as demonstrated by the impaired induction of irreversible G(1) arrest and senescence-associated ß-galactosidase activity by ATRA in the presence of HBx. The anti-senescence effect of HBx was also observed in another human hepatoma cell line, Hep3B, but not in Huh-7 cells in which the p16 and p21 proteins are absent. In addition, HBx suppressed ATRA-mediated induction of p16 and p21 in HepG2 cells via promoter hypermethylation, resulting in inactivation of retinoblastoma protein. Furthermore, the ability of HBx to overcome ATRA-induced cellular senescence almost completely disappeared when the levels of p16 and p21 in the HBx-expressing cells became similar to those in the control cells by complementation in the former by exogenous expression, knockdown of their expression in the latter using specific small interfering RNA or treatment with a DNA methylation inhibitor, 5-Aza-2'-deoxycytidine. These results suggest that HBx executes its potential by downregulating levels of p16 and p21 via DNA methylation. As cellular senescence is a tumour-suppression process, the present study provides a new strategy by which HBV promotes hepatocarcinogenesis.

Genetic Characterization and Pathogenesis of Avian Influenza Virus H7N3 Isolated from Spot-Billed Ducks in South Korea, Early 2019.

Low-pathogenicity avian influenza viruses (LPAIV) introduced by migratory birds circulate in wild birds and can be transmitted to poultry. These viruses can mutate to become highly pathogenic avian influenza viruses causing severe disease and death in poultry. In March 2019, an H7N3 avian influenza virus-A/Spot-billed duck/South Korea/WKU2019-1/2019 (H7N3)-was isolated from spot-billed ducks in South Korea. This study aimed to evaluate the phylogenetic and mutational analysis of this isolate. Molecular analysis revealed that the genes for HA (hemagglutinin) and NA (neuraminidase) of this strain belonged to the Central Asian lineage, whereas genes for other internal proteins such as polymerase basic protein 1 (PB1), PB2, nucleoprotein, polymerase acidic protein, matrix protein, and non-structural protein belonged to that of the Korean lineage. In addition, a monobasic amino acid (PQIEPR/GLF) at the HA cleavage site, and the non-deletion of the stalk region in the NA gene indicated that this isolate was a typical LPAIV. Nucleotide sequence similarity analysis of HA revealed that the highest homology (99.51%) of this isolate is to that of A/common teal/Shanghai/CM1216/2017 (H7N7), and amino acid sequence of NA (99.48%) was closely related to that of A/teal/Egypt/MB-D-487OP/2016 (H7N3). An in vitro propagation of the A/Spot-billed duck/South Korea/WKU2019-1/2019 (H7N3) virus showed highest (7.38 Log10 TCID50/mL) virus titer at 60 h post-infection, and in experimental mouse lungs, the virus was detected at six days' post-infection. Our study characterizes genetic mutations, as well as pathogenesis in both in vitro and in vivo model of a new Korea H7N3 viruses in 2019, carrying multiple potential mutations to become highly pathogenic and develop an ability to infect humans; thus, emphasizing the need for routine surveillance of avian influenza viruses in wild birds.

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 C virus core protein induces epithelial-mesenchymal transition in human hepatocytes by upregulating E12/E47 levels.

Downregulation of E-cadherin is a hallmark of epithelial-mesenchymal transition (EMT), an essential component of cancer progression to more aggressive phenotypes characterized by tumor dedifferentiation, infiltration, and metastasis. However, the underlying mechanism for E-cadherin downregulation in hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) is still unclear. In this study, we found that ectopic expression of HCV core protein or infection with HCV in human hepatocytes upregulated the levels of the transcriptional repressors, E12 and E47, resulting in inactivation of the E-cadherin promoter, containing E-box motifs, and subsequent repression of its expression. E12/E47 knock-down almost completely abolished the potential of HCV core protein to repress E-cadherin expression. HCV core protein inhibited ubiquitin-dependent proteasomal degradation of E12/E47 without affecting their expression at the transcriptional level. E12/E47 upregulation ultimately led to EMT in human hepatocytes, as demonstrated by morphological changes, altered expression levels of EMT markers, including E-cadherin, plakoglobin, and fibronectin, and increased capacity for cell detachment and migration. In conclusion, HCV core protein represses E-cadherin expression by upregulating E12/E47 levels to induce EMT in HCV-associated HCC.

Hepatitis C virus Core protein stimulates cell growth by down-regulating p16 expression via DNA methylation.

Hepatitis C virus Core plays a vital role in the development of hepatocellular carcinoma; however, its action mechanism is still controversial. Here, we showed that Core down-regulated levels of p16, resulting in inactivation of Rb and subsequent activation of E2F1, which lead to growth stimulation of hepatocytes. For this effect, Core inhibited p16 expression by inducing promoter hypermethylation via up-regulation of DNA methyltransferase 1 (DNMT1) and DNMT3b. The growth stimulatory effect of Core was abolished when levels of p16 were restored by either exogenous complementation or treatment with 5-Aza-2'dC, indicating that the effect is critical for the stimulation of cell growth by Core.