Hydrogen Peroxide Inhibits Hepatitis B Virus Replication by Downregulating HBx Levels via Siah-1-Mediated Proteasomal Degradation in Human Hepatoma Cells.

The hepatitis B virus (HBV) is constantly exposed to significant oxidative stress characterized by elevated levels of reactive oxygen species (ROS), such as H2O2, during infection in hepatocytes of patients. In this study, we demonstrated that H2O2 inhibits HBV replication in a p53-dependent fashion in human hepatoma cell lines expressing sodium taurocholate cotransporting polypeptide. Interestingly, H2O2 failed to inhibit the replication of an HBV X protein (HBx)-null HBV mutant, but this defect was successfully complemented by ectopic expression of HBx. Additionally, H2O2 upregulated p53 levels, leading to increased expression of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induced the ubiquitination-dependent proteasomal degradation of HBx. The inhibitory effect of H2O2 was nearly abolished not only by treatment with a representative antioxidant, N-acetyl-L-cysteine but also by knockdown of either p53 or Siah-1 using specific short hairpin RNA, confirming the role of p53 and Siah-1 in the inhibition of HBV replication by H2O2. The present study provides insights into the mechanism that regulates HBV replication under conditions of oxidative stress in patients.

Hydrogen Peroxide Inhibits Hepatitis C Virus Replication by Downregulating Hepatitis C Virus Core Levels through E6-Associated Protein-Mediated Proteasomal Degradation.

Hepatitis C virus (HCV) is constantly exposed to considerable oxidative stress, characterized by elevated levels of reactive oxygen species, including hydrogen peroxide (H2O2), during acute and chronic infection in the hepatocytes of patients. However, the effect of oxidative stress on HCV replication is largely unknown. In the present study, we demonstrated that H2O2 downregulated HCV Core levels to inhibit HCV replication. For this purpose, H2O2 upregulated p53 levels, resulting in the downregulation of both the protein and enzyme activity levels of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b, and activated the expression of E6-associated protein (E6AP) through promoter hypomethylation in the presence of HCV Core. E6AP, an E3 ligase, induced the ubiquitin-dependent proteasomal degradation of HCV Core in a p53-dependent manner. The inhibitory effect of H2O2 on HCV replication was almost completely nullified either by treatment with a representative antioxidant, N-acetyl-L-cysteine, or by knockdown of p53 or E6AP using a specific short hairpin RNA, confirming the roles of p53 and E6AP in the inhibition of HCV replication by H2O2. This study provides insights into the mechanisms that regulate HCV replication under conditions of oxidative stress in patients.

Hepatitis B Virus X Protein Stimulates Hepatitis C Virus (HCV) Replication by Protecting HCV Core Protein from E6AP-Mediated Proteasomal Degradation.

Most clinical and experimental studies have suggested that hepatitis C virus (HCV) is dominant over hepatitis B virus (HBV) during coinfection, although the underlying mechanism remains unclear. In this study, we found that the HBV X protein (HBx) upregulates the levels of the HCV core protein to stimulate HCV replication during coinfection in human hepatoma cells. For this purpose, HBx upregulated both the protein levels and enzyme activities of cellular DNA methyltransferase 1 (DNMT1) and DNMT3b, and this subsequently reduced the expression levels of the E6-associated protein (E6AP), an E3 ligase of the HCV core protein, via DNA methylation. The ubiquitin-dependent proteasomal degradation of the HCV core protein was severely impaired in the presence of HBx, whereas this effect was not observed when E6AP was either ectopically expressed or restored by treatment with 5-aza-2'dC or DNMT1 knockdown. The effect of HBx on the HCV core protein was accurately reproduced in HBV/HCV coinfection systems, which were established by either monoinfection by HCV in Huh7D cells transfected with a 1.2-mer HBV replicon or coinfection by HBV and HCV in Huh7D-Na+-taurocholate cotransporting polypeptide cells, providing evidence for the stimulation of HCV replication by HBx. The present study may provide insights into understanding HCV dominance during HBV/HCV coinfection in patients. IMPORTANCE Hepatitis B virus (HBV) and hepatitis C virus (HCV) are major human pathogens that cause a substantial proportion of liver diseases worldwide. As the two hepatotropic viruses have the same modes of transmission, coinfection is often observed, especially in areas and populations where HBV is endemic. High-risk populations include people who inject drugs. Both clinical and experimental studies have shown that HCV is more dominant than HBV during coinfection, but the underlying mechanism remains unclear. In this study, we show that HBV X protein (HBx) stimulates HCV replication by inhibiting the expression of E6-associated protein (E6AP) via DNA methylation, thereby protecting the HCV core protein from proteasomal degradation, which can contribute to HCV dominance during HBV/HCV coinfection.

Tumor Suppressor p53 Inhibits Hepatitis B Virus Replication by Downregulating HBx via E6AP-Mediated Proteasomal Degradation in Human Hepatocellular Carcinoma Cell Lines.

HBx, a multifunctional regulatory protein, plays an essential role in the replication and pathogenesis of the hepatitis B virus (HBV). In this study, we found that in human hepatoma cells, the tumor suppressor p53 downregulates HBx via ubiquitin-dependent proteasomal degradation. p53 transcriptional activity that results from HBV infection was not essential for this effect. This was shown by treatment with a potent p53 inhibitor, pifithrin-α. Instead, we found that p53 facilitated the binding of E6-associated protein (E6AP), which is an E3 ligase, to HBx and induced E6AP-mediated HBx ubiquitination in a ternary complex of p53, E6AP, and HBx. The ability of p53 to induce E6AP-mediated downregulation of HBx and inhibit HBV replication was demonstrated in an in vitro HBV infection system. This study may provide insights into the regulation of HBx and HBV replication, especially with respect to p53 status, which may also help in understanding HBV-associated tumorigenesis in patients.

Tumour suppressor p53 inhibits hepatitis C virus replication by inducing E6AP-mediated proteasomal degradation of the viral core protein.

The tumour suppressor p53 has been implicated in the host defence system against hepatitis C virus (HCV) infection, although the detailed mechanism remains unknown. Here, we found that p53 inhibits HCV replication by downregulating HCV Core protein levels in human hepatoma cells. For this effect, p53 potentiated the role of E6-associated protein (E6AP) as an E3 ligase to induce ubiquitination and proteasomal degradation of HCV Core. Specifically, p53 facilitated the binding of E6AP to HCV Core through direct interactions with the two proteins. In addition, E6AP failed to induce ubiquitination of HCV Core in the absence of p53, suggesting that p53 increases the E3 ligase activity of E6AP in a triple complex consisting of p53, E6AP and HCV Core.

Hepatitis B virus X protein and hepatitis C virus core protein cooperate to repress E-cadherin expression via DNA methylation.

Dual infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) is closely associated with an increased risk of hepatocellular carcinoma; however, the underlying mechanism is poorly understood. In the present study, we found that HBV X protein (HBx) and HCV core protein work together to inhibit E-cadherin expression in human hepatoma cells. For this effect, they additively increased both the level and activity of enzymes, DNA methyltransferase 1, 3a, and 3b to induce promoter hypermethylation of E-cadherin in a p53-dependent fashion. Their additive effect on E-cadherin levels was reproduced in an in vitro HBV/HCV dual infection system using Huh7D-NTCP cells. As a result, HBV and HCV additively upregulated mesenchymal marker such as N-cadherin, Snail, Twist and Vimentin but cooperatively downregulated epithelial markers such as E-cadherin, Slug and Plakoglobin. In addition, the coinfected cells exhibited faster cell migration and higher invasion ability, as compared with monoinfection, which are hallmarks of epithelial-mesenchymal transition required for the initiation of metastasis in cancer progression.

All-trans retinoic acid inhibits HCV replication by downregulating core levels via E6AP-mediated proteasomal degradation.

Here, we found that all-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, strengthens the anti-viral defense mechanism of E6-associated protein (E6AP) that downregulates hepatitis C virus (HCV) Core levels via ubiquitin-dependent proteasomal degradation. For this effect, ATRA downregulated both protein and enzyme activity levels of DNA methyltransferase 1 and 3b and activated E6AP expression via promoter hypomethylation in HepG2 cells but not in Hep3B cells, in which p53 was absent. Ectopic p53 expression but not E6AP overexpression restored the ability of ATRA to downregulate HCV Core levels in Hep3B cells, suggesting a direct role of p53 in the E6AP-mediated ubiquitination of HCV Core. ATRA also downregulated HCV Core levels during HCV infection in Huh7D cells to inhibit virus replication, providing theoretical basis for the clinical application of ATRA against HCV infection.

Hepatitis C virus core protein inhibits hepatitis B virus replication by downregulating HBx levels via Siah-1-mediated proteasomal degradation during coinfection.

Most clinical and experimental studies have suggested that hepatitis C virus (HCV) is dominant over hepatitis B virus (HBV) during coinfection, although the mechanism remains unclear. Here, we found that HCV core protein inhibits HBV replication by downregulating HBx levels during coinfection in human hepatoma cells. For this effect, HCV core protein increased reactive oxygen species levels in the mitochondria and activated the ataxia telangiectasia mutated-checkpoint kinase two pathway in the nucleus, resulting in an upregulation of p53 levels. Accordingly, HCV core protein induced p53-dependent activation of seven in absentia homolog one expression, an E3 ligase of HBx, resulting in the ubiquitination and proteasomal degradation of HBx. The effect of the HCV core protein on HBx levels was accurately reproduced in both a 1.2-mer HBV replicon and in vitro HBV infection systems, providing evidence for the inhibition of HBV replication by HCV core protein. The present study may provide insights into the mechanism of HCV dominance in HBV- and HCV-coinfected patients.

Proteasomal activator 28 gamma stabilizes hepatitis B virus X protein by competitively inhibiting the Siah-1-mediated proteasomal degradation.

Proteasomal activator 28 gamma (PA28γ) upregulates the levels of HBx, a regulatory protein of hepatitis B virus (HBV) to stimulate HBV replication; however, the detailed mechanism remains unknown. Here, we found that PA28γ impaired the ability of seven in absentia homolog 1 (Siah-1) as an E3 ubiquitin ligase of HBx. PA28γ competitively inhibited the binding of Siah-1 to HBx in human hepatoma cells. Accordingly, PA28γ increased the stability of HBx and decreased HBx ubiquitination, abolishing the potential of Siah-1 to downregulate HBx levels. PA28γ also executed its role as an antagonist of Siah-1 during HBV replication, as demonstrated by an in vitro HBV replication system. The present study may provide insights into the mechanisms underlying the regulation of HBV replication.