All-trans retinoic acid downregulates HBx levels via E6-associated protein-mediated proteasomal degradation to suppress hepatitis B virus replication.

All-trans retinoic acid (ATRA), recognized as the principal and most biologically potent metabolite of vitamin A, has been identified for its inhibitory effects on hepatitis B virus (HBV) replication. Nevertheless, the underlying mechanism remains elusive. The present study reveals that ATRA induces E6-associated protein (E6AP)-mediated proteasomal degradation of HBx to suppress HBV replication in human hepatoma cells in a p53-dependent pathway. For this effect, ATRA induced promoter hypomethylation of E6AP in the presence of HBx, which resulted in the upregulation of E6AP levels in HepG2 but not in Hep3B cells, emphasizing the p53-dependent nature of this effect. As a consequence, ATRA augmented the interaction between E6AP and HBx, resulting in substantial ubiquitination of HBx and consequent reduction in HBx protein levels in both the HBx overexpression system and the in vitro HBV replication model. Additionally, the knockdown of E6AP under ATRA treatment reduced the interaction between HBx and E6AP and decreased the ubiquitin-dependent proteasomal degradation of HBx, which prompted a recovery of HBV replication in the presence of ATRA, as confirmed by increased levels of intracellular HBV proteins and secreted HBV levels. This study not only contributes to the understanding of the complex interactions between ATRA, p53, E6AP, and HBx but also provides an academic basis for the clinical employment of ATRA in the treatment of HBV infection.

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.

All-trans Retinoic Acid Inhibits Hepatitis B Virus Replication by Downregulating HBx Levels via Siah-1-Mediated Proteasomal Degradation.

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to abolish the potential of HBx to downregulate the levels of p14, p16, and p21 and to stimulate cell growth during hepatitis B virus (HBV) infection, contributing to its chemopreventive and therapeutic effects against HBV-associated hepatocellular carcinoma. Here, we demonstrated that ATRA antagonizes HBx to inhibit HBV replication. For this effect, ATRA individually or in combination with HBx upregulated p53 levels, resulting in upregulation of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induces ubiquitination and proteasomal degradation of HBx in the presence of ATRA. The ability of ATRA to induce Siah-1-mediated HBx degradation and the subsequent inhibition of HBV replication was proven in an in vitro HBV replication model. The effects of ATRA became invalid when either p53 or Siah-1 was knocked down by a specific shRNA, providing direct evidence for the role of p53 and Siah-1 in the negative regulation of HBV replication by ATRA.

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.

Hepatitis C virus core protein activates proteasomal activator 28 gamma to downregulate p16 levels via ubiquitin-independent proteasomal degradation.

Proteasomal activator 28 gamma (PA28γ), an essential constituent of the 20S proteasome, is frequently overexpressed in hepatocellular carcinoma. Hepatitis C virus (HCV) core protein is recently known to activate PA28γ expression in human hepatocytes via upregulation of p53 levels; however, its role in HCV tumorigenesis remains unknown. Here, we found that HCV core-activated PA28γ downregulates p16 levels via ubiquitin-independent proteasomal degradation. As a result, HCV core protein activated the Rb-E2F pathway to stimulate cell cycle progression from G1 to S phase, resulting in an increase in cell proliferation. The potential of HCV core protein to induce these effects was almost completely abolished by either PA28γ knockdown or p16 overexpression, confirming the role of the PA28γ-mediated p16 degradation in HCV tumorigenesis.

Hepatitis B virus X protein stimulates cell growth by downregulating p16 levels via PA28γ-mediated proteasomal degradation.

Proteasomal activator 28 gamma (PA28γ), an essential constituent of the 20S proteasome responsible for ubiquitin-independent degradation of target proteins, is frequently overexpressed in hepatocellular carcinoma. Recently, we have reported that hepatitis B virus (HBV) X protein (HBx) activates PA28γ expression in human hepatocytes via upregulation of p53 levels; however, its role in HBV tumorigenesis remains unknown. Here, we found that HBx-activated PA28γ downregulates p16 levels via ubiquitin-independent proteasomal degradation. As a result, HBx activated the Rb-E2F pathway and stimulated G1/S cell cycle progression, resulting in an increase in cell proliferation. The potential of HBx to induce these effects was reproduced in a 1.2-mer HBV replicon and in in vitro HBV infection systems and was almost completely abolished by either PA28γ knockdown or p16 overexpression, demonstrating the critical role of the PA28γ-mediated p16 degradation in HBV tumorigenesis.

HBx natural variants containing Ser-101 instead of Pro-101 evade ubiquitin-dependent proteasomal degradation by activating proteasomal activator 28 gamma expression.

Proteasomal activator 28 gamma (PA28γ) is frequently overexpressed in hepatocellular carcinoma; however, its underlying mechanism and role in hepatitis B virus (HBV) replication are largely unknown. Here, we found that HBV X protein (HBx) natural variants containing Ser-101 instead of Pro-101 increase reactive oxygen species levels in the mitochondria and activate the ataxia telangiectasia mutated/checkpoint kinase 2 pathway in the nucleus, resulting in the phosphorylation of p53 at Ser-15 and Ser-20 and the subsequent upregulation of its protein levels. Therefore, HBx variants containing Ser-101 induced p53-dependent activation of PA28γ expression in human hepatoma cells. The elevated PA28γ levels upregulated HBx levels through the inhibition of seven in absentia homologue 1-dependent proteasomal degradation. The self-amplifying ability of HBx variants containing Ser-101 via a positive feedback loop involving p53 and PA28γ was accurately reproduced in both a 1.2-mer HBV replicon and in vitro HBV infection systems, which also provided evidence for the stimulation of HBV replication by these HBx variants. In conclusion, the ability of HBx to upregulate PA28γ levels via p53 activation, in a Ser-101-dependent pathway, is critical for the stimulation of HBV replication.

Hepatitis B Virus X Protein Stimulates Virus Replication Via DNA Methylation of the C-1619 in Covalently Closed Circular DNA.

Methylation of HBV cccDNA has been detected in vivo and in vitro; however, the mechanism and its effects on HBV replication remain unclear. HBx derived from a 1.2-mer HBV replicon upregulated protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), 3a, and 3b, resulting in methylation of the negative regulatory region (NRE) in cccDNA, while none of these effects were observed with an HBx-null mutant. The HBx-positive HBV cccDNA expressed higher levels of HBc and produced about 4-fold higher levels of HBV particles than those from the HBx-null counterpart. For these effects, HBx interrupted the action of NRE binding protein via methylation of the C-1619 within NRE, resulting in activation of the core promoter. Treatment with 5-Aza-2'dC or DNMT1 knock-down drastically impaired the ability of HBx to activate the core promoter and stimulate HBV replication in 1.2-mer HBV replicon and in vitro infection systems, indicating the positive role of HBx-mediated cccDNA methylation in HBV replication.

Hepatitis B virus X protein activates proteasomal activator 28 gamma expression via upregulation of p53 levels to stimulate virus replication.

Proteasomal activator gamma (PA28γ), frequently overexpressed in hepatocellular carcinoma, is believed to play important roles in tumourigenesis. However, the underlying mechanism of PA28γ overexpression and its possible roles in hepatitis B virus (HBV) replication are largely unknown. In the present study, we found that hepatitis B virus X protein (HBx) activates PA28γ expression by upregulating p53 levels in human hepatoma cells. The elevated PA28γ levels in turn repressed seven in absentia homologue 1 expression via downregulation of p53 levels, thereby inhibiting ubiquitin-dependent proteasomal degradation of HBx, which ultimately led to upregulation of HBx levels. The correlation among HBx, p53 and PA28γ was exactly reproduced in a 1.2-mer HBV replicon system, mimicking the natural course of HBV infection. In particular, knockdown of either p53 or PA28γ in HepG2 cells downregulated HBx levels and thereby inhibited HBV replication, whereas overexpression of p53 or PA28γ in Hep3B cells upregulated HBx levels, which stimulated HBV replication, indicating that p53 and PA28γ act as activators of HBV replication. In conclusion, HBx levels are upregulated via a positive feedback loop involving p53 and PA28γ to stimulate HBV propagation.

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 B virus X protein suppresses all-trans retinoic acid-induced apoptosis in human hepatocytes by repressing p14 expression via DNA methylation.

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to activate p14 expression via promoter hypermethylation to induce p53-dependent apoptosis in human hepatocytes. In this study, we found that the oncogenic hepatitis B virus (HBV) X protein (HBx) of HBV, derived from both overexpression and 1.2-mer replicon systems, suppresses ATRA-induced apoptosis in p53-positive human hepatocytes. For this effect, HBx upregulated both protein and enzyme activity levels of DNA methyltransferase 1, 3a and 3b, in the presence of ATRA and thereby inhibited p14 expression via promoter hypermethylation, resulting in inactivation of the p14-mouse double minute 2 pathway and subsequent downregulation of p53 levels. As a result, HBx was able to impair the potential of ATRA to activate apoptosis-related molecules, including Bax, p53-upregulated modulator of apoptosis, caspase-9, caspase-3 and poly (ADP-ribose) polymerase. In conclusion, the present study provides a new oncogenic action mechanism of HBx, namely by suppressing the anticancer potential of ATRA to induce p53-dependent apoptosis in HBV-infected hepatocytes.

Hepatitis B virus X protein activates E3 ubiquitin ligase Siah-1 to control virus propagation via a negative feedback loop.

The seven in absentia homologue 1 (Siah-1) protein is an E3 ubiquitin ligase that induces ubiquitin-dependent proteasomal degradation of HBx, the principal regulatory protein of hepatitis B virus (HBV); however, its role in HBV propagation remains unknown. Here, we found that HBx upregulates Siah-1 levels in HepG2 but not in Hep3B cells, in which p53 is absent. For this effect, HBx sequentially activated ataxia telangiectasia mutated kinase and checkpoint kinase 2 via phosphorylation at the Ser-1981 and Thr-68 residues, respectively, which led to the activation of p53 via phosphorylation at the Ser-15 and Ser-20 residues. As a result, HBx was heavily ubiquitinated by Siah-1 and degraded by the ubiquitin-proteasome system in HepG2 cells, whereas this effect was marginal or undetectable in Hep3B cells. Knock-down of p53 in HepG2 cells downregulated Siah-1 levels and subsequently upregulated HBx levels, whereas ectopic p53 expression in Hep3B cells upregulated Siah-1 levels and subsequently downregulated HBx levels. In addition, Siah-1 knock-down impaired the ubiquitination and proteasomal degradation of HBx in HepG2 cells, whereas ectopic Siah-1 expression induced ubiquitin-dependent proteasomal degradation of HBx in Hep3B cells. The effects of HBx on p53 and Siah-1 were exactly reproduced in a 1.2-mer HBV replicon system, mimicking the natural course of HBV infection. In particular, Siah-1 knock-down upregulated the levels of HBx derived from the HBV replicon, resulting in an increase in HBV production. In conclusion, HBx modulates its own protein level via a negative feedback loop involving p53 and Siah-1 to control HBV propagation.

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.