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.

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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.

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.

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.