RNAi-based knockdown of HBx mRNA in HBx-transformed and HBV-producing human liver cells.

RNA interference (RNAi) is the process of sequence-specific gene silencing induced by 21-23-nt RNA of small interfering RNA (siRNA). The HBx of hepatitis B virus (HBV) causing human liver diseases has been known as a multifunctional protein which affects transcription, cell growth, and apoptotic cell death. Here, we demonstrate that the HBx-specific siRNA (siRNAx) and short hairpin RNA (shRNAx) effectively induce the degradation of HBx mRNA in HBx-transformed and HBV-producing human liver cells by up to 80-90%. Also, the HBx expression in HBx-transformed cells was continuously silenced by retransformation with the shRNAx expression vector. These results imply that HBx-driven RNAi, either delivery of siRNAx or expression of shRNAx, provides a promising anti-HBV approach to suppress the HBx expression in human hepatoma cells.

Transcriptional activation of gammaherpesviral oncogene promoters by the hepatitis B viral X protein (HBx).

The latent membrane protein-1 (LMP1) of Epstein-Barr Virus (EBV), saimiri transformation protein (STP) of Herpesvirus saimiri (HVS), and K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) are potent gammaherpesvirus oncogenes. To study the possible effects of double viral infection, we investigated the effects of oncogenic early proteins of DNA viruses E1A and E1B (adenovirus-5), E6 and E7 (human papillomavirus-16), HBx (hepatitis B virus), Tag (SV40), and gammaherpesviral oncogene during co-infection in human B-lymphoma (Ramos) and human T-cell leukemia (Jurkat) cell lines. HBx transactivated the promoters of LMP1, STP, and K1 the most, by about six-, three-, and twofold, respectively. Analyses of site-directed mutation and the heterologous promoter system showed that HBx activated the promoter activity of these genes via the NF-kappaB site. These results suggest that HBV (HBx) infection of cells previously infected by gammaherpesviruses transactivates their oncogenes, resulting in possible virus-related disease pathogenesis.

Transcriptional repression of the human p53 gene by hepatitis B viral core protein (HBc) in human liver cells.

Hepatitis B virus (HBV) is a causative agent of chronic and acute hepatitis, and is associated with the development of hepatocellular carcinoma (HCC). We demonstrate here that the Hepatitis B viral core protein (HBc) functions as a repressor on the promoter activity of the human p53 gene. The functional analyses of the promoter of the p53 gene by serial deletion, site-directed mutagenesis, and the heterologous promoter system revealed that the promoter activity was repressed through the E2F1-binding site (nucleotides -28 to -8) by HBc. An electrophoretic mobility shift assay (EMSA) showed that the HBc reduced the DNA-binding ability of E2F1 to the binding site of the p53 promoter. The interaction of HBc with E2F1 was also observed by glutathione S-transferase (GST) fusion protein binding assay. Furthermore, HBc represses the expression of the p53 gene in the human liver cell line HepG2. Finally, HBc and HBx synergistically repress both the promoter activity and the expression of the p53 gene in HepG2 cells. These results, together with our previous study, strongly suggest that HBc, like HBx, represses the expression of the human p53 tumor suppressor gene.

The transcriptional activation of the human copper/zinc superoxide dismutase gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin through two different regulator sites, the antioxidant responsive element and xenobiotic responsive element.

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced during biological oxidations and environmental stress. The most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces SOD1 in human liver cells. Deletion analyses showed that the promoter region between -400 and -239 was responsible for the induction, in which two different characteristic regulatory elements, the antioxidant responsive element (ARE) and xenobiotic responsive element (XRE), are located. When the cells transfected with the plasmid containing those two cis-elements, the transactivation of SOD1 promoter was about 4-fold by TCDD, whereas mutation either on the ARE or XRE elevated the promoter activity by about 2-fold. Functional analyses of these two elements by deletion, mutation in the natural context, heterologous promoter assay, and gel mobility shift assay supported the notion that the activation of the SOD1 promoter was induced by TCDD through these two regulatory elements ARE and XRE. These results alongside our previous data indicate that the induction of SOD1 in response to TCDD is mediated by either Nrf2 protein or Ah receptor protein through ARE and XRE, respectively. These results also imply that the SOD1 can be induced by dioxin either in combination with or independently of these two regulatory elements to effectively defend cells from oxidative stress.

Hepatitis B viral core protein activates the hepatitis B viral enhancer II/pregenomic promoter through the nuclear factor kappaB binding site.

We here demonstrated that the hepatitis B viral (HBV) core protein (HBc) functions as a transcriptional activator on the pregenomic promoter of HBV. Detailed analyses on the HBV pregenomic promoter by serial deletion, mutation, and heterologous promoter system showed that the site responsible for activation was the nuclear factor kappaB (NF-kappaB) binding site (GGGACGTACT, nucleotides 1408-1417) upstream of the enhancer II/pregenomic promoter. The electrophoretic mobility shift assay using the HBc-transfected HepG2 nuclear extracts showed that the HBc enhanced the NF-kappaB DNA-binding ability. These results suggest that the HBc functions as a positive regulator, which may enhance viral replication in hepatocytes.

Activation of the human transforming growth factor alpha (TGF-alpha) gene by the hepatitis B viral X protein (HBx) through AP-2 sites.

The HBx protein is known as a transactivator and potential oncogene, and TGF-alpha as a potent mitogen in hepatocellular carcinoma. By assays of serial deletion of the promoter of TGF-alpha gene and the cotransfection of HBx and AP-2 expression vectors, we observed that the HBx significantly activated the promoter activity through AP-2 sites located in the proximal region of the TGF-alpha promoter (-136 to -30). This effect was also observed in the heterologous promoter assay system containing AP-2 sites. The mutation analyses of three AP-2 sites in the promoter revealed that all three AP-2 sites contributed to the activation of the TGF-a gene in the presence of HBx. Accordingly, the mRNA level of TGF-alpha was significantly elevated in the HBx-expressing cell, HepG2-HBx and the HBV-producing cell, HepG2-K8. These results suggest that the HBx protein could increase the mitogenic effect of TGF-alpha by the transactivation of the gene through AP-2 binding sites and consequently, these interactions may accelerate the process of hepatocarcinogenesis.

Transcriptional activation of the promoter of human cytomegalovirus immediate early gene (CMV-IE) by the hepatitis B viral X protein (HBx) through the NF-kappaB site.

The reactivation of latent cytomegalovirus (CMV) in a human by another viral infection may induce virus-related symptoms. Based on this presumption, we investigated the effect of HBx on the activation of the CMV-IE, which is also known as a transactivator and potential oncogene. The HBx transactivated the CMV-IE promoter by up to 4- and 18-fold factors in human liver HepG2 and lung fibroblast MRC-5 cells, respectively. Cotransfection of HBx with each transcription factor presented in the CMV-IE promoter showed that only NF-kappaB synergistically activated the promoter by up to a 14-fold factor. Serial deletion assays and point mutation analysis showed that the third NF-kappaB site (nt -267 to -258) and the second one (nt -162 to -153) appeared as the major responsible site and minor one, respectively, for the transactivation. These results suggest the possibility that the HBV infection of a cell previously infected by CMV would exert influence on the reactivation of the latent cytomegalovirus in a human to induce virus-related symptoms.

E2F1 activates the human p53 promoter and overcomes the repressive effect of hepatitis B viral X protein (Hbx) on the p53 promoter.

The functional effect of the interaction of E2F1 and hepatitis B virus X protein (HBx) on the promoter of human p53 gene was studied using chloramphenicol acetyl transferase (CAT) assay. E2F1 activated the p53 promoter through E2F1 binding site. As previously reported, HBx repressed the p53 promoter through E-box. When E2F1 was cotransfected with HBx, E2F1 overcame the repressive effect of HBx on the p53 promoter through the E2F1 site. However, in the thymidine kinase (tk) heterologous promoter system with the E2F1 binding sites, cotransfection of E2F1 and HBx showed a strong synergistic activation. An in vitro interaction assay showed that E2F1 and HBx physically bind with each other. Analyses of the interaction domain with the GAL4 fusion protein showed that the pRb-binding domain of E2F1 was necessary for the functional interaction of these two proteins. Taken together, these results imply the functional inhibitory action of E2F1 on the HBV life cycle and HBV-mediated hepatocellular carcinogenesis (HCC). Therefore, the normal or enhanced function of E2F1 gene would be important in controlling the HBx function in HCC.