The Mechanism Of Hepatitis B Virus Encoded Protein Induces Transcription Of Human Inflammatory Gene

Chronic infection with hepatitis B virus (HBV), a hepatotropic DNA virus, remains to be a major health problem worldwide despite the availability of an effective vaccine. It is estimated that 400 million people worldwide are persistently infected with HBV and at risk of developing chronic hepatitis, cirrhosis and hepatocellular carcinoma. HBV-encoding proteins are reported to play important roles in the pathogenesis of liver disease, such as influencing viral replication and regulating host genes expression. Our previous study has demonstrated that Hepatitis B virus core protein (HBc) and HBx but not HBs initiate the transcription of human Fibrinogen-like protein 2 (hfgl2) gene through transcription factor c-Ets-2 and MAPK signal pathway.Hepatocyte damage in human liver diseases commonly results from apoptosis and necrosis. Although apoptosis can be induced by a wide number of stimuli and mechanisms, the hepatocyte is particularly susceptible to apoptosis by death receptors. At least three members of the TNF superfamily are involved in mediating apoptosis of hepatocytes during HBV infection, that is, TNF, Fas ligand (FasL) and TRAIL. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a typeâ…¡transmembrane protein and a member of the TNF family, induces apoptosis in tumor cells by binding to its receptors expressed on the surface of target cells, which subsequently leads to caspase activation with minimal adverse effects to normal cells. Roles of TRAIL in hepatic cell death remain controversial until recently. Initial studies suggest that, unlike TNF and FasL, soluble recombinant TRAIL may not induce death of normal hepatocyte in mice and nonhuman primates. However, other researchers have also demonstrated that TRAIL was able to induce hepatocyte death of virally infected, transformed cells and fatty acid-treated hepatocytes. In the group of chronic hepatitis, the expression level of TRAIL was significantly higher than those of healthy control and HBV carrier. Besides, it was coincident with the status of the disease and was significantly correlated with the level of liver damage.To explore the viral and host factors which involved in the human TRAIL (hTRAIL) transcription. Eukaryotic expression plasmids of HBc, HBs or HBx were cotransfected with an hTRAIL luciferase report construct into HepG2 cells respectively. The result of luciferase assay showed that HBx protein, significantly enhanced transcription activity of hTRAIL promoter in HepG2 cells, wheras neither HBs nor HBc protein displayed transcription activity on hTRAIL. The transcriptional activity was determined by the relative activity of luciferase and pRL-TK Renilla luciferase reporter plasmid served as an internal control. Compared with the control group, relative luciferase activity in HepG2 cells transfected with pcDNA-HBx was at an average of 9.2-fold elevation. By fluorescent quantitation real-time PCR and Western blottingting, we investigated that the level of hTRAIL mRNA and protein also increased significantly compared with the control group. These results indicate that HBx protein activate the hTRAIL gene transcription, wheras HBs and HBc show no effect.A strong regulatory region which be responsible for activation of HBx protein on hTRAIL gene transcription was located on -89 to -29 (relative to the transcriptional starting site). By site-directed mutagenesis, the two cis-elements spl in the region of -89/-29 were demonstrated to play an important role in transcription of hTRAIL gene in response to HBx protein, while one c-myb cis-elements in the same domain was found not responsable for hTRAIL transcription in response to HBx protein. We investigated that an sp1/KLFs (sp1-like and Kruppel-like factors) family member spl bound to the cognate cis-element in hTRAIL promoter,and it was shown to be responsible for hTRAILgene transcription in response to HBx proteins via EMS A assays. Using sp1 monocloned antibody, ChiP assay showed that the hTRAIL promoter DNA fragment bound to spl transcription factor, include the domain between-89 to-29(relative to the transcriptional starting site). The confocal immunofluorescence study proved that spl transcription factor partially translocated into the nucleus of HepG2 cells in response to HBx. Using shRNA interference technique, we found that the inhibition of spl expression showed a 64.8% decreasing of relative luciferase activity of hTRAIL promoter.In conclusion, our studies have demonstrated that HBV-encoded HBx protein initiated the transcription of hTRAIL gene through the specific combination of transcription factor splwith its cis-element. This work provides new evidences on the interaction between HBV virus and host gene hTRAIL expression,and on the apoptois mechanism after virus infected. The transcription factor spl could be a new therapeutic target for diseases intervention such as severe hepatitis.