Innate Immune Evasion By The Hepatitis B Virus-mediated Downregulation Of TRIF

Background:Hepatitis B virus (HBV) infection is one of top ten causes of death in the world and it is well established that the chronic infection with HBV plays an essential role in the aetiology of cirrhosis, and hepatocellular carcinoma (HCC). According to the groble epidemiological data in2002,54.4%of new cases of HCC worldwide in that year could be attributed to HBV infection. Nucleoside analogues and interferon a-based treatment is less-than-ideal on this infection up to now. As the target cell infected by HBV, the host cellular defence in hepatocytes is important to limit viral replication. Although it is not the immune cell in the usual sense, recent researches revealed that a variety of innate immune pathways and original immune surveillance also existed in hepatocytes and proved the effect in the inhibition of HBV replication. For their own survival, HBV develops many corresponding strategies to evade immune clearance. Thus, the identification of molecular mechanisms by which HBV evades the host innate immune system would enrich our knowledge of how the hepatitis B virus infection persists. Moreover, the increasing understanding of these mechanisms would help us to develop the possible new treatment strategy for HBV infection and better prevent cirrhosis and HCC in the etiology. Objective:Hepatocytes are the target cells in the hepatitis B virus infection. More and more attention is paid to the effect of host cellular defense in the inhibition of hepatitis B virus. The current studies found that the innate immune responses in the hepatocytes can effectively defend host cell against the viral infection. As a key adaptor protein in the TLR3signaling pathway, TRIF is often hijacked by virus such as HAV and HCV for the development of innate immune evasion strategies. Therefore, in this sudy, we investigated the HBV expression on the regulation of TRIF protein in hepatocytes and conducted a preliminary exploration of the mechanism that is responsible for the processing of TRIF by HBV. Furthermore, we evaluated that whether the expression and replication of HBV gene can be regulated by TRIF protein and discussed the relevant antiviral molecular mechanism. We wish to increase our understanding of how the hepatitis B virus evades the innate immune responses in host cells and consequently provide a new theoretical strategy selection for the treatment of HBV.Methods:1. qRT-PCR and Western blot were applied to detect the mRNA and protein expression of TRIF in HCC cells that transiently transfected HBV expression plasmid and blank plasmid, in HepG2and HepG2.2.15cells, in HBV-specific siRNA transfected HepG2.2.15cells and control cells and in13HBV-negative and12HBV-positive liver tissue samples.2. We constructed plasmids that can express viral protein X, core, large surfsce protein (LHBs) and polymerase. These expressive constructions and empty vector were transfected into HCC cells followed by Western blot detection of TRIF expression to found which viral protein is responsible for the change of TRIF. Western blot analysis of TRIF protein was also performed to confirm the role of X protein in the regulation of TRIF expression in HCC cells transfected with the wild-type HBV plasmid (HBV), in cells transfected with the X-defective mutant HBV plasmid (HBVX-), in cells transfected with the combination of the HBVX-and X plasmids, in cells transfected with the gradually increased amount of X plasmid, in cells transfected with the combination of constant TRIF plasmid and gradually increased X plasmid, in cells transfected with the X plasmid following the treatment with proteasome inhibitor MG132. Immunoprecipitation was applied to examine the interaction between X and TRIF proteins in HCC cells that were co-transfected with X and TRIF constructions.3. HCC cells were co-transfected with TRIF and HBV plasmid. The HBsAg and HBeAg level in the culture supernatants was determined using ELISA. The HBV replicative intermediate DNA in cellular HBV virus particles was analyzed by quantitative real-time PCR. The level of viral mRNA in transfected HCC cells was assessed by qRT-PCR and the expression of viral protein by Western blot probed with anti-HBc antibodies. In HepG2.2.15cells transfected with TRIF plasmid, HBV expression and replication were detected in the same ways.4. TRIF protein was overexpressed in HCC cells. Transcriptional activation of NF-kB and ISRE and activation of IFN-P promoter induced by TRIF were observed by dual-luciferase reporter assay. The mRNA expressions of TNF-a, ISG15and IFN-β were detected using qRT-PCR. To observe the NF-κB activation in the regulation of HBV survival, we treated the HBV transiently transfected and stably transfected HCC cells with NF-κB inhibitor BAY11-7082. Replication and expression of HBV were determined in the ways mentioned above. After TRIF protein elevated in HCC cells, apoptosis was examined by microscopic observation based on cellular and nuclear morphology and measurement of the activities of caspase-3and-7.Results:1. The relative lower expression of TRIF protein was found in all the HBV-expressing HCC cells and HBV-positive liver tissue samples compared with their respective controls. The relative expression of TRIF mRNA was decreased in HCC cells that stably transfected with HBV and HBV-positive liver tissue samples while no significant change in the in HCC cells that transiently transfected with HBV compared with their respective controls. In the HCC cells with HBV stable transfection, the decreased expression of TRIF protein and similar expression of its mRNA were observed after silence of HBV expression by a HBV-specific siRNA. 2. Using the wild-type HBV plasmid carrying129%length of HBV genome (subtype ayw) as template, we successfully constructed a series of plasmids that can express viral proteins X, Core, LHBs and polymerase. We found that the expression of X protein resulted in decreased level of TRIF protein in transfected HCC cells and this reduction caused by X was in a dose-dependent manner. In addition, we carried an experiment using the wild-type HBV, the X-defective mutant HBV (HBVX-) and the combination of HBVX-and X expression plasmid, and reconfirmed the role of the X protein in the reduction of TRIF. The treatment of proteasome inhibitor MG132rescued the expression of TRIF protein in the background of X expression. Immunoprecipitation failed to find the direct interaction between X and TRIF in the HCC cells co-transfected with these two expressive plasmids.3. Overexpression of TRIF protein dramatically reduced the levels of HBsAg and HBeAg in cell culture supernatants, lower the HBV replicative intermediate DNA in cellular virus particles, decreased the HBc mRNA and protein levels in transfected HCC cells.4. Overexpression of TRIF in HCC cells induced the activation of NF-κB and ISRE and IFN-P promotor. The mRNA expression levels of TNF-a, ISG15and IFN-(3were increased. HBV replication and expression in the HCC cells were significantly enhanced by blocking NF-κB activation. The TRIF expression induced cell apoptosis.Conclusions:1. HBV reduced the expression of TRIF protein. However, it showed differences in the mRNA expression of TRIF between transient transfection model and stable transfection model which represented a persistent statement of HBV infection. Hepatitis B virus X protein was responsible for TRIF downregulation. HBx reduced TRIF protein in a dose-dependent manner and in a proteasome-dependent way.2. Overexpression of TRIF protein not only promoted the activation of NF-κB, ISRE and IFN-β but also induced cell apoptosis. HBV replication and expression in HCC cells were dramatically repressed by TRIF expressing.3. Based on these findings, we revealed a new mechanism that HBV evaded the cellular innate immunity by downregulation of TRIF protein in hepatocyte. It increased our understanding of how HBV persists and provided a possibile strategy for HBV treatment.