| Objective: Hepatitis B virus(HBV)infection can do great harm to human health by causing chronic hepatitis B(CHB),and the innate immune response mechanism plays an important role in resisting HBV infection.Previous research in our group has found that there is a significant difference in the expression of DNA sensor IFI16 in liver tissues and peripheral blood mononuclear cells(PBMCs)between hepatitis B patients and normal controls,and the correlation between IFI16 expression and HBV replication has been initially verified with cells in vitro.However,the molecular mechanism involved remains to be further studied.Here we aimed to determine the role of IFI16 in the process of HBV replication and expression,and the molecular mechanism of IFI16 to induce innate immune response in hepatocytes.That will provide new insights into the search for anti-HBV therapeutic targets and development of new drugs.Methods: Hep G2 cells were co-transfected with p BS-HBV1.3 and IFI16 plasmids,and then HBs Ag and HBe Ag levels in cell culture supernatants were collected and detected by ELISA and HBV m RNA expression was measured by RT-q PCR to verify the inhibitory effect of IFI16 on HBV expression.HBV stable transfection cells Hep AD38 and HBV infection cells Hep G2-NTCP were also used to confirm the above experiments.IFI16 and STING plasmids were cotransfected into Hep G2 cells or Hep AD38 cells,ELISA and RT-q PCR were used to verify whether IFI16 and STING had synergistic effects on HBV inhibition.The activation of STINGTBK1-IRF3-IFN-b pathway under HBV stimulation was tested by RT-q PCR.Protein Coimmunoprecipitation(Co-IP)was used to detect the Interaction between IFI16 and STING moleculars.The HBV infection efficiency of Hep G2-NTCP cells and the IFI16 expression difference between Hep AD38 and Hep G2 cells were measured by immunofluorescence.p Enter-IFI16-?HIN plasmid with HIN domain mutation and p Enter-IFI16-?PYD plasmid with PYD domain mutation were successfully constructed using p Enter-IFI16 as a template,and both of them were used to confirm the importance of IFI16 functional domain in HBV replication and signal transduction.p BS-HBV1.3 was transfected into Hep G2 cells in a dosedependent manner,and the expression of IFI16 was detected by Western Blot to explore the effect of HBV on it.Results: Observe the effect of IFI16 on HBV when it was overexpressed in Hep G2 cells,it was found that IFI16 can inhibit the expression of HBV-associated antigen and the level of HBV m RNA.The experiment of IFI16 and STING plasmids co-transfection revealed a synergistic effect between them on HBV inhibition.RT-q PCR showed that the STING pathway could be activated by HBV stimulation.And then we detected the complex of IFI16 and STING through Co-IP,indicating that there is an interaction between them.Consistent with the results observed in Hep G2 cells above,overexpression of IFI16 can inhibit the levels of HBV antigens and m RNA and can activate innate immune response in Hep AD38 cell lines which stably expressing HBV.The infection efficiency of HBV can reach more than 80% in Hep G2-NTCP cells,and the overexpression of IFI16 can also inhibits the expression of HBV antigens in HBV infection model.The inhibitory effects of IFI16 on HBV antigens and m RNA decrease when its HIN and PYD domains are mutated,and it can hardly activate the STING pathway.We also observed that the levels of IFI16 were significantly different in Hep G2 and Hep AD38 cell lines.Furthermore,the expression of IFI16 was inhibited along with the increase of HBV replication in Hep G2 cells.Conclusions: IFI16 can inhibit the replication and expression of HBV,and induce the production of IFN-? by synergizing STING signal transduction pathway to exert antiviral immunity effects,which may be related to the HIN and PYD domains of IFI16.In addition,bulk replication of HBV can induce the inhibitory effect of IFI16,which may lead to the mechanism of immune escape of HBV. |
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