TRIM22 Inhibits Hepatitis B Virus Gene Expression And Replication Via Transcriptional Repression

Hepatitis B virus(HBV) is an important human pathogen that can cause severe liver diseases,including hepatitis and hepatocellular carcinoma.Worldwide,it is estimated that over 350 million persons are persistently infected with HBV.But until now,there is no efficient therapeutic measure for this infection.The genome of HBV is a 3.2 kb circular and partially double-stranded DNA molecule,carrying four genes named the C, S,X,and P genes.The C gene codes for the core antigen,which forms the nucleocapsid, and the e antigen,which may play an immunoregulatory role during viral infection.The S gene codes for the surface antigens that presumably function in the attachment of the virus to cell surfaces.The X gene codes for a transcriptional transactivator,and the P gene codes for the viral DNA polymerase,which is also a reverse transcriptase.The expression of these viral gene products is controlled by four promoters:two surface promoters,one core promoter,and one X promoter.TRIM22,also named Staf50(stimulating transacting factor,50 kDa),is a member of the tripartite motif(TRIM) family of proteins which contain RING,B-box,and coiled-coil domains in an ordered arrangement from N-terminus to C-terminus.TRIM proteins are involved in diverse cellular processes,including cell apoptosis,proliferation, differentiation,and transcriptional regulation etc.In recent years,some TRIM proteins are found to play important roles in antiviral processes,such as TRIM5α,TRIM19 (PML),TRIM25 and TRIM28 etc.It has also been reported that many TRIM proteins can be upregulated by interferons,supporting their potential role as effectors in the anti-viral cellular response.Thus,it has been speculated that the TRIM proteins may represent a new and widespread class of antiviral molecules involved in innate immunity. Intrahepatic gene expression analysis of acutely infected chimpanzees has shown that the TRIM22 is among the genes that are induced in the liver in correlation with HBV clearance.However,which factors contribute to the induction of TRIM22 in liver,and the role of TRIM22 in the HBV clearance remains unclear.In this study,we will investigate the induction of TRIM22 in hepatocyte-derived cell lines,the anti-HBV ability of TRIM22 in vitro and in vivo and the anti-viral mechanisms of TRIM22.PartⅠThe induction of TRIM22 in hepatocyte-derived cell lines by interferons and p53It is well established that the HBV gene expression and replication can be suppressed efficiently by interferon through a noncytopathic mechanism.To investigate whether typeⅠand typeⅡinterferon can upregulate the TRIM22 expression in hepatocytes,dose response and time course experiments were performed in HepG2 cells, a human hepatocyte-derived cell line,using quantitative PCR.The expression of the TRIM22 gene was low in quiescent HepG2 cells,but was up-regulated significantly by both IFN-αor IFN-γin a dose-dependent manner,although IFN-γwas revealed to be a relatively weaker inducer.Maximal TRIM22 expression induced by IFN-αis at about 24 h,however the induction of TRIM22 by IFN-γis time-dependent.To further confirm this phenomenon,TRIM22 mRNA expression was analyzed in another human hepatocyte-derived cells,Huh7 cells.Similar phenomenon was observed:the mRNA expression of TRIM22 in Huh7 cells was barely detectable without stimulation,but was highly up-regulated in the presence of 1000 U/ml IFN-αor IFN-γfor 24 hours,and IFN-αwas showed to be a relatively stronger inducer.To investigate whether interferon can modulate the TRIM22 expression at the protein level,a polyclonal anti-peptide antibody against TRIM22 was prepared.Western blot analysis showed that TRIM22 expression can be upregulated by both IFN-αand IFN-γin HepG2 cells.TRIM22 is a p53 target gene in some cell lines,and p53 has been shown to be able to inhibit the HBV replication efficiently.In order to investigate whether p53 can upregulate the TRIM22 expression in hepatocytes,we treated HepG2 cells with UV irradiation to induce the expression of endogenous p53,or transfected HepG2 cells with pCMV-p53 to investigate whether overexpression of p53 proteins could stimulate the TRIM22 gene expression.Our results showed that the expression of TRIM22 was upregulated significantly in the presence of both endogenous and exogenous p53 stimulations. PartⅡThe effect of TRIM22 on the HBV gene expression and replicationAs both interferons and p53 could inhibit the HBV gene expression and replication significantly,the interferon-inducible p53 target gene TRIM22 was then examined for its anti-viral activity against HBV by cotransfecting HepG2 cells with TRIM22 expression plasmid and HBV replication competent plasmid.ELISA was used to determine the HBsAg and HBeAg level in the supernanats of cultured cells,Northern blot was used to detected the HBV-RNA transcripts and Southern blot was used to detected the HBV replicative intermediates.Our results showed that overexpression of TRIM22 proteins resulted in a significant reduction of HBsAg and HBeAg secretion:HBsAg was reduced by about 75%and HBeAg by about 60%on day 3 posttransfection.A dose-response analysis showed that TRIM22 could inhibit HBV protein synthesis in a dose-dependent manner.As the inhibition of HBV protein synthesis by TRIM22 could be due to a reduction of viral transcript synthesis,Northern blot was performed to detect the HBV transcripts.As anticipated,the Northern blot analysis showed that overexpression of TRIM22 resulted in a dramastic reduction of HBV transcripts.To determine whether the decrease in viral protein expression and RNA transcription was associated with a change to HBV DNA replicative intermediates,Southern blot was performed to detect the viral replicative intermediates 3 days posttransfection.The results showed TRIM22 could suppress HBV replication significantly.To rule out the possibility that the reduction of HBV protein synthesis is due to the toxity for the transfected cells,the morphology and cell count of transfected cells were examined under microscopy;and the total protein concentration in transfected cells was measured by a bicinchoninic acid(BCA) method. We found that there were no difference in the cell morphology,the cell count and the total protein concentration between TRIM22-transfected cells and control cells.Because TRIM22 showed substantial inhibition on HBV gene expression and replication in cell culture model,further experiments were used to examine whether TRIM22 could also display HBV inhibition in vivo.TRIM22 expression plasmid and HBV replication competent plasmid were co-delivered to mouse liver by hydrodynamic injection.In concordance with findings from cell culture experiments,the HBV protein synthesis was suppressed significantly by TRIM22 in mice.Serum HBsAg in mice treated with TRIM22 was reduced by about 65%and HBeAg by 60%on day 4 when compared with mice without TRIM22 treatment.Immunohistochemical analysis showed that TRIM22 could inhibit the HBcAg expression in mice significantly and Northern blot analysis showed that TRIM22 could suppress the HBV-RNA synthesis dramastically.To rule out the possible toxity contributed by TRIM22,alanine aminotransferase(ALT) levels in sera of mice were determined.No difference in ALT levels was observed between the empty vector treated mice and the TRIM22 expression vector treated mice.As it has been reported that the N-terminal RING domain and the C-terminal SPRY domain are especially important for the proper function of some TRIM proteins, the contribution of RING domain and SPRY domain of TRIM22 to the inhibition of HBV gene expression were examined.A Cysteine to alanine changes(C15A) were introduced into the RING domain of TRIM22 by using the Quick-change mutagenesis kit, and TR1M22 without the spry domain(TRIM22-Δspry) was generated by PCR.Then, pTHBV-1 was cotransfected into HepG2 cells with pTRIM22-WT,pTRIM22-C15A and pTRIM22-Δspry respectively,and the HBsAg and HBeAg in the supernants of transfeted cells was determined 3 days posttransfection.The results showed that a mutation that converts one conserved cysteine of the RING domain to alanine(C15A) or the deletion of SPRY domain will lead to the lose of the anti-HBV ability of TRIM22,which indicated that TRIM22 may serve as an integrated functional structure,rather than a collection of separate modules.To rule out the failure of TRIM22-C15A and TRIM22-Δspry to inhibit the HBV gene expression was not due to the reduced protein expression level,Western blot was performed to determine the expression levels of TRIM22-WT,TRIM22-C15 A and TRIM22-Δspry.The result showed that the protein expression level of TRIM22-C15A and TRIM22-Δspry was comparable to that of TRIM22-WT.PartⅢThe anti-HBV mechanisms of TRIM22To investigate whether TRIM22 can inhibit the activities of HBV promoters,we first constructed four HBV promoter dependent luciferase report vectors(CpLUC, SpLUC,XpLUC and PS(1)pLUC).Then,the wild-type or mutant TRIM22 expression plasmid was cotransfect with the four HBV promoter dependent luciferase reporter plasmids into HepG2 cells,respectively.After transfection for 48 h,cells were harvested with the addition of cell iysis buffer and the luciferase assay was performed.We founded that TRIM22 could downregulate the activity of four HBV promoters to different extents: the surface promoter downregulated by about 75%,core promoter by about 65%,pre-S promoter by about 42%and X promoter by about 28%,while the TRIM22 RING domain cysteine mutants and SPRY domain deletion mutants lost this ability.These data indicate TRIM22 can inhibit the HBV gene expression at the transcriptional level.As it is reported that some cytokines,such as TNFαand interferon can also downregulate the activity of HBV promoter and the activation of NF-κB or IFN-βsignaling pathway is essential for the production of these proinflammatory cytokines,we examined the effect of TRIM22 on the NF-κB and IFN-βpromoter activity.To determine whether TRIM22 could induce NF-κB promoter activity,NF-κB promoter dependent luciferase plasmid was transfected into HepG2 cells along with TRIM22 or RIG I(2CARD) expression plasmid,respectively.After transfection for 42 hours,the medium in TNF-αcontrol wells was replaced with fresh medium supplemented with 1000 U/ml of TNFα.After cultured for additional 6 hours,cells were harvested with the addition of cell lysis buffer and the luciferase assay was performed. The results showed that the NF-κB promoter activity couldn't be induced by TRIM22, but could be upregulated about 15.5 fold by TNF-αand about 2.1 fold by RIG I (2CARD),which suggesting that TRIM22 coul not activate NF-κB signaling pathway.To investigate the effects of TRIM22 on the activity of IFN-βpromoter,IFN-βpromoter-luciferase reporter construct was transfected into HepG2 cells along with TRIM22 or RIG I(2CARD) expression plasmid,respectively.After transfection for 48 hours, cells were harvested with the addition of cell lysis buffer and the luciferase assay was performed.The results showed that TRIM22 didn't induce the IFN-βpromoter activity in HepG2 cells,while RIG I(2CARD) can upregulate the IFN-βpromoter activity in HepG2 cells by about 13.5 fold,suggesting that TRIM22 could not activate IFN-βsignaling pathway.These results indicate that the inhibition of the activities of HBV promoters mediated by TRIM22 may not be due to the increased production of proinflammatory cytokines,such as TNF and IFN.In many instances the intracellular localization of TRIM proteins was found to be crucial for their appropriate biological function.To investigate the intracellular localization of TRIM22 in HepG2 cells,pTRIM22-WT,pTRIM22-C15A and pTRIM22-Δspry was transfected into HepG2 cells using using the calcium phosphate precipitation method.After transfection for 48 hours,indirect immunoflurence analysis was used to determine their intracellular localization.The results showed that TRIM22-WT and TRIM22-C15A were mainly localized to the nuclei of HepG2 cells; however,TRIM22-Δspry was localized exclusively to the cytoplasma of HepG2 cells, indicating SPRY domain is crucial for the correct intracellular localization of TRIM22. In order to investigate the intracellular localization of endougenous TRIM22 in HepG2 cells,the cytoplasmic and nuclear extracts of HepG2 cells were prepared.Then, Western blot was performed to detect endogenous TRIM22 using anti-TRIM22 antibody. It was also found that endogenous TRIM22 was also mainly localized to the nuclei of HepG2 cells,which may be in agreement with the role of TRIM22 to act as a transcriptional suppressor.In summary,TRIM22 was found to be upregulated by interferon or p53 in human hepatocyte-derived cell line,and this interferon inducible and p53 target gene can inhibit HBV gene expression and replication efficiently.TRIM22 can interfere with HBV promoter activity efficiently,and TRIM22 proteins mainly localized to the nuclei of HepG2 cells,which suggesting that TRIM22 may act as a transcriptional repressor in its anti-viral activity against HBV.