E3 Ubiquitin Ligase TRIM21 Restricts Hepatitis B Virus Replication By Promoting Degradation Of HBx

BackgroundHepatitis B virus(HBV)infection causes acute hepatitis B,chronic hepatitis B and cirrhosis,liver fibrosis,and hepatocellular carcinoma in humans,remaining one of the most challenging public health problems in the world.There still emerge one million new Hepatitis B cases in China each year.The current available drugs,such as ?-interferon and nucleoside analog medicines,show limited effects as they can hardly clear the virus in the body.There is an urgent need for developing novel anti-HBV inhibitory molecules.Tripartite motif protein(TRIM)family member,TRIM21,has E3 ubiquitin ligase activity.It participates in a variety of cellular physiological courses.And it plays both a positive and a negative role in regulating the Type ? Interferon pathway during virus infection.It has been reported that TRIM21 limits HBV replication through its cytosolic IgFc receptor function.Whether TRIM21 possesses a direct anti-HBV activity remains unknown.PurposeTo investigate the role and molecular mechanism of TRIM21 in limiting HBV replication in hepatocytes.Method1.HBV-infected hepatocyte model.Hepatoma cell line Huh7 was transfected with 1?g pUC19-HBV1.3.Reverse transcription quantitative PCR was used to detect HBV replication intermediate,RNA,and ELISA was used to detect surface antigen(HBsAg)and e antigen(HBeAg)in the cell supernatant 48 hrs after transfection.2.Establishment of a mouse model for HBV infection.10 ?g pUC19-HBV1.3 DNA was given to C57BL/6 mice through a hydrodynamic injection.4 days later,then hepatic HBV RNA expression,serum HBsAg and HBeAg levels and HBc expression in situ were detected by qRT-PCR,ELISA and immunohistochemistry.3.Knockdown and overexpression of TRIM21.p-Flag-TRIM21 was transfected into hepatoma cells to overexpress TRIM21,and TRIM21-specific siRNA was transfected to knock down endogenous TRIM21 expression.4.Construction of domain-deleted TRIM21 plasmids.The ?RING,?B-BOX,and ?PRY/SPRY deletion mutant plasmids of TRIM21 were respectively constructed by enzyme digestion and ligation.5.TRIM21 activity on HBV replication in vitro.Various TRIM21 constructs were co-transformed with pUC19-HBV1.3 into HepG2 cells.48 hrs later,qRT-PCR was used to detect the HBV RNA.Absolute quantitative PCR was used to detect secreted HBV DNA.Nested PCR was used to detect HBV Precore-core DNA level.6.Co-immunoprecipitation of TRIM21 with HBV target proteins.Myc-tagged HBV protein contracts,p-HBx,p-HBs,and p-HBc were respectively co-transfected with p-Flag-TRIM21 into HEK293T before the SDS-PAGE assay.48 hrs later,the Flag antibody was used to determine the HBV protein targeted by TRIM217.Ubiquitin-proteasome degradation of HBV proteins by TRIM21.p-Flag-TRIM21 was co-transfected with pHBx into HEK293T cells.Or p-Flag-TRIM21,pHBx,and HA-tagged total ubiquitin plasmid were co-transfected into HEK293T.48 hrs later,cells were treated with 20 ?M proteasome inhibitor MG 132 for 4 hrs.Myc antibody was used for co-immunoprecipitation and western blot was used to detect the expression of ubiquitin.8.Transient overexpression of TRIM21 in mice and TRIM21 knockout mice development.C57 female mice were injected with 20 ?g p-Flag-mTRIM21 through hydrodynamic injection.TRIM21 homozygous knockout mice were constructed9.In vivo HBV-inhibitory activity of TRIM21.20 ?g p-Flag-mTRIM21 and 10?g pUC19-HBV1.3 were co-injected into C57 mice through hydrodynamic injection.Or pUC19-HBV1.3 was injected into TRIM21-KO mice.4 days later,serum HBeAg and HBsAg levels,hepatic HBV RNA levels were detected by ELISA and qRT-PCR.Immunohistochemistry was performed to analyze hepatic HBcAg and HBxAg expression.Results1.HBV up-regulates TRIM21 expression in hepatocytes.In hepatocytes,HBV infection induced TRIM21 upregulation at the mRNA and protein levels.2.TRIM21 potently inhibits HBV replication and expression in vitro.In Huh7 and HepG2,overexpression of TRIM21 significantly inhibited HBV RNA replication and HBeAg/HBsAg expression.Knock down of TRIM21 significantly increased HBV RNA,HBeAg,and HBsAg expression.3.The antiviral activity of TRIM21 depends on the RING domain.The ?RING and ?PRY-SPRY of TRIM21 constructs blocked the anti-HBV effect of TRIM21.4.TRIM21 interacts with HBx protein.Co-immunoprecipitation asssy confirmed that TRIM21 intensively interacted with HBx and moderately interacted with HBc.There was no interaction between TRIM21 and HBsAg.Immunofluorescent staining also confirmed the interaction of TRIM21 with HBx.5.TRIM21 degrades HBx through the ubiquitin-proteasome pathway.After co-transfection with HBV protein plasmids,TRIM21 directly inhibited HBx,but not HBc protein expression in HEK293T cells.MG132 treatment reversed the inhibitory effect.By co-transfection with ubiquitin plasmids,TRIM21 promoted ubiquitination modifications of HBx protein.6.TRIM21 markedly inhibits the HBV replication in mice.C57 mice were in vivo co-transfected with pUC19-HBV1.3 and pTRIM21 plasmids by hydrodynamic injection.4 days after that,over-expressed TRIM21 significantly reduced hepatic HBV RNA,serum HBeAg and HBsAg expression.Liver tissue immunohistochemistry revealed that TRIM21 remarkably inhibited HBcAg and HBxAg expression.Compared with WT mice,TRIM21 KO mice significantly increased HBV RNA,serum HBeAg and HBsAg,hepatic HBcAg and HBxAg expression,demonstrating a significant anti-HBV function of TRIM21 in vivoConclusion:This study clarifies the role and molecular target of E3 ubiquitin ligase TRIM21 in HBV infection of human hepatocytes.HBV infection upregulates hepatic TRIM21 expression,which potently inhibits HBV replication both in vivo and in vitro via its RING domain and the PRY-SPRY domain.TRIM21 directly interacts with HBx and degrades HBx through ubiquitin-proteasome pathway thereby inhibiting HBV replication.Our data reveal a direct antiviral activity of TRIM21 on HBV through targeting HBx.This may shed new light on the anti-HBV mechanism of TRIM21 and provides new candidate molecular targets for the treatment of Hepatitis B.