TRIM21 Promotes Ubiquitination Of HBV DNA Polymerase And Inhibits HBV Replication

Objective:Hepatitis B virus(HBV)infection is a major health problem in the world,which can cause severe liver diseases,such as chronic hepatitis,cirrhosis,fibrosis and is also a risk factor for hepatocellular carcinoma.accounting for about 800%.HBV is a small hepatotropic DNA virus with envelope,whose genome is partially double stranded,relaxed,circular DNA.But it replicates via reverse transcription,which is completed by HBV DNA polymerase(HBV DNA Pol).HBV DNA Pol initiates the replication of HBV by recognizing and binding to ε element of HBV pregenomic RNA(pgRNA),and formation of complex packing into the nuclecapsid with pgRNA,then catalyzes the reverse transcription of pgRNA into DNA with its reverse transcriptase activity,it also has the activity of DNA polymerase,completes the formation of double strand of HBV DNA.Therefore,the study on the regulation of HBV DNA Pol is helpful to understand the process of HBV replication.The tripartite motif protein(TRIM)family has three conserved domains(RING,B-box and coil-coil domain)and a variable C terminal.The members of TRIM family participate in many biological processes,such as cell differentiation,proliferation,development,apoptosis and so on.Part of TRIM proteins have antiviral functions,especially for retroviruses,they are also involved in the innate immune pathways.As a member of TRIM family,TRIM21 is an intracellular IgG Fc receptor.It degrades virus particles rapidly through ubiquitination and degradation of target protein,which provides a kind of antibody dependent,non cytotoxic mechanism on virus.In this study,we found that TRIM21 may be one of the proteins interacting with HBV DNA Pol,and affected the expression of HBV DNA Pol by affinity purification and mass spectrometry.However,there are few researches on the impact of TRIM21 on HBV replication,the purpose of this study is to explore the role and mechanism of TRIM21 in HBV replication.Method:First,we utilized the affinity purification and mass spectrometry to screen proteins that might interact with HBV DNA Pol,identified TRIM21 as the candidate through querying gene functions and related literatures on Gene and Pubmed websites,then constructed its over expression and knockdown plasmid.The interaction and subcellular localization between HBV DNA Pol and TRIM21 were confirmed by CoIP and the image analysis of immunofluorescence combined confocal microscopy.Western blot was performed to examine the effect of TRIM21 on the expression of HBV DNA Po1,lysosome and proteasome inhibitors were used to investigate the degradation pathway of HBV DNA Pol.The effects of TRIM21 and its E3 ligase activity deficient mutants on the ubiquitination level of HBV DNA Pol were detected.We investigated the effect of TRIM21 on the half-life of HBV DNA Pol with cycloheximide.UbPred and CKSAAP were used for predicting the potential ubiquitination sites of HBV DNA Pol,then we constructed the mutants and identified the sites of HBV DNA Pol that TRIM21 mediated ubiquitination.Meanwhile,in Huh7 and HepG2.2.15 cells,we analyzed the effects of TRIM21 on HBeAg and HBsAg secretion by ELISA,and RT-qPCR assay was used to study the effects of TRIM21 on HBV DNA,cccDNA,pgRNA and total RNA level.Results:The affinity purification and mass spectrometry analysis identified TRIM21 was a HBV DNA Pol-associated protein.Co-immunoprecipitation and immunofluorescence confocal microscopy showed that HBV DNA Pol can interact with TRlM21 and colocalization of HBV DNA Pol and TRIM21 in cytoplasm.TRIM21 decreased the expression level of HBV DNA Pol,but this regulation was weakened by MG132.Overexpression of TRIM21 promoted the ubiquitination of HBV DNA Pol on the K260 and K283 sites,and shortens the proteins’ half-life.But the TRIM21 mutant lacking E3 ubiquitin ligase activity losted the ability of accelerating the ubiquitination of HBV DNA Pol.TRIM21 inhibited the secretion of HBeAg and HBsAg,the expression of HBV DNA and cccDNA,and the level of pgRNA and total RNA.Conclusions:In this study,we confirmed the interaction between TRIM21 and HBV DNA Pol and found that TRIM21 promoted the degradation of HBV DNA Pol through the ubiquitin proteasome pathway,thereby reducing the protein expression of HBV DNA Pol in HCC cells and inhibiting the replication of HBV.