| Background: Hepatitis B virus(HBV)infection is the most important cause of liver cirrhosis and hepatocellular carcinoma in humans,which is a serious threat to human health.Covalently closed circular DNA(cccDNA)of HBV is the only viral replication template,which cannot be effectively inhibited and cleared by current treatments of type I interferon and nucleotide analogues for HBV infection.And the persistent transcription of cccDNA is the root cause that current treatments rarely lead to cure.Thus,there is an urgent need to study the mechanisms of HBV cccDNA formation,transcription,stability,and to develop highlypotent drugs that inactivate or clear cccDNA.Recently,the structural maintenance of chromosomes 5/6 complex(Smc5/6)was identified as a host restriction factor of HBV.HBV replication and cccDNA transcription were inhibited by Smc5/6,however HBV antagonizes this restriction by expressing HBx which targets Smc5/6 for degradation via the Cullin-4 RING ubiquitin ligase complex(CRL4)depending on DNA damage-binding protein 1(DDB1).The mechanisms of how Smc5/6 inhibits the transcription and replication of HBV and how HBx interacts with Smc5/6 and whether HBx interacts with Smc5/6 in a DDB1-independent manner remain elusive.Objective: In this study,we will investigate the interaction of Smc5/6-HBx-DDB1 axis in detail,and study the effect of blocking HBx-Smc5/6 interaction on HBV replication,and explore the molecular mechanism of Smc5/6 inhibition of cccDNA transcription,laying the foundation for the development of drugs that functionally cure HBV.Methods: The interaction of Smc5/6-HBx-DDB1 axis was investigated by in vitro binding assay,alanine scanning mutagenesis strategy and Co-immunoprecipitation.The effects of mutation of the key residues of HBx that interact with Smc5/6 on degradation of Smc5/6 and transcription of extrachromosomal luciferase reporter genes in hepatoma cells,as well as on HBV replication in hepatoma cells and in mice model.The effects of Smc5/6 on the epigenetic modifications of HBV cccDNA were examined by chromatin immunoprecipitation assay.The Smc5/6 complex was expressed and purified by Insect Baculovirus Expression Vector System,and the effects of Smc5/6 on circular DNA transcription were detected by in vitro transcription and translation system.Results: HBx interacts with multiple subunits of Smc5/6 in a DDB1-independent manner in vitro pull-down assay.The HBx C-terminal is sufficient to interact with Smc5/6.Residue F132 of HBx is critical for interaction with Smc5/6.The reaction kinetics of Smc5/6 degradation by HBx exhibited a “hook effect”.HBx F132 A mutation impaired the function of HBx to degrade Smc5/6 and to stimulate the extrachromosomal reporter transcription in hepatoma cells,and also significantly inhibited HBV replication in cells and mouse models,and F132 A mutation significantly inhibited the H3K27 ac modification of the HBV cccDNA.Smc5/6 suppresses circular DNA transcription in vitro.Conclusion: Collectively,this study identifies a key residue F132 on HBx for Smc5/6 interaction and HBV replication.Based on these findings,drugs that disrupt the HBx-Smc5/6 interaction will be potential for achieving functional cure of HBV infection.The HBx peptide emcompassing residue F132 will be potential therapeutic agents for HBV.The reaction kinetics of Smc5/6 degradation by HBx exhibited a “hook effect”,indicating that HBx can degrade Smc5/6 efficiently by this way.The HBx F132 A mutant inhibited the H3K27 ac modification of the HBV cccDNA,suggesting that Smc5/6 may inhibit cccDNA transcription by regulating the epigenetic modifications of cccDNA.HBx F132 A will lay the foundation for establishing a model to study the mechanism of HBV replication regulated by HBx and Smc5/6 complex. |
