Construction Of HBV CccDNA Model And Interaction Proteomics Analysis

Hepatitis B virus(HBV)is a member of the Hepadnaviridae family with strict host species specificity and hepatocellular tropism,leading to acute or chronic hepatitis,liver cirrhosis and even hepatocellular carcinoma.Statistical data from World Health Organization illustrated that 296 million people worldwide were chronically infected with HBV in 2019,and about 820,000 people died from liver cirrhosis and liver cancer caused by HBV infection.Despite of the application of the effective HBV vaccine,there is no efficient treatment that can completely eliminate the virus in patients with HBV infection.Therefore,it is of great translational value and social significance to further elucidate the pathway and regulatory mechanism of HBV replication in hepatocytes and to seek the potential intervention targets.Covalently closed circular DNA(cccDNA)is the only template for HBV transcription and the reservoir of virus persistence due to its high stability and half-life of several months to years.Therefore,probing into the biological characteristics of HBV cccDNA and its interaction with host factors might provide a potential target for clearing up HBV infection.However,the abundance of cccDNA in HBV infected hepatocytes is extremely low,and the structure and composition of cccDNA microchromosomes are very similar to the host cell genome,which makes it difficult to screen the host factors interacting with cccDNA under natural life infection.Although HBV plasmids have been widely used in the study of virus replication and regulation,these plasmids usually carry exogenous sequences and can not well simulate the natural infection state.In the present study,we established minicircle plasmid containing HBV genome to support HBV replication and produce cccDNA,and initically screened the host proteins interacting with cccDNA.Methods and results1.Construction and validation of mMC-HBVTo construct the minicircle plasmid containing HBV genome,we first used HBV prcccDNA as a template to amplify complete HBV genome,which was then ligated with skeleton part required of pCMV-MC to form pmini-HBV parent.pmini-HBV parent was transformed into ZYCY10P3S2T strains and then the recombination was induced by arabinose to produce the minicircle plasmid MC-HB V.However,transfection of the original version of MC-HBV did not produce the spliced pgRNA and the nascent cccDNA.Analysis of RNA structure showed that the secondary structure of intron region was a hairpin structure,not a simple rod-like structure.We then optimized the secondary structure of intron sequence by adding some extra sequences to construct modified HBV minicircle plasmid(mMC-HBV).After transfection of mMC-HBV plasmid into hepatocellular carcinoma cell lines,high levels of HBsAg and HBeAg were detected in the supernatant,and spliced pgRNA and nascent cccDNA were also produced in the cells.More importantly,the progeny viruses produced by mMC-HBV transfected cells could successfully infect Huh7-NTCP and HepG2-NTCP cells.These results suggest that mMC-HBV can support HBV replication in vitro.2.mMC-HBV produces cccDNA with typically epigenetic characteristicsIt is well-known that epigenetic modification of cccDNA is critical for cccDNA transcription.Thus,we turned to detect the epigenetic status of HBV cccDNA.We transfected mMC-HBV into HepG2 cells or infected HepG2-NTCP cells with the supernatant from mMCHBV transfected cells.ChIP-qPCR assay,disclosed that acetylated or methylated histones and acetyltransferases was enriched in cccDNA.Over expression of HBx enhanced the enrichment level of AcH4 in cccDNA,while IFN-α treatment showed an inhibitory effect.These results suggest that mMC-HBV can produce cccDNA with epigenetic characteristics.3.Establishment of mMC-HBV mouse modelFor establishing an in vivo HBV model,C57BL/6 mice were hydrodynamically injected with different doses of mMC-HBV,and blood samples were collected weekly’ to detect serum HBsAg levels.Results showed that high levels of HBsAg could be detected in mice for 4 weeks after injection.The results indicate that mMC-HBV can be used to establish acute HBV infection model.4.Biotinylated labeling of mMC-HBV and establishment of pull-down systemIn order to screen the candidate interaction molecules of cccDNA,we labeled mMC-HBV minicircle plasmids with biotin in vitro.Then,Biotin-mMC-HBV was pulled down with streptavidin-coupled magnetic beads.Dot blot assay verified that the minicircle plasmids were successfully labeled.5.Screening of cccDNA interaction proteins by using Biotin-mMC-HBVNext,we incubated Biotin-mMC-HBV,non-labeled mMC-HBV,or linearized BiotinmMC-HBV with HepG2 nuclear proteins,pull-down and mass spectrometry assay were performed to obtain the potential binding proteins with cccDNA.It was found that the pulled-down proteins were significantly enriched in DNA replication,chromatin remodeling,gene expression regulation,epigenetic regulation,virus life cycle and other biological processes.Further protein interaction analysis revealed a chromosomal separation interaction network consisting of six subunits of the mucin complex(SMC3,SMC1A,RAD21,PDS5A,PDS5B and STAG2)and two key regulatory molecules(NIPBL and WAPL).These results suggest that Biotin-mMC-HBV can be used to study the interaction proteomics of cccDNA.Innovations and significancesIn the present study,we construct a minicircle plasmid model of HBV cccDNA to simulate the natural infection state,and verify that it can support HBV replication both in vitro and in vivo.Moreover,HBV minicircle plasmid model can be applied for screening cccDNA interacting proteins,providing a useful tool for further delineating HBV replication process and screening antiviral therapeutic strategies.