The Effect Of NIRF On The Acetylation Status Of HBV CccDNA Bound-H3in HepG2Cells

Objective：NIRF (Np95/ICBP-90like RING finger protein) ICBP90molecularstructure is similar to the human and mouse NP95protein which is newlydiscovered in2002located on human chromosome3p23-24.1protein. Itincluding five Tudor, PhD, SRA/YDG and RING-finger domains, andprocessed802amino acid residues. HBV is addicted to sunset family ofDNA viruses the lysogenic virus genome was slack circulardouble-stranded structure, which contains special replication properties.The current study demonstrates that the HBV concluded the structurewhich is similar to the eukaryotic cells chromatin. It produced HBVcccDNA (covalently closed circular HBV DNA) during the replication ofthe virus, the nucleus cccDNA and histone、 non-histone proteins gettogether to form the virus micro-chromosomes, which is, the HBVreplication intermediates midbody. Early studies have shown that NIRFprotein combined with deacetylase were involved in the virus histonemodifications. And previous studies in our laboratory have confirmed thatNIRF combined with HBc (hepatitis B virus core protein) caused HBcubiquitination and degradation. To determine the effect of NIRF protein(Np95/ICBP-90like RING finger protein) on the repilcation of HBV andthe acetylation of cccDNA-bound H3histone at various times aftertransient transfection of linear HBV DNA into human hepatoma HepG2 cells.Methods：HepG2cells were infected with FLAG-NIRF、 pGEM-HBV1.3plasmids. The transfection of HepG2cells was completed withLipofectamine2000following the manufacturer as a protocol. Eachtransfected cell culture supernatant HBsAg and HBeAg secretion wasdetected by ELISA at12hours,24hours,48hours and72hours. Theexpression of HBV mRNA were examined by RT-PCR at12h、24h、48hand72h. and NIRF protein was detected by western-blot respectivel after3days. Finally the levels of the HBV cccDNA-bound H3histone and theHBV cccDNA-bound acetylated H3histone were identified byChIP(Chromatin Immunoprecipitation) at12h、24h、48h and72h.Results:Our results demonstrated that NIRF can inhibted the antigensconnected HBeAg、 HBsAg in HepG2cells. and the acetylation ofcccDNA-bound H3histone were associated with the level of HBVreplication. NIRF can also inhibit the levels of cccDNA-bound H3andacetylated H3in HepG2cells, which may play roles on a betterunderstanding of the mechanisms of HBV and confirm a new therapeuticstrategies against hepatitis B virus.Conclusion：The results confirmed that NIRF can inhibit the replication of HBVin liver cancer cells in vitro, at the same time this inhibition may alsoparticipate in the assembly of the HBV virus in a host cell, proteindegradation, and the phenomenon of external secretion of virus. AndNIRF can also down-regulate the expression of cccDNA-bound H3and cccDNA-bound acetylated H3in HepG2cells, We’re looking forward thatNIRF can provide theoretical support for the subsequent pathogenesisresearch of HBV replication and the development of the effective antiviraldrugs.