The Effect And Mechanism Of Nirf Protein On HBV Core Protein

ObjectiveTo study the effect of NIRF on HBc and its mechanism. To determine NIRF is an E3ligase of HBc and the interaction between NIRF and HBc in vivo. To explore whether NIRF promotes ubiquitination of HBc in vivo and the effect of NIRF on replication and secretion of HBV.Methods1. The complete coding sequence of NIRF gene was amplified from pIRES2-EGFP-NIRF by PCR using designed specific primers and inserted into eukaryotic expression vector pCMV-Myc. The constructed recombinant plasmid pCMV-Myc-NIRF was transfected into HEK293cells and expression of NIRF protein was determined by Western blotting. HBc gene was amplified from pCMV-Core by PCR using designed specific primers and inserted into eukaryotic expression vector pcDNA3. The constructed recombinant plasmid pcDNA3-HBc was transfected into HEK293cells and expression of HBc protein was determined by Western blotting. To construct plasmid pcDNA3-UB by total gene synthesis.2. The immunofluorescence assay of NIRF and HBc was used to find out the co-localization of NIRF and HBc in HEK293cells or HepG2.2.15cells.3. Immunoprecipitation was used to find out the interaction between NIRF and HBc in HEK293cells or HepG2.2.15cells.4. Degradation Kinetics analysis was performed in HEK293cells. Ubiquitination assay was used in order to verify the degradation was ubiquitination.5. NIRF was up-regulated and down-regulated in HepG2.2.15cells. Western blotting was used to detect the level of HBc. Real-time fluorescent PCR was performed to analyse replication and secretion of HBV.Result1. Both restriction analysis and sequencing proved that recombinant plasmid pCMV-Myc-NIRF, pcDNA3-HBc and pcDNA3-UB were constructed correctly.2. NIRF and HBc co-located in HEK293cells or HepG2.2.15cells.3. Immunoprecipitation was certified that NIRF binded to HBc in vivo. The level of NIRF and HBc increased when MG-132was added.4. NIRF promoted the degradation of HBc and the degradation was ubiquitination.5. The level of HBc and HBV decreased when NIRF was up-regulated. The level of HBc and HBV increased when NIRF was down-regulated. ConclusionThe eukaryotic expression plasmid pCMV-Myc-NIRF, pcDNA3-HBc and pcDNA3-UB were constructed successfully. Exogenous NIRF and HBc or endogenous NIRF and HBc co-located in vivo. NIRF is an E3ligase of HBc. It can bind to HBc and promote ubiquitination of HBc in vivo. NIRF is a negative regulator of HBc. NIRF regulates replication and secretion of HBV negatively.