Heparinsulfate-D-Glucosaminyl-3-0-Sulfotra-Nferase 3B1 Downregulates HBV Replication In HepG2 And HepG2.H7 Cells

Objective: Hepatitis B Virus replication is subjected to various factors of host cells. The aim of this study is to investagate the influence of the host factor HS3ST3B1 on HBV replication in vitro and possible mechanism underlied these actions.Methods: To detect alteration of both HBV DNA level and HBV total RNA level in HepG2 cells contransfected with HS3ST3B1 and pCH9-HBV, using Southern blot and Real-time PCR techniques; Meanwhile, to detect alteration in the level of both HBV DNA and HBV total RNA when the overexpressed HS3ST3B1 is interfered with; To detect the change of HBV DNA level when the endogenous HS3ST3B1 is interfered with and detect the level of HBV DNA in HBV stable cell line HepG2.H7,which is transfected with different dose of HS3ST3B1. In addition, activity of HBV promoters is detected when HepG2 is contransfected with HS3ST3B1 and pCH9-HBV and when the overexpressed HS3ST3B1 is knock-down through RNAi.Result: Southern blot reveals the level of HBV DNA is sharply decreased when HS3ST3B1 was contranfected with pCH9-HBV in HepG2 cells, whereas the level of HBV DNA is restored when the overexpressed HS3ST3B1 is interfered with; Meanwhile, the level of HBV DNA is upregulated when endogenous HS3ST3B1 is silenced. The same result can be abtained in HBV stable cell line. In addition, the downregulation effect of HS3ST3B1 on the HBV replication is dose-dependent. Real-time PCR data reveals HBV total RNA is obviously downregulated when HS3ST3B1 is cotransfected with pCH9-HBV in HepG2 cells, and the downregualation effect can be restored by the interference of HS3ST3B1. Dual-Luciferase?Reporter Assay System data reveal the activity of HBV promoters are not changed when HS3ST3B1 was contransfected with pCH9-HBV. The activity of HBV promoters are also not changed when the overexpressed HS3ST3B1 is interfered with.Conclusion: HS3ST3B1 has an downregulation effect on the level of both HBV DNA and HBV total RNA, but the downregulation of HBV total RNA is not achieved through inhibiting HBV promoters by HS3ST3B1. Objective: To construct methods to amplify redundant region of cccDNA and rcDNA positive strain specificly, and HBV1.1×genome which has mutation in corresponding site of redundant region, laying a foudation for the research on the removal of redundant region.Methods: 1. Extract rcDNA from culture suppernatant of HBV stable cell line HepG2, and design corresponding primers, then construct a method to specificly amplify redundant region of rcDNA positive strain; 2. Extract cccDNA from nucleus of HBV stable cell line HepG2.H7, and design corresponding primers, then construct a method to specificly amplify redundant region of cccDNA. 3. Design mutant primers which correspond to redundant region of HBV, and construct HBV1.1×genome mutant, which can cause a mutation in the redundant region of rcDNA.Result: Successfully construct methods which can specificly amplify redundant region of HBV cccDNA and rcDNA positive strain, and HBV1.1×genome mutants were also constructed. So the foundation was laid to construct mutant HBV stable cell line and identify which redundant region of HBV negetive strain is removed in the process of HBV cccDNA biogenesis.