The Preliminary Study Of Epiregulin, Fibronectin And Asgpr-Mediated Entry Of Hepatitis B Virus Into Liver Cells

Hepatitis B virus (HBV) infection is the major health concern around the world, and no effective treatment has been developed till now. The crux of the matter, limited the development of anti-HBV drugs, is due to the smaller viral genomes and the higher mutation. Several recent examples show that inhibiting host-cell proteins can prevent viral infection. The human genomes might contain more genes related to HBV infection and replication in comparing with the viral genome. A systematic approach to find these potential cellular antiviral targets is host gene expression analysis using DNA microarrays. We have previously found three molecules that are closely related to HBV infection,which is Epiregulin(EREG),Fibronectin(FN) and Asialoglycoprotein recertor(ASGPR).In this paper,We examined interaction of surface HBV antigen(sHBsAg)with this three molecules and studied the role of the three molecules in HBV infection according to DNA microarrays analysis in order to analyze the mechanism of HBV endocytosis and promote the development of anti-HBV drugs based on the interaction between HBV and host cells.Our results shows that EREG can interact with HBsAg in vivo and in vitro,and EREG can help HBsAg bind to hepatic cells. It is concluded that EREG act as a soluble and subsidiary receptor that helps HBV attach to hepatic cells and benefit viral particles enter into cells. We confirmed the interaction between HBsAg, FN and ASGPR using co-immunoprecipitation technology. To screen HBsAg binding domains of FN and ASGPR,we cloned the genes of fragments of FN and ASGPR into expression vector pGEX4T-1. The constructed expression vectors were transformed into E.coli and induced to express the recombinant proteins. After purification,the fusion proteins were used in GST-pull down experiment to screen HBsAg interactional domains.The results shows that FN may interact with HBsAg by its N-Terminus of cell binding domain and Heparinâ…¡binding domain, and interact with ASGPR by its cell binding domain with 120kD molecular mass. The results also indicate that ASGPR may interact with FN by its carbohydrate recognition domain, but it is not sure that which domain of ASGPR interact with HBsAg because the domains expressed in E.coli lack modification or the conditions used in experiment are not fit for the interaction between domains of ASGPR and HBsAg.In conclusion, we verify that EREG,FN and ASGPR can interact with HBsAg by co-immunoprecipitation technology , and screen the interactional domains between FN ,ASGPR and HBsAg by GST-pull down technology. We also analyze the role of EREG in HBV infection and binding to hepatic cells, our studies provide the evidences that EREG may function as a soluble and subsidiary receptor during HBV adhension to hepatic cells.