Isolation Of Cellular Specific Receptors For Hepatitis B Virus Envelope Protein And Identifaction Of Its Binding Site To The Pres1 Domain

The life cycle of virus is belived to begin when virus attatches to the host cell membrane via its envelope proteins. The process of the attatchment and the following the fusion of a viral envelope and cell membrane is thought to be very important for its successfully infecting host cells. Receptors on host cell membrane have the characters of specificity, high affinity, limited binding sites and other biological effects. Identifaction of the cellular macromolecules responsible for virus attachment is useful to understand its life cycle and pathogenesis, to take measure of prevention and therapy of virus disease.Hepatitis B virus (HBV) infection is a major worldwide public health problem. Despite considerable advances in the understanding of natural history of HBV disease, the process of its replication and antigenic structure, most of the early steps in the virus life cycle remain unclear for lack of an in vitro infection system. HBV attatching to permissive cells, fusing and penetrating through cell membranes and releasing subsequent genome are largely a mystery. Current knowledge on the early steps of HBV infection shows that HBV binding to the special recptors on human hepatocyte membrane via preS1 domain of large surface protein (LHBs) and then fusing with cell membrane trigger the viral infection. Thus the special recptors binding to HBV is thought to be a potential target for the development of novel antiviruals. Though many cellular molecules have been identified as putative receptors for HBV attatchment, the cellular protein that is responsible for this binding has not been found yet.In an attempt to identify a cell surface receptor for HBV, purified recombinant fusion proteins of preS1 domain of HBV with glutathione S- transferase (GST) and FLAG tags were expressed in prokaryon cells and eucaryou cells, and then used as ligands. The results of the bionomics study of the fusion proteins showed the recombinant proteins had good antigenicity and immogenicity, and had the capacity of inhibiting the binding of HBV virion to anti-preS1 and specific binding to HepG2. It is suggested that the recombinant proteins mimic native functions of preS1 in HBV particles and may be valuable candidates for studying the receptors of HBV permissive cells. An approximately 110-kDa protein (p110) on human hepatoma cell line HepG2 plasma membrane was shown to bind specifically to the preS1 domain of the fusion proteins by pull down test and co-immunoprecipitation. Analysis of the tissue and species specificity of p110 expressed in several human cell lines of different tissue origin and primary cultured mouse hepatocytes showed that p110 expression was restreicted to human hepatoma cell line HepG2. The receptor binding assay using serially or internally deleted segments of preS1 showed that amino acid residues 21-33 was essential for the binding of preS1 to p110. And DMSO treated HepG2 did not increase the expression of p110.Taken together the results suggest that p110 may be a crucial molecule in the viral entry machinery.