The surface antigen (HBsAg) of hepatitis B virus (HBV) is the major coat protein of the virion. It is essential for infectivity and the target of neutralizing antibodies produced in response to infection. Unlike most viral coat proteins, HBsAg is unusual in being a secretory protein. Previous studies have shown that the major polypeptide (p24) of HBsAg is secreted without cleavage of N. terminal amino acid sequences. In addition, we and others have shown that several larger polypeptides (p31 and p40) related to HBsAg are synthesized and secreted in vivo. We propose to examine the mechanism by which p24 is secreted, and to determine if sequences important in p24 secretion also affect p31 and p40 export. Transport of HBsAg polypeptides will be studied in an in vitro translation system programmed with mRNA transcribed from recombinant plasmids bearing the HBsAg coding region downstream of a phage SP6 promoter. Translation of this synthetic mRNA in the presence of added microsomal membranes results in transmembrane transport and glycosylation of the protein. In-phase deletion mutants located throughout the gene will be tested to determine which sequences contain topogenic signals. We will also create gene fusions between p24 and a nonexported protein (chimpanzee alpha-globin) to further search for sequences which will direct protein translation. Mutations which ablate p24 secretion will be crossed onto plasmids encoding p31 and p40 and their effects on the export of these proteins also assayed in vitro. Tests of the secretory phenotype of these mutants in vivo will be performed by examination of mouse L cells stably transformed by mutant DNA's. The impact of the mutations on self-assembly of HBsAg polypeptides will be determined by equilibrium density centrifugation in CsCl. Finally, the impact of secretory mutants on viral morphogenesis and replication in vivo will be examined in an animal model of HBV, the ground squirrel hepatitis virus (GSHV).
| Simon, K; Lingappa, V R; Ganem, D (1988) Secreted hepatitis B surface antigen polypeptides are derived from a transmembrane precursor. J Cell Biol 107:2163-8 |
| Ganem, D; Varmus, H E (1987) The molecular biology of the hepatitis B viruses. Annu Rev Biochem 56:651-93 |
