Two distinct segments of the hepatitis B virus surface antigen contribute synergistically to its association with the viral core particles

The long surface antigen polypeptide (L-HBsAg) of hepatitis B virus (HBV) i s believed to mediate contact between the virus envelope and nucleocapsid p rotein (HBcAg). The N and C termini of L-HBsAg were shortened progressively in order to define the minimum contiguous sequence of amino acids that con tains the residues necessary for association with HBcAg. The resulting muta nts were expressed in rabbit reticulocyte lysates and their interaction wit h HBcAg was examined with an immunoprecipitation assay and an equilibrium b inding assay in solution to give relative dissociation constants. Binding o f HBcAg particles by L-HBsAg displayed two widely differing dissociation co nstants, indicating two distinct binding sites between the molecules. The t wo distinct sites, one located between residues 24 and 191 and the other be tween residues 191 and 322 of L-HBsAg, contribute synergistically to high-a ffinity binding to HBcAg, but disruption of either of these segments result ed in a much weaker interaction showing only one dissociation constant. Inh ibition of the interaction by peptides that bind to the tips of the nucleoc apsid spikes differentiated contacts in HBcAg for the two binding domains i n L-HBsAg and implied that the amino-terminal binding domain contacts the t ips of the HBcAg spikes. Analysis of specific single amino acid mutants of L-HBsAg showed that Arg92 played an important role in the interaction. (C) 1999 Academic Press.