HEPATITIS CORE ANTIGEN PRODUCED IN ESCHERICHIA-COLI - SUBUNIT COMPOSITION, CONFORMATIONAL-ANALYSIS, AND IN-VITRO CAPSID ASSEMBLY

The production and biochemical and physicochemical analysis are descri bed of recombinant produced hepatitis B virus core antigen (HBcAg caps id) and the corresponding particle produced by a deletion mutant missi ng the C-terminal 39 residues (HBeAg). Conditions for producing HBeAg from HBcAg capsids by in vitro proteolysis are also described. The mor phology and masses of these capsids were determined by scanning transm ission electron microscopy. Both HBcAg and HBeAg capsids comprise two size classes that correspond to icosahedral lattices with triangulatio n numbers (T) of 3 and 4, containing 180 and 240 subunits per capsid, respectively: This dimorphism was confirmed by sedimentation equilibri um and sedimentation velocity measurements on a Beckman Optima XL-A an alytical ultracentrifuge. More than 60% of HBcAg capsids were T = 4, w hereas only 15-20% of HBeAg capsids were of this size class: the remai nder, in each case, were T = 3. Circular dichroism and Raman spectrosc opy were used to determine the overall secondary structures of HBcAg a nd HBeAg capsids. Both have high alpha-helical contents, implying that this capsid protein does not conform to the canonical beta-barrel mot if seen for all plant and animal icosahedral viral capsids solved to d ate. We suggest that the C-terminal domain of HBcAg has a random coil conformation. In vitro dissociation of HBeAg capsids under relatively mild conditions yielded stable dimers. The reassociation of HBeAg dime rs into capsids appears to be driven by hydrophobic processes at neutr al pH. Capsid assembly is accompanied by little change in subunit conf ormation as judged by circular dichroism and fluorescence spectroscopy . The thermal stability of HBcAg capsids was compared calorimetrically with that of in vitro assembled HBeAg capsids. Both have melting temp eratures >90 degrees C, implying that the C-terminal region makes Litt le difference to the thermal stability of HBcAg: nevertheless, we disc uss its possible role in facilitating disassembly and the release of v iral nucleic acid.