HBcAg has been expressed in E.coli were it assembles in the bacterial cytoplasm into icosahedral capsids, which contain bound host nucleic acid. Deletion of the polybasic C-terminal 34 residues (protamine domain) also produces assembly competent protein. The capsids from C-terminal truncated protein (Cp149) do not contain nucleic acid and their structure has been previously determined by cryo-electron microscopy and image analysis and by X-ray crystallography. Native HBeAg is also C-terminally truncated at position 149 and in addition contains a 10 residue N-terminal extension derived from partial processing of precursor protein (pre-C). Although detailed knowledge of the function and structure of HBeAg are unknown it has clinical importance as a serological marker.? Using surface plasmon resonance (Biacore) to measure antibody antigen interactions, a kinetic-affinity map of a panel of monoclonal antibodies (mAbs) against HBV nucleocapsid proteins was determined. Monoclonal antibodies binding to the assembled (HBcAg) and non-assembled forms of the capsids (HBeAg) were identified and new combinations useful for clinical diagnosis described. Previous, structural determinations of nucleocapsid-antibody complexes by cryo-electron microscopy helped to more clearly explain the immunological distinction between the assembled HBcAg and unassembled HBeAg antigen. This work was extended by preparing immune complexes of HBeAg suitable for crystallization. This will potentially lead to detailed structural information on the secreted from of the HBV capsid protein. In addition, the structure of an immune complex of capsids and an antibody representative of the surface immunoglobulin from naive B-cells was determined. The binding of HBV capsids to these naive B-cells results in their activation and T-cell interaction. The binding involves a novel interaction involving Fab framework sequences and may be a mechanism whereby the virus down-regulates the cytotoxic T-cell response. This work represents the first structural model of a naive, low-affinity, receptor immunoglobin binding to a viral capsid the first step in the process of antigen internalization and B cell activation. This work may have therapeutic applications. ? ? Knowledge of the biophysical properties of the HBV capsid protein is limited due to the size of the multiprotein megadalton complex. In collaboration with Albert Heck (Utrecht University) macromolecular tandem and ion mobility mass spectrometry was used to study the stability and conformational diversity of HBV capsids. Very precise mass measurements were made and the exact molecular stoichiometries of HBV nucleocapsid complexes determined for the first time. The prospect of characterizing other viruses and macromolecular assemblies both native and synthetic - in a similar manner opens up a new horizon in biomolecular analysis. Examples include the study of HBV assembly and the detection of minor proteins that may be present in only a few copies per virion but are nevertheless of functional importance.
