The HBV core gene codes for precore protein (pre-C) which is either partially processed to form a secreted non-particulate protein called e-antigen (HBeAg) or fully processed to produce core antigen (HBcAg). HBcAg is a 183-residue protein which assembles to form a nucleocapsid which encapsulates a RNA-reverse transcriptase complex (HBV polymerase). 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: residues 1-149) 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 pre-C. Although the function and structure of HBeAg are unclear it is an important serological marker. ? Using surface plasmon resonance (Biacore), a kinetic-affinity map of a panel of monoclonal antibodies (mAbs) against HBV nucleocapsid proteins revealed a range of binding affinities. Monoclonal antibodies binding to the assembled and non-assembled forms of the capsids were identified. These findings more clearly define monoclonal antibody combination useful for clinical diagnosis. Some of the HBV nucleocapsid-antibody complexes were characterized further by cryo-electron microscopy (A. Steven, NIAMS). The results revealed a greater number of discontinuous epitopes than had been described previously. The findings help explain the immunological distinction between the assembled HBcAg and unassembled HBeAg antigen. A review article was published in 2005 summarizing these findings. Structural studies were extended to include an immune complex of capsids and an antibody representative of the surface immunoglobulin from nave B-cells. The binding of HBV capsids to these nave B-cells results in their activation and T-cell interaction. The binding is low affinity and involves a novel interaction involving fab framework sequences and may be a mechanism whereby the virus down-regulates the cytotoxic T-cell response. ? Biophysical and structural studies have been performed on the low molecular weight HBeAg incorporating mutations which improve its biophysical characteristics, especially solubility. These mutants have been used to prepare immune complexes with monoclonal antibodies selected by phage display technology and expressed in bacteria. Immune complexes have produced crystals which may be suitable for structural determination using X-ray crystallography.? Studies on the HBV protein X, a key regulatory protein of HBV that is at the intersection of HBV infection, replication, pathogenesis, and possibly carcinogenesis. The protein appears natively disordered and we have prepared binary complexes with pyrin-related domains which have stabilized the structures suitable for structural investigation.
