HBcAg (resides 1-183) has been expressed in E.coli were it assembles in the bacterial cytoplasm into icosahedral capsids. Deletion of the polybasic C-terminal 34 residues (protamine domain) produces assembly competent protein (residues 1- 149) which is suitable for structural analysis. The structure of the capsids has been previously determined by cryo-electron microscopy and by X-ray crystallography. The closely related HBeAg is a secreted soluble protein which is thought to modulate both the innate and adaptive immune responses so as to favor persistent or chronic infection as well being an important clinical marker. This protein is truncated at position 149 and in addition contains a 10 residue N-terminal extension derived from partial processing of precursor protein (residues (-10) to 149). The HBeAg-specific Fab e6, was found to form a stable complex with the recombinant produced HBeAg protein. The immune complex was crystallized and the structure of HBeAg was determined. The structure clarifies how the short N-terminal propeptide (10 residues), locked into place through formation of intramolecular disulfide bridges, induces a radically altered mode of dimerization relative to HBcAg (140 degree rotation). This modified structure precludes capsid assembly and forms a distinct antigenic repertoire, explaining why HBcAg and HBeAg are cross-reactive at the T-cell level (through sequence identity) but not at the B-cell level (through conformation). Previous structural determinations of nucleocapsid-antibody immune complexes by cryo-electron microscopy were performed with a panel of murine antibodies. This work has been extended to included human antibodies from clinical samples. The results indicate binding to distinct regions on the capsid surface (epitopes) which we had previously identified using the murine antibodies. New studies have focused on specific antibody binding to HBcAg capsids which may contribute to acute liver failure (ALF). The monoclonal antibody E1 Fab was generated from previous work (R.H. Purcell, NIAID) based on immunological and molecular biological studies of tissue from patients with ALF. The E1 Fab binds with high affinity to HBcAg core particles in a unique tangential manner. The binding orientation of the Fab to the HBcAg particles may cluster the membrane bound B-cell receptors, leading to the immunological crisis associated with ALF.

Project Start
Project End
Budget Start
Budget End
Support Year
17
Fiscal Year
2012
Total Cost
$802,171
Indirect Cost
Name
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Department
Type
DUNS #
City
State
Country
Zip Code
Bereszczak, Jessica Z; Watts, Norman R; Wingfield, Paul T et al. (2014) Assessment of differences in the conformational flexibility of hepatitis B virus core-antigen and e-antigen by hydrogen deuterium exchange-mass spectrometry. Protein Sci 23:884-96
Bereszczak, Jessica Z; Havlik, Marlene; Weiss, Victor U et al. (2014) Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry: morphology selective binding of Fabs to hepatitis B virus capsids. Anal Bioanal Chem 406:1437-46
DiMattia, Michael A; Watts, Norman R; Stahl, Stephen J et al. (2013) Antigenic switching of hepatitis B virus by alternative dimerization of the capsid protein. Structure 21:133-142
Bereszczak, Jessica Z; Rose, Rebecca J; van Duijn, Esther et al. (2013) Epitope-distal effects accompany the binding of two distinct antibodies to hepatitis B virus capsids. J Am Chem Soc 135:6504-12
Wu, Weimin; Chen, Zhaochun; Cheng, Naiqian et al. (2013) Specificity of an anti-capsid antibody associated with Hepatitis B Virus-related acute liver failure. J Struct Biol 181:53-60
Kandiah, Eaazhisai; Watts, Norman R; Cheng, Naiqian et al. (2012) Cryo-EM study of Hepatitis B virus core antigen capsids decorated with antibodies from a human patient. J Struct Biol 177:145-51
Watts, Norman R; Conway, James F; Cheng, Naiqian et al. (2011) Role of the propeptide in controlling conformation and assembly state of hepatitis B virus e-antigen. J Mol Biol 409:202-13
Uetrecht, Charlotte; Watts, Norman R; Stahl, Stephen J et al. (2010) Subunit exchange rates in Hepatitis B virus capsids are geometry- and temperature-dependent. Phys Chem Chem Phys 12:13368-71
Watts, Norman R; Vethanayagam, Joe G; Ferns, R Bridget et al. (2010) Molecular basis for the high degree of antigenic cross-reactivity between hepatitis B virus capsids (HBcAg) and dimeric capsid-related protein (HBeAg): insights into the enigmatic nature of the e-antigen. J Mol Biol 398:530-41
Watts, Norman R; Cardone, Giovanni; Vethanayagam, Joe G et al. (2008) Non-canonical binding of an antibody resembling a naive B cell receptor immunoglobulin to hepatitis B virus capsids. J Mol Biol 379:1119-29

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