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 (murine origin) 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 immune complexes by cryo-electron microscopy helped to more clearly explain the immunological distinction between the assembled HBcAg and unassembled HBeAg antigen. This work has been extended to included human antibodies and the preliminary results indicate binding to distinct regions on the capsid surface (epitopes) which we had previously identified using the model murine antibodies. This work, together our previous description of an immune complex of capsids and an antibody representative of the surface immunoglobulin from naive B-cells, provide one of the most complete structural pictures of the interaction of antibodies with a viral protein system related to an important human disease. Using a rabbit antibody library, monoclonal antibodies (mAbs) were selected then humanized to produce chimeric mAb fragment antigen binding portions (Fab). Fabs against the HBV capsid proteins were selected for high affinity binding to the capsid subunits. The binding of the antibodies prevent the assembly of the capsids (a central functional and structural component of the HBV virus) and may with development provide useful anti-HBV reagents. Direct determination 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. This work was extended to include measurements of stability and elasticity of the capsids which resulted in the detection of conformational heterogeneity in the capsid population. Also, the stability of the capsids was addressed by the direct determination of the slow exchange of constituent subunits using isotopically labeled protein. The methods and approaches used have general applicability to the study of large molecular assemblies used in nano- and biotechnology

Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2009
Total Cost
$716,592
Indirect Cost
Name
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Department
Type
DUNS #
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Country
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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
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
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
Uetrecht, Charlotte; Versluis, Cees; Watts, Norman R et al. (2008) Stability and shape of hepatitis B virus capsids in vacuo. Angew Chem Int Ed Engl 47:6247-51

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