Abstract

The first goal of this project is to determine the structures of the surface proteins of the hepatitis B virus, referred to as the hepatitis B surface antigen (HBsAg), and to determine the relationship between their structures and functions both as essential viral proteins and as the important host targets for prevention of the viral disease. HBsAg is produced in large amounts during the normal course of HBV infection, a fact which has been extensively exploited: HBsAg serves as the basis for the diagnosis of HBV infection and also as the current vaccine against HBV. HBsAg consists of a """"""""nested"""""""" set of three proteins, referred to as the S, M, and L proteins, each of which occurs in two forms due to further post-translational modifications. The structure of these will be determined following isolation of the protein from naturally infected human plasma, or following expression of the relevant protein by cloned viral DNA in eukaryotic or prokaryotic expression vectors, using a combination of physical (circular dichroism, FTIR, mass spectrometry, ultracentrifugation), chemical (Edman degradation, carboxypeptidase digestion, reduction and alkylation), and immunological (monoclonal antibody binding studies, studies of immunogenicity) techniques. The function of each of these proteins as binding proteins to cell receptors and polymerized albumin will be examined. The antigenic activity of these proteins will be examined by site specific mutagenesis, chemical modification, use of anti- synthetic peptide and monoclonal antibodies. The second goal of this project will be to utilize the information gained from the above studies to identify specific antigenic domains of the surface proteins (S,M, and L) for incorporation into novel chimeric proteins containing the cholera B toxin subunit for investigation as a potential oral immunogen against HBV. This will be done utilizing synthetic DNA fused to the Cholera B subunit gene, and expression by appropriate prokaryotic expression vectors.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI015955-12
Application #
3126512
Study Section
Virology Study Section (VR)
Project Start
1991-06-01
Project End
1996-05-31
Budget Start
1993-06-01
Budget End
1994-05-31
Support Year
12
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
Virginia Commonwealth University
Department
Type
Schools of Medicine
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298

  Publications

Yelamos, B; Nunez, E; Gomez-Gutierrez, J et al. (2001) Urea equilibrium unfolding of the major core protein of the retrovirus feline immunodeficiency virus and its tryptophan mutants. Biochim Biophys Acta 1546:87-97
Nath, M D; Peterson, D L (2001) In vitro assembly of feline immunodeficiency virus capsid protein: biological role of conserved cysteines. Arch Biochem Biophys 392:287-94
Nunez, E; Wei, X; Delgado, C et al. (2001) Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus. Protein Expr Purif 21:183-91
Rodriguez-Crespo, I; Yelamos, B; Albar, J P et al. (2000) Selective destabilization of acidic phospholipid bilayers performed by the hepatitis B virus fusion peptide. Biochim Biophys Acta 1463:419-28
Rodriguez-Crespo, I; Nunez, E; Yelamos, B et al. (1999) Fusogenic activity of hepadnavirus peptides corresponding to sequences downstream of the putative cleavage site. Virology 261:133-42
Yelamos, B; Nunez, E; Gomez-Gutierrez, J et al. (1999) Circular dichroism and fluorescence spectroscopic properties of the major core protein of feline immunodeficiency virus and its tryptophan mutants. Assignment of the individual contribution of the aromatic sidechains. Eur J Biochem 266:1081-9
Birkett, A J; Yelamos, B; Rodriguez-Crespo, I et al. (1997) Cloning, expression, purification, and characterization of the major core protein (p26) from equine infectious anemia virus. Biochim Biophys Acta 1339:62-72
Rodriguez-Crespo, I; Gomez-Gutierrez, J; Encinar, J A et al. (1996) Structural properties of the putative fusion peptide of hepatitis B virus upon interaction with phospholipids. Circular dichroism and Fourier-transform infrared spectroscopy studies. Eur J Biochem 242:243-8
Jin, L; Wei, X; Gomez, J et al. (1995) Use of alpha-N,N-bis[carboxymethyl]lysine-modified peroxidase in immunoassays. Anal Biochem 229:54-60
Milich, D R; Peterson, D L; Schodel, F et al. (1995) Preferential recognition of hepatitis B nucleocapsid antigens by Th1 or Th2 cells is epitope and major histocompatibility complex dependent. J Virol 69:2776-85

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