Structure of hepatitis B virus core and e-antigen. A single precore amino acid prevents nucleocapsid assembly.
The hepatitis B virus core gene codes for two polypeptides: the core protein, which assembles to form particles (HBcAg), and the secreted precore protein (HBeAg). Expression vectors directing the synthesis in Escherichia coli of a recombinant HBeAg corresponding in sequence to serum-derived HBeAg encompassing the 10 precore amino acids remaining after cleavage of the precursor and residues 1-149 of HBcAg (PC-HBeAg) were constructed. Recombinant PC-HBeAg, HBcAg, and C-terminally truncated HBcAg were isolated from E. coli and analyzed by sucrose velocity sedimentation, electron microscopy, anti-HBc/e specific monoclonal antibody analysis, and for immunogenicity. HBcAg and truncated HBcAg formed 27-nm particles and displayed HBc antigenicity. In contrast, PC-HBeAg was nonparticulate and did not band in sucrose gradients. PC-HBeAg was recognized efficiently by HBeAg-specific antibodies and displayed little HBc antigenicity. Immunogenicity studies including T and B cell recognition confirmed that PC-HBeAg demonstrates HBe antigenicity. The presence of the 10 precore amino acids therefore prevented particle formation. To analyze which precore amino acids might be responsible for the prevention of particle formation a cysteine to glutamine substitution at amino acid position -7 was introduced into PC-HBeAg (-7C-->Q)PC-HBeAg. This single amino acid change at position -7 restored particle formation and HBc antigenicity. The evolutionarily conserved cysteine at position -7 thus appears responsible for the prevention of particle assembly in the HBeAg biosynthesis pathway.
Schödel
F
F
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Peterson
D
D
Zheng
J
J
Jones
J E
JE
Hughes
J L
JL
Milich
D R
DR
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D002213:Capsid / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D005822:Genetic Vectors; D006512:Hepatitis B Core Antigens / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D008819:Mice, Nude; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology*; D014758:Viral Core Proteins / Q000235:genetics* / Q000378:metabolism
D006512:Hepatitis B Core Antigens; D006513:Hepatitis B e Antigens; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D011994:Recombinant Proteins; D014758:Viral Core Proteins