Secretion and antigenicity of hepatitis B virus small envelope proteins lacking cysteines in the major antigenic region.
Disulfide bonds are of crucial importance for the structure and antigenic properties of the hepatitis B virus (HBV) envelope. We have evaluated the role of the eight highly conserved cysteines of the major antigenic region for assembly, secretion, and antigenicity of the envelope proteins. Mutants carrying single or multiple substitutions of alanine for cysteine were analyzed using epitope tagging and transient expression in COS-7 cells. The only single cysteines found to be indispensable for efficient secretion were Cys-107 and Cys-138, but double mutation of Cys-137 and Cys-139 also created a block to secretion. Poorly secreted mutants formed aberrant oligomeric structures. The antigenicity of the secreted or intracellularly retained mutants was analyzed using a panel of six monoclonal antibodies recognizing group- and subtype-specific determinants. We demonstrate that Cys-107 is critical for the structure of the group determinant a, whereas Cys-147, previously implicated in intramolecular disulfide bonding, is dispensable. Mutant proteins lacking Cys-121 and -124, -137, -147, or -149 have grossly distorted structures of the y subtype determinant. Our data raise the possibility that HBV strains carrying cysteine mutations are nonreactive in hepatitis B surface antigen-specific immunoassays.
Mangold
C M
CM
Institut für Medizinische Mikrobiologie, Johannes Gutenberg-Universität Mainz, Germany.
Unckell
F
F
Werr
M
M
Streeck
R E
RE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine / Q000032:analysis*; D004220:Disulfides / Q000737:chemistry; D006031:Glycosylation; D006509:Hepatitis B / Q000175:diagnosis; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D006515:Hepatitis B virus / Q000276:immunology / Q000378:metabolism*; D007118:Immunoassay; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004220:Disulfides; D006514:Hepatitis B Surface Antigens; D016376:Oligonucleotides, Antisense; D014759:Viral Envelope Proteins; D003545:Cysteine