Mutational analysis of the cysteine residues in the hepatitis B virus small envelope protein.
The small envelope protein of hepatitis B virus is the major component of the viral coat and is also secreted from cells as a 20-nm subviral particle, even in the absence of other viral proteins. Such empty envelope particles are composed of approximately 100 copies of this polypeptide and host-derived lipids and are stabilized by extensive intermolecular disulfide cross-linking. To study the contribution of disulfide bonds to assembly and secretion of the viral envelope, single and multiple mutants involving all 14 cysteines in HepG2 and COS-7 cells were analyzed. Of the six cysteines located outside the region carrying the surface antigen, Cys-48, Cys-65, and Cys-69 were each found to be essential for secretion of 20-nm particles, whereas Cys-76, Cys-90, and Cys-221 were dispensable. By introduction of an additional cysteine substituting serine 58, the yield of secreted particles was increased. Of four mutants involving the eight cysteines located in the antigenic region, only the double mutant lacking Cys-121 and Cys-124 was secreted with wild-type efficiency. Secretion-competent envelope proteins were intracellularly retained by secretion-deficient cysteine mutants. According to alkylation studies, both intracellular and secreted envelope proteins contained free sulfhydryl groups. Disulfide-linked oligomers were studied by gel electrophoresis under nonreducing conditions.
Institut für Medizinische Mikrobiologie, Johannes Gutenberg-Universität Mainz, Germany.
C M
CM
Mangold
R E
RE
Streeck
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D003545:Cysteine; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014162:Transfection; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D003545:Cysteine