Mangold C M, Unckell F, Werr M, Streeck R E
Institut für Medizinische Mikrobiologie, Johannes Gutenberg-Universität Mainz, Germany.
Virology. 1995 Aug 20;211(2):535-43. doi: 10.1006/viro.1995.1435.
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.
二硫键对于乙型肝炎病毒（HBV）包膜的结构和抗原特性至关重要。我们评估了主要抗原区域的八个高度保守的半胱氨酸对包膜蛋白的组装、分泌和抗原性的作用。使用表位标签和在COS-7细胞中的瞬时表达分析了携带丙氨酸对半胱氨酸的单取代或多取代的突变体。发现对于有效分泌必不可少的唯一单个半胱氨酸是Cys-107和Cys-138，但Cys-137和Cys-139的双突变也导致分泌受阻。分泌不良的突变体形成异常的寡聚结构。使用一组识别组特异性和亚型特异性决定簇的六种单克隆抗体分析分泌的或细胞内保留的突变体的抗原性。我们证明Cys-107对于组决定簇a的结构至关重要，而先前涉及分子内二硫键形成的Cys-147是可有可无的。缺乏Cys-121、-124、-137、-147或-149的突变蛋白具有严重扭曲的y亚型决定簇结构。我们的数据增加了携带半胱氨酸突变的HBV毒株在乙型肝炎表面抗原特异性免疫测定中无反应性的可能性。
