Long D, Wilcox W C, Abrams W R, Cohen G H, Eisenberg R J
Department of Microbiology, University of Pennsylvania, Philadelphia 19104-6003.
J Virol. 1992 Nov;66(11):6668-85. doi: 10.1128/JVI.66.11.6668-6685.1992.
Glycoprotein D (gD) is a structural component of the herpes simplex virus envelope which is essential for virus penetration. The function of this protein is highly dependent on its structure, and its structure is dependent on maintenance of three intact disulfide bonds. gD contains six cysteines in its ectodomain whose spacing is conserved among all its homologs in other alphaherpesviruses as well as Marek's disease virus. For other proteins, conservation of cysteine spacing correlates with conservation of disulfide bond structure. We have now solved the disulfide bond structure of gD-1 and gD-2 of herpes simplex virus types 1 and 2, respectively. Two approaches were used. First, we constructed 15 double-Cys mutants of gD-1, representing all possible disulfide pairs. In each case, codons for cysteines were changed to serine. We reasoned that if two cysteines normally form a disulfide bond, double mutations which eliminate one proper bond should be less harmful to gD structure than double mutations which eliminate two disulfide bonds. The mutated genes were cloned into a eucaryotic expression vector, and the proteins were expressed in transiently transfected cells. Three double mutations, Cys-1,5, Cys-2,6, and Cys-3,4 permitted gD-1 folding, processing, transport to the cell surface, and function in virus infection, whereas 12 other double mutations each produced a malfolded and nonfunctional protein. Thus, the three functional double-Cys mutants may represent the actual partners in disulfide bond linkages. The second approach was to define the actual disulfide bond structure of gD by biochemical means. Purified native gD-2 was cleaved by CNBr and proteases, and the peptides were separated by high-performance liquid chromatography. Disulfide-linked peptides were subjected to N-terminal amino acid sequencing. The results show that cysteine 1 (amino acid [aa] 66) is bonded to cysteine 5 (aa 189), cysteine 2 (aa 106) is bonded to cysteine 6 (aa 202), and cysteine 3 (aa 118) is bonded to cysteine 4 (aa 127). Thus, the biochemical analysis of gD-2 agrees with the genetic analysis of gD-1. A similar disulfide bond arrangement is postulated to exist in other gD homologs.
糖蛋白D（gD）是单纯疱疹病毒包膜的一种结构成分，对病毒穿透至关重要。该蛋白的功能高度依赖于其结构，而其结构又依赖于三个完整二硫键的维持。gD在其胞外区含有六个半胱氨酸，其间距在其他甲型疱疹病毒以及马立克氏病病毒的所有同源物中都是保守的。对于其他蛋白质，半胱氨酸间距的保守性与二硫键结构的保守性相关。我们现在分别解析了1型和2型单纯疱疹病毒gD-1和gD-2的二硫键结构。采用了两种方法。首先，我们构建了gD-1的15个双半胱氨酸突变体，代表了所有可能的二硫键对。在每种情况下，半胱氨酸的密码子都被替换为丝氨酸。我们推断，如果两个半胱氨酸通常形成一个二硫键，消除一个正确二硫键的双突变对gD结构的损害应该小于消除两个二硫键的双突变。将突变基因克隆到真核表达载体中，蛋白质在瞬时转染的细胞中表达。三个双突变，即半胱氨酸1,5、半胱氨酸2,6和半胱氨酸3,4，允许gD-1折叠、加工、转运到细胞表面并在病毒感染中发挥功能，而其他12个双突变各自产生了错误折叠且无功能的蛋白质。因此，这三个功能性双半胱氨酸突变体可能代表了二硫键连接中的实际配对。第二种方法是通过生化手段确定gD的实际二硫键结构。纯化的天然gD-2用溴化氰和蛋白酶切割，肽段通过高效液相色谱分离。对二硫键连接的肽段进行N端氨基酸测序。结果表明，半胱氨酸1（氨基酸[aa]66）与半胱氨酸5（aa189）相连，半胱氨酸2（aa106）与半胱氨酸6（aa202）相连，半胱氨酸3（aa118）与半胱氨酸4（aa127）相连。因此，gD-2的生化分析与gD-1的遗传分析一致。推测其他gD同源物中存在类似的二硫键排列。
