Long D, Cohen G H, Muggeridge M I, Eisenberg R J
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6003.
J Virol. 1990 Nov;64(11):5542-52. doi: 10.1128/JVI.64.11.5542-5552.1990.
We previously constructed seven mutations in the gene for glycoprotein D (gD) of herpes simplex virus type 1 in which the codon for one of the cysteine residues was replaced by a serine codon. Each of the mutant genes was cloned into a eucaryotic expression vector, and the proteins were transiently expressed in mammalian cells. We found that alteration of any of the first six cysteine residues had profound effects on protein conformation and oligosaccharide processing. In this report, we show that five of the mutant proteins exhibit temperature-sensitive differences in such properties as aggregation, antigenic conformation, oligosaccharide processing, and transport to the cell surface. Using a complementation assay, we have now assessed the ability of the mutant proteins to function in virus infection. This assay tests the ability of the mutant proteins expressed from transfected plasmids to rescue production of infectious virions of a gD-minus virus, F-gD beta, in Vero cells. Two mutant proteins, Cys-2 (Cys-106 to Ser) and Cys-4 (Cys-127 to Ser), were able to complement F-gD beta at 31.5 degrees C but not at 37 degrees C. The rescued viruses, designated F-gD beta(Cys-2) and F-gD beta(Cys-4), were neutralized as efficiently as wild-type virus by anti-gD monoclonal antibodies, indicating that gD was present in the virion envelope in a functional form. Both F-gD beta(Cys-2) and F-gD beta(Cys-4) functioned normally in a penetration assay. However, the infectivity of these viruses was markedly reduced compared with that of the wild type when they were preincubated at temperatures above 37 degrees C. The results suggest that mutations involving Cys-106 or Cys-127 in gD-1 confer a temperature-sensitive phenotype on herpes simplex virus. These and other properties of the cysteine-to-serine mutants allowed us to predict a disulfide bonding pattern for gD.
我们先前在单纯疱疹病毒1型糖蛋白D（gD）基因中构建了7个突变体，其中一个半胱氨酸残基的密码子被丝氨酸密码子取代。每个突变基因都被克隆到真核表达载体中，并且这些蛋白质在哺乳动物细胞中瞬时表达。我们发现前六个半胱氨酸残基中的任何一个发生改变都会对蛋白质构象和寡糖加工产生深远影响。在本报告中，我们表明其中五个突变蛋白在聚集、抗原构象、寡糖加工以及转运至细胞表面等特性方面表现出温度敏感性差异。使用互补试验，我们现在评估了突变蛋白在病毒感染中发挥功能的能力。该试验检测从转染质粒表达的突变蛋白拯救gD缺陷病毒F-gDβ在Vero细胞中产生感染性病毒粒子的能力。两个突变蛋白，Cys-2（Cys-106突变为Ser）和Cys-4（Cys-127突变为Ser），在31.5℃时能够互补F-gDβ，但在37℃时则不能。拯救的病毒，命名为F-gDβ（Cys-2）和F-gDβ（Cys-4），被抗gD单克隆抗体中和的效率与野生型病毒一样，这表明gD以功能形式存在于病毒粒子包膜中。F-gDβ（Cys-2）和F-gDβ（Cys-4）在穿透试验中均正常发挥功能。然而，当它们在高于37℃的温度下预孵育时，这些病毒的感染性与野生型相比明显降低。结果表明，gD-1中涉及Cys-106或Cys-127的突变赋予单纯疱疹病毒温度敏感表型。半胱氨酸到丝氨酸突变体的这些以及其他特性使我们能够预测gD的二硫键模式。
