Sodora D L, Eisenberg R J, Cohen G H
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104.
J Virol. 1991 Aug;65(8):4432-41. doi: 10.1128/JVI.65.8.4432-4441.1991.
Glycoprotein D (gD) is an envelope component of herpes simplex virus essential for virus penetration. gD contains three sites for addition of asparagine-linked carbohydrates (N-CHO), all of which are utilized. Previously, we characterized mutant forms of herpes simplex virus type 1 gD (gD-1) lacking one or all three N-CHO addition sites. All of the mutants complemented the infectivity of a gD-minus virus, F-gD beta, to the same extent as wild-type gD. Here, we show that recombinant viruses containing mutations in the gD-1 gene which eliminate the three N-CHO signals are viable. Two such viruses, called F-gD(QAA)-1 and F-gD(QAA)-2, were independently isolated, and the three mutations in the gD gene in one of these viruses were verified by DNA sequencing. We also verified that the gD produced in cells infected by these viruses is devoid of N-CHO. Plaques formed by both mutants developed more slowly than those of the wild-type control virus, F-gD(WT), and were approximately one-half the size of the wild-type. One mutant, F-gD(QAA)-2, was selected for further study. The QAA mutant and wild-type gD proteins extracted from infected cells differed in structure, as determined by the binding of monoclonal antibodies to discontinuous epitopes. However, flow cytometry analysis showed that the amount and structure of gD found on infected cell surfaces was unaffected by the presence or absence of N-CHO. Other properties of F-gD(QAA)-2 were quite similar to those of F-gD(WT). These included (i) the kinetics of virus production as well as the intracellular and extracellular virus titers; (ii) the rate of virus entry into uninfected cells; (iii) the levels of gB, gC, gE, gH, and gI expressed by infected cells; and (iv) the turnover time of gD. Thus, the absence of N-CHO from gD-1 has some effect on its structure but very little effect on its function in virus infection in cell culture.
糖蛋白D（gD）是单纯疱疹病毒包膜的一个组成部分，对病毒穿透至关重要。gD含有三个用于添加天冬酰胺连接碳水化合物（N-CHO）的位点，且全部被利用。之前，我们对缺乏一个或全部三个N-CHO添加位点的1型单纯疱疹病毒gD（gD-1）突变形式进行了表征。所有突变体对缺失gD的病毒F-gDβ的感染性互补程度与野生型gD相同。在此，我们表明，gD-1基因中含有消除三个N-CHO信号突变的重组病毒是可行的。独立分离出了两种这样的病毒，称为F-gD(QAA)-1和F-gD(QAA)-2，其中一种病毒gD基因中的三个突变通过DNA测序得到了验证。我们还证实，受这些病毒感染的细胞中产生的gD不含N-CHO。两种突变体形成的噬斑比野生型对照病毒F-gD(WT)的噬斑发展得更慢，且大小约为野生型的一半。选择了一种突变体F-gD(QAA)-2进行进一步研究。从感染细胞中提取的QAA突变体和野生型gD蛋白在结构上有所不同，这是通过单克隆抗体与不连续表位的结合确定的。然而，流式细胞术分析表明，感染细胞表面gD的数量和结构不受N-CHO存在与否的影响。F-gD(QAA)-2的其他特性与F-gD(WT)非常相似。这些特性包括：（i）病毒产生的动力学以及细胞内和细胞外病毒滴度；（ii）病毒进入未感染细胞的速率；（iii）感染细胞表达的gB、gC、gE、gH和gI的水平；以及（iv）gD的周转时间。因此，gD-1中不存在N-CHO对其结构有一定影响，但对其在细胞培养中病毒感染中的功能影响很小。
