Chiang H Y, Cohen G H, Eisenberg R J
School of Dental Medicine, University of Pennsylvania, Philadelphia 19104.
J Virol. 1994 Apr;68(4):2529-43. doi: 10.1128/JVI.68.4.2529-2543.1994.
Glycoprotein gD is a component of the herpes simplex virus (HSV) envelope essential for virus entry into susceptible cells. Previous studies using deletion and point mutations identified a functional domain of HSV-1 gD (gD-1) from residues 231 to 244. However, many of the deletion mutations had global effects on gD-1 structure, thus precluding assessment of the functional role of large portions of the protein. In this study, we constructed a large panel of linker-insertion mutants in the genes for gD-1 and HSV-2 gD (gD-2). The object was to create mutations which would have only localized effects on protein structure but might have profound effects on gD function. The mutant proteins were expressed in transiently transfected L cells. Monoclonal antibodies (MAbs) were used as probes of gD structure. We also examined protein aggregation and appearance of the mutant glycoproteins on the transfected cell surface. A complementation assay measured the ability of the mutant proteins to rescue the infectivity of the gD-null virus, FgD beta, in trans. Most of the mutants were recognized by one or more MAbs to discontinuous epitopes, were transported to the transfected cell surface, and rescued FgD beta virus infectivity. However, some mutants which retained structure were unable to complement FgD beta. These mutants were clustered in four regions of gD. Region III (amino acids 222 to 246) overlaps the region previously defined by gD-1 deletion mutants. The others, from 27 through 43 (region I), from 125 through 161 (region II), and from 277 to 310 (region IV), are newly described. Region IV, immediately upstream of the transmembrane anchor sequence, was previously postulated to be part of a putative stalk structure. However, residues 277 to 300 are directly involved in gD function. The linker-insertion mutants were useful for mapping MAb AP7, a previously ungrouped neutralizing MAb, and provided further information concerning other discontinuous epitopes. The mapping data suggest that regions I through IV are physically near each other in the folded structure of gD and may form a single functional domain.
糖蛋白gD是单纯疱疹病毒（HSV）包膜的一个组成部分，对于病毒进入易感细胞至关重要。先前使用缺失和点突变的研究确定了HSV-1 gD（gD-1）从第231位至244位残基的一个功能结构域。然而，许多缺失突变对gD-1结构有全局性影响，因此无法评估该蛋白大部分区域的功能作用。在本研究中，我们构建了一大组gD-1和HSV-2 gD（gD-2）基因的接头插入突变体。目的是产生仅对蛋白质结构有局部影响但可能对gD功能有深远影响的突变。突变蛋白在瞬时转染的L细胞中表达。单克隆抗体（MAb）用作gD结构的探针。我们还检测了突变糖蛋白在转染细胞表面的蛋白质聚集和出现情况。互补试验测定了突变蛋白在反式中拯救gD缺失病毒FgDβ感染性的能力。大多数突变体被一种或多种针对不连续表位的MAb识别，被转运到转染细胞表面，并拯救了FgDβ病毒的感染性。然而，一些保留结构的突变体无法互补FgDβ。这些突变体聚集在gD的四个区域。区域III（氨基酸222至246）与先前由gD-1缺失突变体定义的区域重叠。其他区域，从第27位至43位（区域I），从第125位至161位（区域II），以及从第277位至310位（区域IV），是新描述的。区域IV，紧接跨膜锚定序列上游，先前被假定为推定的茎结构的一部分。然而，第277位至300位残基直接参与gD功能。接头插入突变体对于定位MAb AP7（一种先前未分类的中和MAb）很有用，并提供了有关其他不连续表位的进一步信息。定位数据表明，在gD的折叠结构中，区域I至IV在物理上彼此靠近，可能形成一个单一的功能结构域。
