Gallagher John R, Atanasiu Doina, Saw Wan Ting, Paradisgarten Matthew J, Whitbeck J Charles, Eisenberg Roselyn J, Cohen Gary H
Department of Microbiology, School of Dental Medicine, University of Pennsylvania Philadelphia, Pennsylvania, United States of America.
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
PLoS Pathog. 2014 Sep 18;10(9):e1004373. doi: 10.1371/journal.ppat.1004373. eCollection 2014 Sep.
Entry of herpes simplex virus (HSV) into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP) throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy.
单纯疱疹病毒（HSV）进入靶细胞需要病毒糖蛋白gD、gH/gL和gB进行复杂的相互作用和构象变化。在病毒进入过程中，gB从融合前构象转变为融合后构象，驱动病毒包膜与宿主细胞膜融合。虽然融合后gB的结构已知，但gB的融合前构象仍然难以捉摸。由于gB的融合前构象是中和抗体的关键靶点，我们着手通过在整个gB胞外域进行荧光蛋白（FP）的基因插入来描述其结构。我们创建了在gB的三个球状结构域中每个结构域都有FP插入的gB构建体。在21个FP插入构建体中，我们发现有8个构建体能够使gB保持膜融合活性。由于FP的大小，gB中能够耐受FP插入的区域必须暴露于溶剂中。两个FP插入突变体在细胞表面表达但无功能，而位于冠部的FP插入则未在表面表达。这是首次在功能性病毒融合蛋白的结构域内进行荧光蛋白插入的报道，我们的结果与基于融合前水泡性口炎病毒G晶体结构构建的融合前HSV gB模型一致。此外，我们发现可以将来自两个不同结构域的功能性FP插入组合起来，创建一个同时标记有CFP和YFP的功能性gB形式。用这个构建体测量了荧光共振能量转移（FRET），我们发现当与gH/gL共表达时，来自gB的FRET信号与仅含有CFP的构建体以及在多核巨细胞中发现的gB有显著差异，这表明这个构建体以及其他类似设计的构建体可能是在任何可通过光学显微镜观察的模型系统中监测gB构象的强大工具。
