Keil W, Wagner R R
Department of Microbiology and Cancer Center, University of Virginia School of Medicine, Charlottesville 22908.
Virology. 1989 Jun;170(2):392-407. doi: 10.1016/0042-6822(89)90430-3.
Deletion mutants and chimeras of the glycoprotein (G) genes of vesicular stomatitis virus serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ) were cloned in plasmids and vaccinia virus vectors under control of the bacteriophage T7 polymerase promoter for expression in CV-1 cells co-infected with a T7 polymerase-expressing vaccinia virus recombinant. Truncated and chimeric G proteins expressed by these vectors were tested for their capacity to react with VSV-Ind and VSV-NJ epitope-specific monoclonal antibodies (MAbs) by Western blot analysis for those antigenic determinants not affected by disulfide-bond reducing conditions or by immuno dotblot analysis for those that are. These experiments allowed us to create putative epitope maps for glycoproteins of both serotypes based on binding affinity and cross-reactivity of VSV-Ind and VSV-NJ MAbs for truncated or chimeric G proteins of known amino acid sequences. Seven of the 9 VSV-NJ G epitopes, including all 4 epitopes involved in virus neutralization by MAbs, mapped to the center (amino acid sequence 193-289) of the 517 amino acid VSV-NJ G protein. Four of the 11 VSV-Ind G epitopes, including 2 neutralizable epitopes, mapped to the cysteine-rich amino-terminal domain (amino acid sequence 80-183) of the 511 amino acid VSV-Ind G protein; the remaining 7 VSV-Ind G epitopes, including 2 involved in virus neutralization, were clustered in the cysteine-poor carboxy-terminal domain (amino acid sequence 286-428). In site-specific mutants of the VSV-Ind G gene defective in one or both glycosylation sites, only the amino-terminal epitopes of the VSV-Ind G protein were affected by deletion of the carbohydrate chain at residue 179; deletion of the carbohydrate chain at residue 336 did not alter reactivity of the G protein with any of the relevant monoclonal antibodies. These results are discussed in relation to earlier attempts to map the antigenic determinants of VSV-NJ and VSV-Ind G proteins by proteolysis of the G protein and by sequencing the G genes of mutant viruses selected for their resistance to neutralization by epitope-specific monoclonal antibodies.
水泡性口炎病毒印第安纳型（VSV-Ind）和新泽西型（VSV-NJ）糖蛋白（G）基因的缺失突变体和嵌合体被克隆到质粒和痘苗病毒载体中，置于噬菌体T7聚合酶启动子的控制下，以便在与表达T7聚合酶的痘苗病毒重组体共感染的CV-1细胞中表达。通过蛋白质印迹分析测试这些载体表达的截短和嵌合G蛋白与VSV-Ind和VSV-NJ表位特异性单克隆抗体（MAb）反应的能力，以检测那些不受二硫键还原条件影响的抗原决定簇；对于那些受影响的抗原决定簇，则通过免疫斑点印迹分析进行检测。这些实验使我们能够根据VSV-Ind和VSV-NJ MAb对已知氨基酸序列的截短或嵌合G蛋白的结合亲和力和交叉反应性，为两种血清型的糖蛋白创建推定的表位图。VSV-NJ G蛋白的9个表位中的7个，包括所有4个参与MAb介导的病毒中和的表位，定位于517个氨基酸的VSV-NJ G蛋白的中心（氨基酸序列193-289）。VSV-Ind G蛋白的11个表位中的4个，包括2个可中和表位，定位于511个氨基酸的VSV-Ind G蛋白富含半胱氨酸的氨基末端结构域（氨基酸序列80-183）；其余7个VSV-Ind G表位，包括2个参与病毒中和的表位，聚集在半胱氨酸含量低的羧基末端结构域（氨基酸序列286-428）。在一个或两个糖基化位点有缺陷的VSV-Ind G基因的位点特异性突变体中，只有VSV-Ind G蛋白的氨基末端表位受到179位残基碳水化合物链缺失的影响；336位残基碳水化合物链的缺失并没有改变G蛋白与任何相关单克隆抗体的反应性。结合早期通过G蛋白的蛋白水解以及对因对表位特异性单克隆抗体的中和作用具有抗性而选择的突变病毒的G基因进行测序来绘制VSV-NJ和VSV-Ind G蛋白抗原决定簇图谱的尝试，对这些结果进行了讨论。
