Gombold J L, Hingley S T, Weiss S R
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076.
Adv Exp Med Biol. 1993;342:157-63. doi: 10.1007/978-1-4615-2996-5_25.
Primary mouse glial cell cultures were infected with mouse hepatitis virus strain A59 (MHV-A59) and maintained over an 18 week period. Viruses isolated from these cultures 16-18 weeks postinfection produce small plaques on fibroblasts and cause only minimal levels of cell-to-cell fusion at times when wild type causes nearly complete cell fusion. However, when mutant-infected cultures were examined 24-36 hours postinfection approximately 90% of the cells were in syncytia showing that the fusion defect is not absolute but rather delayed. Addition of trypsin to mutant-infected cultures enhanced cell fusion a small (2- to 5-fold) but significant degree. Sequencing of portions of the spike genes of six fusion-defective mutants revealed that all contained the same single nucleotide mutation resulting in a substitution of aspartic acid for histidine in the spike cleavage signal. Mutant virions contained only the 180 kDa form of spike protein suggesting that this mutation prevented the normal proteolytic cleavage of the 180 kDa protein into the 90 kDa subunits. Examination of revertants of the mutants supports this hypothesis. Replacement of the negatively-charged aspartic acid with either the wild type histidine or a non-polar amino acid was associated with the restoration of spike protein cleavage and cell fusion.
将原代小鼠神经胶质细胞培养物用A59株小鼠肝炎病毒（MHV - A59）感染，并维持18周。在感染后16 - 18周从这些培养物中分离出的病毒，在成纤维细胞上形成小噬斑，并且在野生型病毒导致几乎完全细胞融合的时候，仅引起最低水平的细胞间融合。然而，当在感染后24 - 36小时检查突变体感染的培养物时，约90%的细胞处于多核体中，这表明融合缺陷不是绝对的，而是延迟的。向突变体感染的培养物中添加胰蛋白酶可使细胞融合增强少量（2至5倍）但显著的程度。对六个融合缺陷突变体的刺突基因部分进行测序显示，所有突变体都含有相同的单核苷酸突变，导致刺突裂解信号中的组氨酸被天冬氨酸取代。突变体病毒粒子仅含有180 kDa形式的刺突蛋白，这表明该突变阻止了180 kDa蛋白正常蛋白水解切割成90 kDa亚基。对突变体回复体的检测支持了这一假设。用野生型组氨酸或非极性氨基酸取代带负电荷的天冬氨酸与刺突蛋白切割和细胞融合的恢复相关。
