Molecular biological studies on the neurovirulence of murine coronavirus

一株高度嗜神经的小鼠冠状病毒JHM株已被研究为引起急性和亚急性脑脊髓炎脱髓鞘的病毒剂模型。作为一种病毒因子参与神经毒力。包括病毒粒子表面投射的尖峰蛋白(S)被认为是深度参与的。为了研究JHM株神经毒力的机制，我们对表现不同神经毒力的JHM病毒变异株的S蛋白进行了分子生物学分析。我们首先分离出了一种对大鼠具有高度神经毒性的变异体CL-2。与其他无毒变异体相比，该变异体含有更大的S蛋白。我们已经从编码较大的S蛋白的mRNA3中分离出基因，序列分析表明，较大的S蛋白有141个氨基酸簇，这是无毒病毒的一个小蛋白S所缺失的。141个氨基酸簇可能与高神经毒力有关，尽管氨基酸的确切作用尚不清楚。我们进一步研究了利用重组痘苗病毒在哺乳动物细胞中表达较大的S蛋白。表达的S蛋白与原核表达的S蛋白在分子大小、免疫原性、抗原性等方面均无明显差异。此外，表达的S蛋白在各种培养细胞中具有融合性。这些结果表明，重组痘苗病毒表达的S蛋白适合于S蛋白的生物学活性研究。用含S大蛋白基因的重组痘苗病毒观察JHMV的神经毒力的实验正在进行中。

A highly neurotropic murine coronavirus JHM strain has been studied as a model of viral agent to cause acute and subacute encephalomyelitis with demyelination. As a viral factor involving in the neurovirulence. the spike(S)protein comprising the projections on the virion surface is considered to be deeply involved. To study the mechanisms by which JHM strain exhibits the neurovirulence, we have done the molecular biological analysis of S protein of variant JHM viruses showing different neurovirulence. We first of all have isolated a highly neurovirulent variant, cl-2, for rats. This variant has been shown to have a larger S protein as compared with other variants with avirulence. We have isolated the cDNA from mRNA3 encoding the larger S protein and sequence analysis has identified that the larger S protein has 141 amino acid clusters which is missing in a small S protein of avirulent viruses. The 141 amino acid cluster may be implicated with high neurovirulence, although the precise role of the amino acids is yet unclear. We further studied on the larger S protein by expressing in mammalian cells with recombinant vaceinia viruses. The S protein expressed showed no difference in molecular size, immunogenicity or antigenicity as compared with the authentic S protein. Furthermore, the expressed S protein exhibited the fusionability in various cultured cells. These results suggest that the S protein expressed by recombinant vaceinia virus is appropriate for the study of biological activities of the S protein. Experiments are now in progress to see the neurovirulence of JHMV using the recombinant vaccinia viruses with large S protein cDNA.