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Identification of receptor-binding site of the mouse hepatitis virus spike protein

小鼠肝炎病毒刺突蛋白受体结合位点的鉴定

基本信息

批准号：
07660413
负责人：
TAGUCHI Fumihiro
金额：
$ 1.47万
依托单位：
National Institute of Neuroscience, NCNP
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660413/
关键词：
mouse hepatitis virus coronavirus spike protein receptor-binding site

项目摘要

We have previously shown that the N terminal region composed of 330 amino acids of MHV S protein (S1N330) is important for receptor-binding activity, since the S1N330 protein expressed by recombinant vaccina virus binds to the MHV-receptor protein prepared on the membrane paper by Western blot in a similar manner as whole S protein binds to the receptor protein. The aim of this experimetn is to identify the amino acids that participate in the receptor-binding activity in S1N330. We have found that 7MHV strains can use mmCGM1 as a functional receptor. In the S1N330 of these 7 MHV strains, we found 3 major consercative regions which include more than 10 consective amiano acids identical among these 7 MHV strains. We have introduced the mutations in some of amino asids in these regions and analyzed the receptor-binding activity of such mutant proteins. The mutant S1N330 protein with a mutation at amino acid 62 from the N terminus failed to bind to the receptor protein. The receptor binding og a mutant with mutations at 212,214, and 216 was remarkably reduced. These results imply that amino acids at 62,212,214 and 216 play important role for the receptor-binding activity of the MHV s protein. Furthermore, it is suggested that secondary or tertiary structure of SIN330 is necessary for receptor-binding activity, since the region containing amino acid 62 is located apart from a region including amino acids 212,214 and 216 in primary MHV s protein sturucture.

我们先前已经证明，由MHV S蛋白(S1N330)的330个氨基酸组成的N末端区域对于受体结合活性是重要的，因为重组痘苗病毒表达的S1N330蛋白与Western印迹法制备的MHV-受体蛋白结合的方式类似于整个S蛋白与受体蛋白的结合方式。本实验的目的是确定参与S1N330受体结合活性的氨基酸。我们发现7株MHV毒株可以利用MMCGM1作为功能性受体。在这7株MHV的S1N330中，我们发现了3个主要的融合区域，其中含有这7株MHV相同的10多个结合氨基酸。我们已经在这些区域引入了一些氨基酸的突变，并分析了这些突变蛋白的受体结合活性。突变型S1N330蛋白在N端62位氨基酸发生突变，不能与受体蛋白结合。具有212、214和216位突变的突变体的受体结合显著降低。这些结果表明，62、212、214和216位氨基酸对甲型肝炎病毒S蛋白的受体结合活性起重要作用。此外，我们认为SIN330的二级或三级结构是与受体结合活性所必需的，因为含有62个氨基酸的区域与MHV S蛋白一级结构中包含212、214和216个氨基酸的区域不同。