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

我们之前已经证明，由330个氨基酸组成的MHV S蛋白（S1N330）的N端区域对受体结合活性很重要，因为重组疫苗病毒表达的S1N330蛋白与Western blot在膜纸上制备的MHV受体蛋白结合的方式类似于整个S蛋白与受体蛋白结合的方式。本实验的目的是鉴定参与S1N330受体结合活性的氨基酸。我们发现7MHV毒株可以使用mmCGM1作为功能性受体。在这7株MHV的S1N330中，我们发现了3个主要的保守区，包括10多个连续的氨基酸，这些氨基酸在这7株MHV中相同。我们介绍了这些区域的一些氨基酸突变，并分析了这些突变蛋白的受体结合活性。突变体S1N330蛋白在N端第62个氨基酸处发生突变，无法与受体蛋白结合。在212,214和216位点突变的突变体中，受体结合明显减少。这些结果表明，62,212,214和216位氨基酸对MHV蛋白的受体结合活性起重要作用。此外，我们认为SIN330的二级或三级结构对于受体结合活性是必要的，因为在初级MHV的蛋白质结构中包含氨基酸62的区域与包含氨基酸212,214和216的区域分开。

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.