Identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants
Identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants
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DOI：
10.1128/jvi.71.12.9024-9031.1997
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发表时间：
1997-12-01
影响因子：
5.4
通讯作者：
Taguchi, F
中科院分区：
文献类型：
--
作者：
Saeki, K;Ohtsuka, N;Taguchi, F
我们先前通过位置定向诱变分析证明了小鼠肝炎病毒（MHV）尖峰（S）蛋白在62和214至216位的氨基酸残基对受体结合活性很重要（H. suzuki和F. Taguchi，J。Virol。为了进一步识别负责活性的残基，我们分离了未用MHV受体蛋白的可溶性形式中和的突变病毒，因为预计该突变体在负责受体结合活性的氨基酸中具有突变，五个可溶性可溶性 - 受体受体受体 - 隔离的耐药（SRR）突变体在三个不同位置的单个氨基酸中具有突变：一个位于65位（Leu to His） （SRR11）在S1亚基中，三个位于1114（LEU至PHE）（SRR3，SRR4和SRR7），其中一个位于S2亚基的位置1163（CYS至PHE）（SRR18）。通过病毒叠加蛋白印迹测定和共免疫沉淀测定法检查的受体结合活性表明，SRR11蛋白的结合活性极低，而SRR7和SRR18蛋白具有与野生型CL-2蛋白相似的结合活性。但是，当使用细胞表面受体进行结合测定时，所有SRR突变体的活性与野生型相似或仅略有降低活性，这些结果以及我们先前的观察结果表明，位于62至65位的位置S1的S1（在检查的MHV菌株中保守的区域）对于受体结合活性很重要，我们还讨论了S2中具有突变的SRR突变体对S2中具有高电阻的机制尽管有足够的结合与可溶受体的结合，但通过可溶性受体进行了中和。
We previously demonstrated by site-directed mutagenesis analysis that the amino acid residues at positions 62 and 214 to 216 in the N-terminal region of mouse hepatitis virus (MHV) spike (S) protein are important for receptor-binding activity (H. Suzuki and F. Taguchi, J. Virol. 70:2632-2636, 1996). To further identify the residues responsible for the activity, we isolated the mutant viruses that were not neutralized with the soluble form of MHV receptor proteins, since such mutants were expected to have mutations in amino acids responsible for receptor-binding activity, Five soluble-receptor-resistant (srr) mutants isolated had mutations in a single amino acid at three different positions: one was at position 65 (Leu to His) (srr11) in the S1 subunit and three were at position 1114 (Leu to Phe) (srr3, srr4, and srr7) and one was at position 1163 (Cys to Phe) (srr18) in the S2 subunit. The receptor-binding activity examined by a virus overlay protein blot assay and by a coimmunoprecipitation assay showed that srr11 S protein had extremely reduced binding activity, while the srr7 and srr18 proteins had binding activity similar to that of wild-type cl-2 protein. However, when cell surface receptors were used for the binding assay, all srr mutants showed activity similar to that of the wild type or only slightly reduced activity, These results, together with our previous observations, suggest that amino acids located at positions 62 to 65 of S1, a region conserved among the MHV strains examined, are important for receptor-binding activity, We also discuss the mechanism by which srr mutants with a mutation in S2 showed high resistance to neutralization by a soluble receptor, despite their sufficient level of binding to soluble receptors.