A structure-functional analysis of the paramyxovirus M protein by using a virus recovery system from cDNA

使用 cDNA 病毒回收系统对副粘病毒 M 蛋白进行结构功能分析

• 批准号: 10670286
• 负责人: SAKAGUCHI Takemasa
• 金额: $ 1.66万
• 依托单位: Hiroshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670286/
• 关键词: paramyxovirus; M protein; virus recovery; cysteine; morphogenesis; reverse genetics; システイン残基; ジスルフィド結合; パラミクソウィルス; ウィルス回収系

The matrix (M) protein of Sendai virus (SeV) has five cysteine residues, at positions 83, 106, 158, 251 and 295. The fast migration of the M protein in SDS-PAGE in a non-reducing condition suggests that it forms a specific structure. The structure is considered to depend on cysteine residues, since mutations of the cysteine residues affect migration in SDS-PAGE. To determine the cysteine-dependent structure of the M protein in viral replication, we tried to recover virus from mutant genomic cDNA possessing a substitution to serine at one of the cysteine residues or at all of the cysteine residues. SeV M-CィイD283ィエD2S, SeV M-CィイD2106ィエD2S, and SeV M-CィイD2295ィエD2S were successfully recovered from cDNA, while recombinant SeVs possessing M-CィイD2158ィエD2S, M-CィイD2251ィエD2S, and M-C(-) mutations were not, suggesting the importance of the cysteine residues at positions 158 and/or 251 in virus replication. SeV M-CィイD283ィエD2S and SeV M-CィイD2106ィエD2S had smaller virus particles than did the wild-type SeV, whereas SeV M-CィイD2295ィエD2S had larger and heterogeneous particles. Furthermore, SeV M-CィイD2106ィエD2S had a significant amount of empty particles lacking the viral genome. These results indicate that a single-point mutation at the cysteine residues of the M protein affects virus morphology and genome incorporation. SeV M-CィイD283ィエD2S and SeV M-CィイD2106ィエD2S exhibited a lower virus growth in cultured cells and mouse lungs, leading to a lower pathogenicity to mice compared with the wild-type virus, while the SeV M-CィイD2295ィエD2S mutant grew as efficiently as the wild-type virus. The infection of cultured cells with SeV M-CィイD283ィエD2S and SeV M-CィイD2106ィエD2S suggested that virus assembly and/or budding steps might be abrogated.

仙台病毒（SeV）的基质（M）蛋白在第83、106、158、251和295位上有5个半胱氨酸残基。M蛋白在非还原条件下的SDS-PAGE中的快速迁移表明它形成了特定的结构。该结构被认为取决于半胱氨酸残基，因为半胱氨酸残基的突变影响 SDS-PAGE 中的迁移。为了确定病毒复制中M蛋白的半胱氨酸依赖性结构，我们尝试从在一个半胱氨酸残基或所有半胱氨酸残基处具有丝氨酸取代的突变体基因组cDNA中回收病毒。 SeV M-CィイD283ィエD2S、SeV M-CィイD2106ィエD2S和SeV M-CィイD2295ィエD2S已成功从cDNA中回收，而重组SeV拥有M-CィイD2158ィエD2S， M-CィイD2251ィエD2S 和 M-C(-) 突变没有，这表明了 病毒复制中第 158 和/或 251 位的半胱氨酸残基。 SeV M-CィイD283ィエD2S和SeV M-CィイD2106ィエD2S具有比野生型SeV更小的病毒颗粒，而SeV M-CィイD2295ィエD2S具有更大且异质的颗粒。此外，SeV M-CィイD2106ィエD2S 具有大量缺乏病毒基因组的空颗粒。这些结果表明，M 蛋白半胱氨酸残基的单点突变会影响病毒形态和基因组掺入。 SeV M-CィイD283ィエD2S和SeV M-CィイD2106ィエD2S在培养细胞和小鼠肺部表现出较低的病毒生长，导致与野生型病毒相比对小鼠的致病性较低，而SeV M-CィイD2295ィエD2S突变体的生长效率与野生型病毒相同。用SeV M-CィイD283ィエD2S和SeV M-CィイD2106ィエD2S感染培养细胞表明病毒组装和/或出芽步骤可能被废除。