Molecular anatomy of Sendai virus

仙台病毒的分子解剖学

• 批准号: 08457093
• 负责人: KATO Atsushi
• 金额: $ 4.99万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457093/
• 关键词: SENDAI VIRUS; PARAMYXOVIRUS; RIVERSE GENETICS; V PROTEIN; CPROTEIN; PATHOGENESIS; 粒子形成

Sendai virus (SeV) is a member of the recently renamed Respirovirus genus of the Paramyxovirinae. SeV P gene gives use to two accessory proteins, V and C, in addition to the phospho (P) protein. While the P protein has been identified to be a modulator protein essential for viral RNA synthesis, the roles of the V and C proteins in viral replication and pathogenesis have remained unclear. It has not even been established whether these proteins are essential or simply auxiliary for viral replication. These issues are now being clarified by SeV reverse genetics.While the exact copy of the P gene encodes the P protein, the cotranscriptionally edited mRNA directs the V protein. This editing event involves pseudotemplated insertion of one G residue to the nascent chain at a specific site in the template, which shifts the reading frame register form the P ORF to access the -1 ORF encoding a cysteine-rich protein. By disrupting the editing site or introducing nonsense mutations just downstream … More of the editing site in a cDNA plasmid generating a full-length copy of the SeV antigenome, we created mutant viruses totally defective in V gene expression (V(-)) or with truncation of die C-terminal half (VDELTAC). Their characterization led to the conclusions that, although completely dispensable for viral replication in tissue cultured cells, the V protein was essential for maintaining a high viral load in mice to cause pneumonia and that. this "luxury" iii vivo function was encoded primarily by the cysteine-rich C-terminal half. The V protein appeared to be essential for SeV to cope with some early host response(s) recruited to clear the virus.The Sendai C protein is expressed as a nested set of proteins, C', C, Y1 and Y2, from both the unedited P mRNA and the edited V mRNA using the +1 frame relative to the P ORF and starting at different initiation codons. Among them, C protein is the major species expressed in infected cells at molar ratio several-fold higher than the other three. We first silenced C' and C proteins and found that the recovered C/C'(-) viruses were impaired in RNA synthesis and severely attenuated in replication in tissue cultured cells. More notably, the C/C'(-) viruses were almost totally incapable of growing productively in mice and, hence, nonpathogenic for the mice. Silencing all four C proteins was also possible, giving rise to a still more impaired but viable clone, the 4C(-) virus. Thus, it was concluded that SeV C proteins are categorically nonessential gene products but greatly contribute to full replication capability in vitro and are indispensable for in vivo multiplication and pathogenesis. Less

仙台病毒 (SeV) 是最近更名为副粘病毒科呼吸道病毒属的成员，除了磷酸 (P) 蛋白外，P 基因还使用两种辅助蛋白：V 和 C。尽管V和C蛋白是病毒RNA合成所必需的调节蛋白，但它们在病毒复制和发病机制中的作用仍不清楚，甚至还不清楚这些蛋白对于病毒复制是否是必需的或只是辅助的。 SeV 反向遗传学现在正在澄清这些问题。虽然 P 基因的精确副本编码 P 蛋白，但共转录编辑的 mRNA 指导 V 蛋白。这一编辑事件涉及在特定位置将一个 G 残基假模板插入到新生链中。模板中的位点，通过破坏编辑位点或在 cDNA 中的编辑位点下游引入无义突变，将阅读框寄存器从 P ORF 转移到编码富含半胱氨酸的蛋白质的 -1 ORF。质粒生成 SeV 反基因组的全长副本，我们创建了 V 基因表达完全缺陷的突变病毒 (V(-)) 或 C 端半截短的突变病毒 (VDELTAC)。 V蛋白对于组织培养细胞中的病毒复制来说是完全可有可无的，但它对于维持小鼠体内高病毒载量以引起肺炎至关重要，并且这种“豪华”体内功能主要由富含半胱氨酸的编码。 C 末端一半似乎对于 SeV 应对一些早期宿主反应（招募来清除病毒）至关重要。仙台 C 蛋白表达为一组嵌套的蛋白质，C'、C、Y1 和Y2，来自未编辑的 P mRNA 和编辑的 V mRNA，使用相对于 P ORF 的 +1 框架并从不同的起始密码子开始其中，C 蛋白是在感染细胞中以高几倍的摩尔比表达的主要种类。比其他三个。我们首先沉默了C'和C蛋白，发现回收的C/C'(-)病毒的RNA合成受到损害，并且在组织培养细胞中的复制严重减弱。更值得注意的是，C/C'(-)病毒几乎被破坏。完全无法在小鼠体内有效生长，因此对小鼠来说不具有致病性，这也是可能的，从而产生更受损但具有活力的克隆，即 4C(-) 病毒。 C蛋白是绝对不是必需的基因产物，但对体外的完全复制能力有很大贡献，并且对于体内增殖和发病机制是不可或缺的。

项目成果

A.Kato, Y.Sakai, T.Shioda, T.Kondo, M.Nakanishi and Y.Nagai.: "Initiation of Sendai virus multiplication from transfectied cDNA or RNA with negative or positive sense." Genes Cells. 1. 569-579 (1996)

A.Kato、Y.Sakai、T.Shioda、T.Kondo、M.Nakanishi 和 Y.Nagai.：“从转染的具有负义或正义的 cDNA 或 RNA 启动仙台病毒的增殖。”

Atsushi Kato: "Importance of the cysteine-rich carboxyl-terminal half of V protein for Sendai virus pathogenesis." J.Virol. 71. 7266-7272 (1997)

Atsushi Kato：“V 蛋白富含半胱氨酸的羧基末端一半对于仙台病毒发病机制的重要性。”

Takemasa Sakaguchi: "Phosphorylation of the Sendai virus M protein is not essential for virus replication either in vitro or in vivo." Virology. 235. 360-366 (1997)

Takemasa Sakaguchi：“无论是在体外还是在体内，仙台病毒 M 蛋白的磷酸化对于病毒复制都不是必需的。”

Atsushi Kuronati: "The paramyxovirus, Sendai virus, C proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis." Genes Cells. 3. 111-124 (1998)

Atsushi Kuronati：“副粘病毒、仙台病毒、C 蛋白绝对是非必需的基因产物，但沉默它们的表达会严重损害病毒复制和发病机制。”

T.Sakaguchi 他: "Phosphorylation of the Sendai virus M protein is not essential for virus replication either in vitro orin vivo." Virology,. 235. 360-366 (1997)

T. Sakaguchi 等人：“无论是在体外还是在体内，仙台病毒 M 蛋白的磷酸化对于病毒复制都不是必需的。”235. 360-366 (1997)