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）是副粘病毒亚科最近重新命名的呼吸道病毒属的成员。SeV P基因除了磷酸（P）蛋白外，还使用两种辅助蛋白V和C。虽然P蛋白已被鉴定为病毒RNA合成所必需的调节蛋白，但V和C蛋白在病毒复制和发病机制中的作用仍不清楚。甚至还没有确定这些蛋白质是病毒复制所必需的还是仅仅是辅助性的。这些问题现在正被SeV反向遗传学所澄清。虽然P基因的精确拷贝编码P蛋白，但共转录编辑的mRNA指导V蛋白。该编辑事件涉及将一个G残基假模板化插入到模板中特定位点处的新生链中，这将阅读框寄存器从PORF移位到编码富含半胱氨酸的蛋白质的-1 ORF。通过破坏编辑位点或在下游引入无义突变， ...更多信息 我们利用产生SeV反基因组的全长拷贝的cDNA质粒中的编辑位点的突变，产生了V基因表达完全缺陷（V（-））或具有C-末端一半截短（VDELTAC）的突变病毒。他们的表征导致的结论是，虽然完全排除了病毒在组织培养细胞中的复制，但V蛋白对于维持小鼠体内高病毒载量以引起肺炎是必不可少的。这种“奢侈”的体内功能主要由富含半胱氨酸的C-末端的一半编码。仙台C蛋白表达为一套嵌套的蛋白质，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表达的主要种类，其摩尔比是其他三种蛋白的几倍。我们首先沉默C'和C蛋白，发现回收的C/C'（-）病毒在RNA合成中受损，在组织培养细胞中复制严重减弱。更值得注意的是，C/C '（-）病毒几乎完全不能在小鼠体内繁殖，因此对小鼠无致病性。也有可能使所有四种C蛋白沉默，从而产生一种受损更严重但仍能存活的克隆，即4C（-）病毒。因此，可以得出结论，SeV C蛋白是绝对非必需的基因产物，但大大有助于在体外的完整复制能力，是不可缺少的体内增殖和发病机制。少

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。”

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 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。”

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)

加藤 篤 他: "ウイルス最前線、『センダイウイルスエンジニアリング』" 永井美之、野本明男、山本直樹偏 羊土社刊(分担執筆), (1997)

加藤厚等人：“病毒前线，‘仙台病毒工程’”永井义之、野本昭夫、山本直树 淀社出版（合著者），（1997 年）