Molecular anatomy of Sendai virus

仙台病毒(Sendai Virus，SeV)是新近更名的副粘病毒亚科呼吸道合胞病毒属的成员。SEVP基因除了利用磷酸(P)蛋白外，还利用两种辅助蛋白：V和C。虽然P蛋白已被确定为病毒RNA合成所必需的调节蛋白，但V和C蛋白在病毒复制和致病机制中的作用尚不清楚。甚至还没有确定这些蛋白质是病毒复制所必需的，还是仅仅是辅助的。这些问题现在正通过SeV反向基因得到澄清。当P基因的精确拷贝编码P蛋白时，共转录编辑的mRNA指导V蛋白。该编辑事件涉及在模板中的特定位置将一个G残基假模板插入到新生链上，从而将读数框寄存器从P ORF移位以访问编码富含半胱氨酸的蛋白质的ORF。通过扰乱编辑站点或在…下游引入无意义突变在产生全长SeV反基因组拷贝的cDNA质粒中更多的编辑位点，我们创造了V基因表达完全缺陷(V(-))或C末端一半截断(VDELTAC)的突变病毒。结果表明，尽管V蛋白对于组织培养细胞中的病毒复制来说是完全不必要的，但对于在小鼠体内维持高病毒载量以引起肺炎和肺炎是必不可少的。这种“奢侈”的III活体功能主要由富含半胱氨酸的C-末端部分编码。V蛋白似乎是SeV应对早期宿主反应(S)清除病毒所必需的。仙台C蛋白是一组嵌套的蛋白质，C‘，C，Y1和Y2，来自未经编辑的P基因和编辑的V基因，使用相对于P ORF的+1框架，从不同的起始密码子开始。其中，C蛋白是感染细胞表达的主要蛋白，其摩尔比是其他三种蛋白的数倍。我们首先沉默了C‘和C’蛋白，发现回收的C/C‘(-)病毒在组织培养细胞中RNA合成受到损害，复制能力严重减弱。更值得注意的是，C/C‘(-)病毒几乎完全不能在小鼠体内高效生长，因此对小鼠没有致病性。使所有四种C蛋白沉默也是可能的，从而产生了一种更受损但仍有生存能力的克隆，4C(-)病毒。因此，我们得出结论：SeV C蛋白是绝对非必需的基因产物，但对体外的完全复制能力有很大贡献，对体内增殖和致病是必不可少的。较少

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