Mutational analysis of the Sendai virus V protein: Importance of the conserved residues for Zn binding, virus pathogenesis, and efficient RNA editing
Mutational analysis of the Sendai virus V protein: Importance of the conserved residues for Zn binding, virus pathogenesis, and efficient RNA editing
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DOI：
10.1006/viro.2002.1516
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发表时间：
2002-08-01
期刊：
影响因子：
3.7
通讯作者：
Sakaguchi, T
中科院分区：
文献类型：
--
作者：
Fukuhara, N;Huang, C;Sakaguchi, T
仙台病毒的V蛋白（SeV）是病毒在细胞培养中复制所不必需的，但对于病毒在小鼠中的致病性是必不可少的。在V蛋白的C末端，存在在副粘病毒亚科的成员中保守的氨基酸残基，其聚集在三个区域中：区域1，就在RNA编辑位点的下游;以及区域11和111，富含半胱氨酸的锌指样区域。在本研究中，我们将突变引入保守氨基酸中，并产生了9个突变病毒。所有病毒在小鼠肺中的病毒复制受损，在小鼠中的毒力减弱。此外，在溶液中的Zn-65结合测定中，与谷胱甘肽-S-转移酶融合且在区域I、II或III中具有突变的C-末端多肽均具有受损的Zn结合。这些结果表明，保守的氨基酸是重要的V蛋白功能，可能通过蛋白质构象依赖于锌结合。一种突变体SeV V-H318 N具有低效的RNA编辑，表明作为编码位置318处组氨酸的密码子的一部分的核苷酸对于RNA编辑机制是保守的。此外，为了确定在最近的毒力田间分离株中不翻译的V蛋白的C-末端延伸的功能，引入翻译终止密码子以产生相应的短V蛋白。突变体病毒表现出相似的病毒繁殖和致病性，表明V蛋白的C-末端延伸与病毒致病性无关。(C)2002 Elsevier Science（美国）。
The V protein of Sendai virus (SeV) is nonessential for virus replication in cell culture but indispensable for viral pathogenicity in mice. At the C terminus of the V protein, there are amino acid residues conserved among the members of the Paramyxiovinae subfamily that are clustered in three regions: region 1, just downstream of the RNA editing site; and regions 11 and 111, cysteine-rich zinc-finger-like regions. In the present study, we introduced mutations into the conserved amino acids and generated nine mutant viruses. All of the viruses had impaired virus replication in mouse lungs and attenuated virulence in mice. Furthermore, the C-terminal polypeptides fused with glutathione-S-transferase with a mutation in region I, II, or III all had impaired Zn binding in a Zn-65-binding assay in solution. These results demonstrate that the conserved amino acids are important for V protein function, probably via protein conformation dependent on Zn binding. One mutant, SeV V-H318N, had inefficient RNA editing, indicating that the nucleotide that is a part of the codon encoding histidine at position 318 is conserved for the RNA editing machinery. In addition, to determine the function of the C-terminal extension of the V protein, which is not translated in recent virulent field isolates, a translational stop codon was introduced to generate the corresponding short V protein. The mutant virus showed similar virus propagation and pathogenicity, indicating that C-terminal extension of the V protein is not relevant to virus pathogenesis. (C) 2002 Elsevier Science (USA).