The effect of the regulatory mechanism of splicing on influenza C virus replication

剪接调控机制对丙型流感病毒复制的影响

• 批准号: 17590413
• 负责人: HONGO Seiji
• 金额: $ 2.24万
• 依托单位: Yamagata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590413/
• 关键词: influenza C virus; splicing; NS gene; regulation of gene expression

We recently established reverse genetics of influenza C virus with seven Pol I plasmids expressing PB2, PB 1, P3, HE, NP, M and NS viral RNA and nine plasmids expressing PB2, PB1, P3, HE, NP, M1, CM2, NS1 and NS2 proteins and succeeded in generation of a recombinant influenza C virus. To determine whether NS gene products of influenza C virus play a role in pre-mRNA splicing, we attempted to obtain the virus, in which Leu 20 was substituted into termination codon to rescue the virus with little expressing of NS1 and NS2. However, the virus was not rescued, which suggested that NS gene products, NS1 and/or NS2, are required for virus replication. In the transient expression system, coexpression of M gene cDNA with NS1 protein showed that splicing rate of M pre-mRNA increased by 30%, suggesting that NS1 may have the activity to enhance splicing. We then investigated whether C-terminus region (63-246 residues) specific for NS1 possesses the functional domain to enhance splicing. First, we obtained the recombinant virus which lost almost 70% of C-terminus by substituting Phe at residue 91 into termination codon (F91 stop). HMV-II cells infected with the recombinant virus containing F91 stop mutation showed that the ratio of M1 derived from spliced M mRNA / CM2 from unspliced M mRNA was reduced by 20%, a finding which suggests that the functional domain may be present in C-terminal region. The functional domain for enhancing splicing remains to be elucidated by generation NS1 mutants containing the various deletions in C-terminus.

我们最近建立了丙型流感病毒的反向遗传学，其中七个表达PB2、PB 1、P3、HE、NP、M和NS病毒RNA的Pol I质粒和九个表达PB2、PB1、P3、HE、NP、M1、CM2、NS1的质粒和 NS2 蛋白，并成功产生重组丙型流感病毒。为了确定丙型流感病毒的NS基因产物是否在mRNA前体剪接中发挥作用，我们尝试获得将Leu 20替换为终止密码子的病毒，以拯救NS1和NS2表达很少的病毒。然而，病毒并未被拯救，这表明NS基因产物NS1和/或NS2是病毒复制所必需的。在瞬时表达体系中，M基因cDNA与NS1蛋白共表达，发现M前体mRNA的剪接率增加了30%，提示NS1可能具有增强剪接的活性。然后我们研究了 NS1 特异性的 C 末端区域（63-246 个残基）是否具有增强剪接的功能域。首先，我们通过将残基91处的Phe替换为终止密码子（F91终止），获得了丢失了近70%C末端的重组病毒。感染含有F91终止突变的重组病毒的HMV-II细胞显示，来自剪接的M mRNA的M1/来自未剪接的M mRNA的CM2的比例降低了20%，这一发现表明该功能域可能存在于C-终端区域。增强剪接的功能域仍有待通过生成包含 C 末端各种缺失的 NS1 突变体来阐明。

项目成果

Conformational maturation of the nucleoprotein synthesized in influenza C virus-infected cells.

丙型流感病毒感染细胞中合成的核蛋白的构象成熟。

• 发表时间: 2006
• 期刊: Virus Res. 122
• 作者: Umene K;et al.;Sugawara K
• 通讯作者: Sugawara K

An outbreak of measles virus infection due to a genotype D9 at a junior high school in Yamagata, Japan in 2004.

2004 年，日本山形市一所初中爆发了 D9 基因型麻疹病毒感染。

• 发表时间: 2005
• 期刊: Jpn. J. Infec. Dis. 58
• 作者: Takamasa Ueno;Masafumi Takiguchi;梅根健一;Matsumoto K;Tanaka T.;上野貴将;Nakashima K;Mizuta K
• 通讯作者: Mizuta K