CONTROL OF INFLUENZA VIRUS PATHOGENICITY BY PKR INHIBITOR PROTEIN AND ITS APPLICATION

PKR抑制蛋白控制流感病毒致病性及其应用

• 批准号: 12670279
• 负责人: FUKUDA Ryuji
• 金额: $ 2.18万
• 依托单位: KANAZAWA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670279/
• 关键词: INFLUENZA VIRUS; PROTEIN KINESE R; NS1 PROTEIN; PHOSPHORYLATION; PATHOGENICITY; プロテインキナーゼR; NS1プロテイン

The influenza virus nonstructural protein NS1 has been reported to have many functions. We previously demonstrated that it has an RNA binding activity, by which it inhibits the PKR activation in the infected cell. In addition, its RNA binding activity has been recently shown to participate in the resistance against interferon in influenza virus infection, underlining NS1 as a determinant for this virus pathogenicity. In this study, we analyzed the RNA binding ability of NS1 in more detail, and related it to the activation state of PKR, which is one of the indicators of this virus pathogenicity.(1) Effect of C-terminal region of NS1 on its inhibition of PKR activation. The region of NS1 essential for its RNA binding was the N-terminal 82 araino acid sequence, which strongly inhibits the activation of PKR. This truncated NS1 in the presence of the following more than 60.araino acid sequence, and also the full-length NS1, attenuated this activity, and lost its poly A binding activity. It was newly disclosed that NS1'longer than N-terminal 82 amino acid,sequence has a binding ability to U clusters, and bound to the 5-6 U cluster at the 5'-terminal region of Vrna.(2) Effect of phosphorylation of NS1 on its functions. We identified several Ser/Thr residues which are phosphorylated in the infected cells.The effects of NS1 phosphorylation at each of these residues in vitro and in vivo are now investigated by constructing Ala or Asp substitution mutants of these residues. Phosphorylation of NS1 in vitro attenuated its RNA binding activity, but had little effect on its inhibition of the PKR activation. NS1 in the infected cells is distributed in the nucleus and the cytoplasm, and is associated with VRNP and ribosomes. The phosphorylation level of NS1 of these fractions were separately determined without appreciable difference among them.

据报道，流感病毒非结构蛋白NS 1具有多种功能。我们先前证明它具有RNA结合活性，通过该活性它抑制感染细胞中的PKR活化。此外，其RNA结合活性最近已显示参与流感病毒感染中对干扰素的抗性，强调NS 1是该病毒致病性的决定因素。在本研究中，我们更详细地分析了NS 1的RNA结合能力，并将其与PKR的激活状态联系起来，PKR是该病毒致病性的指标之一。(1)NS 1蛋白C端在其抑制PKR活化中的作用NS 1与RNA结合所必需的区域是N-末端82氨基酸序列，该序列强烈抑制PKR的活化。这种截短的NS 1在以下60个以上氨基酸序列以及全长NS 1的存在下减弱了这种活性，并丧失了其多聚腺苷酸结合活性。新发现的NS 1 '比N-末端82个氨基酸长，序列具有与U簇结合的能力，并与Vrna 5'-末端区域的5-6个U簇结合。(2)NS 1磷酸化对其功能的影响。我们鉴定了几个在感染细胞中被磷酸化的Ser/Thr残基，通过构建这些残基的Ala或Asp取代突变体，在体外和体内研究了这些残基上的NS 1磷酸化的影响。NS 1的磷酸化在体外减弱了其RNA结合活性，但对其抑制PKR活化的影响不大。在感染细胞中，NS 1分布于细胞核和细胞质中，并与VRNP和核糖体相关。这些组分的NS 1磷酸化水平分别测定，它们之间没有明显的差异。

项目成果

E.HATADA, R.FUKUDA: "(論文投稿中)"

E.HATADA、R.FUKUDA：“（论文提交中）”