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

据报道，流感病毒非结构蛋白NS1具有多种功能。我们之前证明了它具有RNA结合活性，通过它可以抑制感染细胞中的PKR激活。此外，其RNA结合活性最近被证明参与流感病毒感染中对干扰素的抗性，强调NS1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NS1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) NS1 c端区对PKR激活的抑制作用。NS1与RNA结合所必需的区域是n端82 araino酸序列，该序列强烈抑制PKR的激活。这截断NS1在存在以下60多个。araino氨基酸序列和全长NS1使其活性减弱，失去了poly - A结合活性。新发现的ns1′′比n端82个氨基酸长，序列具有与U簇的结合能力，并在Vrna的5′端区域与5 ~ 6个U簇结合。(2) NS1磷酸化对其功能的影响。我们发现了几个丝氨酸/苏氨酸残基在感染细胞中被磷酸化。通过构建这些残基的Ala或Asp取代突变体，研究了NS1磷酸化对这些残基在体外和体内的作用。体外磷酸化NS1可降低其RNA结合活性，但对PKR激活的抑制作用不大。感染细胞中的NS1分布于细胞核和细胞质中，与VRNP和核糖体相关。分别测定各组NS1磷酸化水平，各组间无明显差异。

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.