BACKGROUND Nonstructural protein 1 (NS1) is a virulence factor encoded by influenza A virus (IAV) that is expressed in the nucleus and cytoplasm of host cells during the earliest stages of infection. NS1 is a multifunctional protein that plays an important role in virus replication, virulence and inhibition of the host antiviral immune response. However, to date, the phosphorylation sites of NS1 have not been identified, and the relationship between phosphorylation and protein function has not been thoroughly elucidated. METHOD In this study, potential phosphorylation sites in the swine influenza virus (SIV) NS1 protein were bioinformatically predicted and determined by Phos-tag SDS-PAGE analysis. To study the role of NS1 phosphorylation sites, we rescued NS1 mutants (Y73F and S83A) of A/swine/Shanghai/3/2014(H1N1) strain and compared their replication ability, cytokine production as well as the intracellular localization in cultured cells. Additionally, we used small interfering RNA (siRNA) assay to explore whether changes in the type I IFN response with dephosphorylation at positions 73 and 83 were mediated by the RIG-I pathway. RESULTS We checked 18 predicted sites in 30 SIV NS1 genes to exclude strain-specific sites, covering H1N1, H1N2 and H3N2 subtypes and identified two phosphorylation sites Y73 and S83 in the H1N1 SIV protein by Phos-tag SDS-PAGE analysis. We found that dephosphorylation at positions 73 and 83 of the NS1 protein attenuated virus replication and reduced the ability of NS1 to antagonize IFN-β expression but had no effect on nuclear localization. Knockdown of RIG-I dramatically impaired the induction of IFN-β and ISG56 in NS1 Y73F or S83A mutant-infected cells, indicating that RIG-I plays a role in the IFN-β response upon rSIV NS1 Y73F and rSIV NS1 S83A infection. CONCLUSION We first identified two functional phosphorylation sites in the H1N1 SIV protein: Y73 and S83. We found that dephosphorylation at positions 73 and 83 of the NS1 protein affected the antiviral state in the host cells, partly through the RIG-I pathway.

中文翻译：

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H1N1猪流感病毒NS1蛋白的酪氨酸73和丝氨酸83去磷酸化可减弱病毒复制并诱导高水平的β干扰素。

背景技术非结构蛋白1（NS1）是由甲型流感病毒（IAV）编码的毒力因子，在感染的最早阶段在宿主细胞的细胞核和细胞质中表达。NS1是一种多功能蛋白，在病毒复制，毒力和抑制宿主抗病毒免疫反应中起重要作用。然而，迄今为止，尚未鉴定出NS1的磷酸化位点，并且尚未完全阐明磷酸化与蛋白质功能之间的关系。方法在本研究中，通过Phos-tag SDS-PAGE分析以生物信息学方法预测并确定了猪流感病毒（SIV）NS1蛋白中潜在的磷酸化位点。要研究NS1磷酸化位点的作用，我们拯救了A /猪/上海/ 3/2014（H1N1）菌株的NS1突变体（Y73F和S83A），并比较了它们在培养细胞中的复制能力，细胞因子产生以及细胞内定位。此外，我们使用小干扰RNA（siRNA）分析来探讨I-IFN反应在73和83位上具有去磷酸化的变化是否是由RIG-I途径介导的。结果我们检查了30个SIV NS1基因中的18个预测位点，以排除菌株特异性位点，涵盖H1N1，H1N2和H3N2亚型，并通过Phos-tag SDS-PAGE分析鉴定了H1N1 SIV蛋白中的两个磷酸化位点Y73和S83。我们发现，NS1蛋白第73和83位的去磷酸化作用减弱了病毒的复制，降低了NS1拮抗IFN-β表达的能力，但对核定位没有影响。RIG-1的敲低显着削弱了NS1 Y73F或S83A突变感染细胞中IFN-β和ISG56的诱导，表明RIG-I在rSIV NS1 Y73F和rSIV NS1 S83A感染后的IFN-β反应中发挥作用。结论我们首先在H1N1 SIV蛋白中鉴定了两个功能性磷酸化位点：Y73和S83。我们发现NS1蛋白的73和83位上的去磷酸化部分地通过RIG-I途径影响了宿主细胞中的抗病毒状态。