A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid-inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function.

中文翻译：

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流感NS1蛋白通过与RIG-I的第二个CARD的菌株特异性直接相互作用来调节RIG-I的激活。

甲型流感病毒非结构蛋白1（NS1）的关键作用是拮抗宿主细胞的抗病毒应答。NS1通过与宿主蛋白（包括细胞质病原体识别受体，视黄酸诱导型基因I（RIG-I））的大量相互作用来实现这一作用。尽管已经研究了这种相互作用的后果，但尚不清楚NS1拮抗RIG-1信号转导的完整机制。先前我们证明了NS1 RNA结合结构域（NS1RBD）与RIG-1的第二个半胱天冬酶激活和募集结构域（CARD）直接相互作用。我们还发现，NS1RBD（R21Q）中的单个菌株特异性多态性完全消除了这种相互作用。在这里，我们调查了R21Q突变对NS1拮抗RIG-I信号传导能力的功能后果。我们观察到，在NS1中带有R21Q突变的流感病毒会导致RIG-I信号显着上调。为此，我们确定NS1中的R21Q突变导致NS1拮抗TRIM25介导的RIG-I CARD泛素化的能力明显下降，这是RIG-I激活的关键步骤。我们还观察到，WT NS1能够直接与串联RIG-I CARD结合，而NS1中的R21Q突变则显着抑制了这种相互作用。此外，我们确定R21Q突变不会阻碍NS1和TRIM25之间的相互作用或NS1RBD结合RNA的能力。此处提供的数据为RIG-I的NS1拮抗作用提供了重要见解，并说明了了解特定于NS1功能的菌株特异性多态性的作用的重要性。