流感病毒和宿主先天免疫系統的交互作用是複雜的。在細胞被RNA流感病毒感染時，RIG-I是一個位於細胞質且可以偵測5端帶有triphosphate官能基的病毒RNA的重要蛋白質，進而引起第一型干擾素等抗病毒的先天免疫反應。在這其中，流感病毒發展了各式各樣的策略，以利於逃脫宿主抗病毒機制。其中最著名的策略之一是流感病毒的NS1蛋白會與RIG-I競爭病毒RNA的結合並阻斷活化下游產生第一型干擾素的訊息傳遞路線。 然而，新興的證據以及我們實驗室初步的發現都顯示著流感病毒的NS1蛋白可以並非藉由競爭病毒RNA的方式去阻斷RIG 所主導產生第一型干擾素的訊息傳遞路線。TRAF3是位於RIG-I所媒介產生第一型干擾素的訊息傳導路徑中一個重要的E3泛素連接酶。我們實驗室發現NS1蛋白利用它的effector區域 (ED)去結合RIG-I 下游的訊息傳導分子TRAF3以抑制第一型干擾素的產生。在本篇論文中，我們著重於研究NS1如何抑制TRAF3活化的分子機制。經由共軛焦螢光顯微鏡的分析結果顯示在HEK293 細胞中NS1蛋白會與內生性的TRAF3有colocalization 的現象。這顯示著NS1有可能會以TRAF3為標的蛋白。緊接著，我們進一步剖析NS1和TRAF3 交互作用中所需的蛋白質區域。經由共同免疫沉澱法分析結果顯示位於NS1的ED 的羧基端區域 (NS1-ED-C, a.a.125-230)和位於TRAF3羧基端的TRAF區域(a.a. 340-568) 是主要參於此交互作用的區段。在RIG-I活化後TRAF3會被行K63位泛素化的轉譯後修飾，已變成活化態的TRAF3。在外給具有活化下游能力的RIG-I 片段後，經由偵測TRAF3的K63位泛素化結果說明了NS1的ED-C 對於抑制TRAF3的K63位泛素化是重要的。並且NS1的ED-C在抑制經由表現RIG-I 片段或是IPS-1蛋白所持續活化干擾素β的啟動子活性也扮演重要角色。進一步經由共同免疫沉澱法分析結果顯示著NS1-ED 可能會和IPS-1去競爭TRAF3的結合。在病毒感染時，病毒的NS1會與內生性的TRAF3有colocalization 的現象。RIG-I片段蛋白所持續活化TRAF3的K63位泛素化在病毒感染時是被抑制的。總結而言，我們的研究在流感病毒NS1藉由抑制TRAF3活化而抑制了RIG-I所媒介的第一型干擾素的產生中，揭開了新的一面。而第一型干擾素在抗流感病毒的先天免疫中扮演不可或缺的角色。

The interaction between the host innate immune system and influenza A virus (IAV) infection is complicated. During IAV infection, a key cytosolic sensor RIG-I detects 5’ triphosphate viral RNA to trigger type I IFN-mediated antiviral responses. Meanwhile, IAV develops the multiple strategies to evade the host antiviral responses. One of the prominent examples is that IAV non-structural protein 1 (NS1) competes with RIG-I for IAV viral dsRNA binding, thereby blocking RIG-I sensing and activation of the downstream pathway to type I IFN production. However, emerging evidence and our preliminary findings suggest that IAV NS1 protein blocks RIG-I-mediated IFN-β activation through an RNA binding-independent manner. Particularly, our data showed that NS1 employed its effector domain (ED) to target a RIG-I downstream mediator TRAF3, which is an E3 ubiquitin ligase essential for IFN-β activation. In this study, we focus on the molecular mechanisms of how IAV NS1inhibits TRAF3 activation. Results from the confocal microscopy analysis showed the colocalization of IAV NS1 and endogenous TRAF3 in HEK293 cells. It suggests that TRAF3 is a potential target of IAV NS1. Subsequently, we dissected the interaction domains between NS1 and TRAF3. The co-immunoprecipitation analyses showed that the C-terminal region of effector domain (NS1-ED-C, a.a.125-230) of NS1 and the C-terminal TRAF domain (a.a. 340-568) of TRAF3 were the main regions involved in this interaction. The in vivo ubiquitination assays indicated that ED-C of NS1 was important for inhibiting the K63-linked poly-ubiquitination of TRAF3 upon RIG-I stimulation. Reporter assays indicated that ED-C of NS1 played an important role in blocking the IFN-β activation upon RIG-I or IPS-1 stimulation. Furthermore, results from the co-immunoprecipitation analyses suggest During IAV infection, NS1 was also shown to be colocalized with TRAF3. Furthermore, the K63-linked poly-ubiquitination of TRAF3 stimulated by an active mutant RIG-I-CARD was inhibited during IAV infection. Collectively, our study provides a novel insight into how IAV NS1 ED functions to block TRAF3 ubiquitination to intercept RIG-I signaling to type I IFN production, which is essential for mounting antiviral immune responses.

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