流行性感冒病毒A型(IAV)每年會感染數百萬人引起呼吸道疾病。流行性感冒病毒A型感染時，病毒RNA 的感應器，包括TLR3、TLR7與RIG-I，都已經被證實可以偵測病毒的RNA 進而活化第一型干擾素相關的抗病毒防禦。同時，IAV也會發展出許多的策略對抗第一型干擾素的表現，使病毒在宿主細胞中成功複製。病毒的非結構蛋白1 (NS1) 已經被證實會透過競爭RIG-I與病毒的RNA結合，阻礙RIG-I下游的第一型干擾素活化。我們實驗室先前的研究發現病毒的NS1會透過RNA結合非依賴性機制拮抗RIG-I下游的E3泛素連接酶TRAF3，阻礙下游的第一型干擾素活化。因此，我研究的具體目標1，主要更進一部探討流行性感冒病毒A型病毒的非結構蛋白1在RIG-I訊號傳遞過程中，究竟是如何拮抗TRAF3並損害它的泛素化及活化。透過序列比對分析的方式，在非結構蛋白1 C端的effector domain有一個重要的TRAF3-結合序列(TRAF3-binding motif, TIM)。在這個重要的TIM產生一個非結構蛋白1的突變體，以測試它拮抗TRAF3的影響。我們的結果顯示非結構蛋白1-TIM突變體顯著性地消除它與TRAF3的相互作用，並因此降低它阻礙RIG-I 訊號傳遞產生第一型干擾素的能力。此外，帶有非結構蛋白1-TIM突變體的重組流行性感冒病毒A型在阻礙第一型干擾素活化是有缺陷的並且在小鼠伴隨著致病性的降低。我們的研究結果顯示新穎的TIM在非結構蛋白1負責拮抗TRAF3，引起抑制RIG-I 訊號傳遞產生第一型干擾素。我們發表的研究顯示，TAPE (TBK1-associated protein in endolysosomes)是一個轉接子涉及在調控TLR-3, TLR-4和RIG-I訊號傳遞路徑產生第一型干擾素。我們的實驗室有興趣的進一步探討TAPE在抗病毒先天免疫中的體內角色。因此，我的目標2，我主要探討TAPE在防禦對抗流行性感冒病毒A型感染的功能性角色。ELISA結果顯示，在流行性感冒病毒A型感染的期間，與對照組傳統的樹突狀細胞(cDCs)相比，TAPE缺乏的cDCs會損害第一型干擾素的產生。我們先前的研究顯示，就體重減輕和存活率方面，TAPE 條件性剔除小鼠對於流行性感冒病毒A型感染更敏感。我的論文更進一步證實，流行性感冒病毒A型病毒量在TAPE 條件性剔除小鼠的肺比起對照組小鼠的肺來的更高。總之，結果不僅證實TAPE在cDCs對於流行性感冒病毒A型感染的期間產生第一型干擾素是重要的，而且也是保護體內對抗流行性感冒病毒A型感染重要的調控因子。

Influenza A virus (IAV) infects millions of people to cause respiratory diseases every year. Viral RNA sensors including TLR3, TLR7 and RIG-I are shown to detect IAV RNA species to trigger type I interferons (IFNs)-mediated antiviral defenses. Meanwhile, IAV develops strategies to antagonize type I IFN expression for its successful replication in host cells. The IAV non-structural protein 1 (NS1) is shown to counteract RIG-I signaling to type I IFN induction through competing with RIG-I for viral RNA binding. Previous work from the lab revealed a viral RNA binding-independent mechanism for NS1 to counteract RIG-I signaling to type I IFN induction through targeting an E3 ubiquitin ligase TRAF3. Thus, the specific aim 1 in my current study is to further clarify the mechanism of how IAV NS1 targets TRAF3 to impair its ubiquitination and activation during RIG-I signaling. Through the sequence analysis, a potential TRAF3-binding motif (TIM) was found in the C-terminal effector domain of NS1. A NS1 mutant in this potential TIM was generated to test its effect on targeting TRAF3. Our results showed that the NS1-TIM mutant abolished its interaction with TRAF3 significantly, and consequently reduced its ability to block RIG-I signaling to type I IFN induction. Furthermore, a recombinant IAV harboring a NS1-TIM mutant was defective in blocking type I IFN induction and concomitantly reduced pathogenicity in mice. Our findings reveal a novel TIM in NS1 responsible for targeting TRAF3, leading to the suppression of RIG-I signaling to IFN-β production. Our published studies have shown that TBK1-associated protein in endolysosomes (TAPE) is an adaptor implicated in regulating the TLR-3, TLR-4, and RIG-I like receptors pathways to type I IFN activation. Our lab has an interest to further explore in vivo role of TAPE in antiviral innate immunity. Thus, in my aim 2, I focus on studying the functional role of TAPE in defending against IAV infection. Results from ELISA showed that TAPE-deficient cDCs compared to control cDCs were impaired in IFN-β cytokines production upon IAV infection. Our previous study revealed that TAPE condition knockout (cKO) mice are more susceptible to IAV infection in terms of the body weight loss and survival rate. My thesis further demonstrated that IAV titers in lungs of TAPE condition knockout (cKO) mice were much higher than those of Tapef/f littermates in vivo. Together, the results not only demonstrate that TAPE is essential for triggering type I IFN production in primary cDCs in response to IAV infection but also is an important regulator to protect against IAV infection in vivo.

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