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研究生： 林峻暘
Lin, Chun-Yang
論文名稱： 宿主先天性免疫與流行性感冒病毒A型之交互作用
The interactions between the host innate immune system and influenza A virus
指導教授： 凌斌
Ling, Pin
學位類別： 碩士
Master
系所名稱： 醫學院 - 微生物及免疫學研究所
Department of Microbiology & Immunology
論文出版年： 2012
畢業學年度： 100
語文別： 英文
論文頁數： 42
中文關鍵詞： 先天性免疫 、流感病毒 、NS1蛋白 、第一型干擾素
外文關鍵詞： innate immunity, TAPE, influenza A virus, NS1, type I interferon
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    • 宿主與流感病毒(IAV)之間的先天性免疫反應為複雜的訊息網，最終可能導致臨床上病情的好轉與惡化。RIG-I是細胞內主要偵測IAV感染的胞內受器。然而，對於IAV感染如何活化RIG-I的詳細機制仍尚未釐清。TAPE是目前我們發現新的先天性免疫調控分子，已知能夠調控TLR3/TLR4以及RIG-I like receptor。我們將專注於兩大主軸：第一、研究TAPE在因應IAV感染時調控RIG-I訊息路徑的腳色；第二、研究IAV NS1蛋白干擾RIG-I活化第一型干擾素之路徑的分子機制。抑制TAPE在人類肺臟上皮細胞A549的表現會導致IAV感染時干擾素b的分泌下降。TAPE基因剔除小鼠之纖維母細胞對於IAV感染所活化的干擾素b啟動子與mRNA表現也相較於正常小鼠來得低。此外，利用siRNA降解或基因剔除使細胞內的TAPE缺乏會導致流感病毒感染初期的病毒RNA複製速率上升。另外，在先前的研究已經證明IAV的NS1蛋白會與RIG-I競爭病毒RNA的結合並阻斷為活化RIG-I所進行的ubiquitination。為了更細分探討IAV NS1不同蛋白區域與宿主先天性免疫之間交互作用的詳細機制，我們製造出表現兩種NS1區域的質體：RNA binding區域(RBD)以及effector區域(ED)。利用干擾素b啟動子報導基因分析發現NS1- RBD只能阻斷IAV病毒RNA所刺激的干擾素b啟動子活化，而在poly I:C(另一雙股RNA類似物)刺激之下則沒有此抑制效果。這結果暗指出NS1-RBD能夠辨認IAV病毒RNA上的5’-triphosphate的官能基以避免RIG-I的偵測。此外，NS1全長或NS1-ED則能夠用不以RNA結合的方式阻斷RIG-I所媒介的干擾素b活化。為了探討NS1-ED抑制第一型干擾素的潛在腳色，我們利用免疫共沉降法發現NS1-ED能夠與ubiquitin E3連接酶─TRAF3，此連接酶對於許多先天性免疫偵測蛋白至第一型干擾素的活化所必需。NS1-ED也能抑制由△RIG-I過度表現所活化的TRAF3造成的K63位點的多重泛素化(polyubiquitination)。總結而言，我們的結果顯示TAPE是病毒感染所活化RIG-I訊息中的重要調控蛋白，並且IAV NS1具有以特殊抑制能活化第一型干擾素的TRAF3達到能不以RNA結合的方式阻斷干擾素產生。我們的研究提供了TAPE於先天性免疫對抗IAV病毒感染的重要性，並發現NS1針對TRAF3以阻斷先天性免疫反應的新的機制。

    Host innate immune responses to influenza A virus (IAV) are complex networks, which may ameliorate or exacerbate clinical outcome. Retinoic acid-inducible gene I (RIG-I) is a major cytosolic sensor for sensing influenza virus infection. However, the detailed mechanism of RIG-I activation in response to IAV infection is still unclear. TAPE (TBK1-associated protein in endolysosomes) is a novel innate immune regulator, which modulates the TLR3/TLR4 and RIG-I-like receptor pathways. We focus on two key aspects (1) to study the role of TAPE in regulating the RIG-I pathway in response to IAV infection, and (2) to study molecular mechanisms of how IAV NS1 protein counteracts the RIG pathway to type I IFN production. Knockdown of TAPE in a human lung epithelial cell line A549 led to the impairment of IFN-b production upon IAV infection. TAPE-/- mouse embryonic fibroblasts (MEFs) compared to wild type MEFs also showed the decreased induction of IFN-b promoter and mRNA expression upon IAV infection. Furthermore, both knockdown and knockout of TAPE led to enhance the IAV mRNA replication at the early stage of IAV infection. Previous studies indicated that IAV NS1 competes with RIG-I for binding viral RNA and blocks RIG-I ubiquitination for activation. To further dissect the mechanisms by which IAV NS1 protein counteracts the host innate immune responses, we generated two truncated NS1 proteins: the RNA binding domain (RBD) and the effector domain (ED) for this purpose. Results from reporter assays showed that NS1-RBD blocked RIG-I-mediated IFN-b promoter activation induced by IAV viral RNA but not poly I:C (a dsRNA analog), suggesting that NS1-RBD is responsible for recognizing the 5’- triphosphate moiety on IAV viral RNA to avoid RIG-I sensing. Moreover, expression of NS1-full length or its ED blocked RIG-I mediated IFN-b induction in an RNA binding-independent manner. To explore the potential targets of NS1-ED in the RIG-I pathway to type I interferon induction, we screened several RIG-I downstream mediators for interaction with NS1-ED. Co-immunoprecipitation analyses showed that NS1-ED interacted with TRAF3 ubiquitin E3 ligase, which is essential for linking several sensors to type I interferon induction. K63-linked poly-ubiquitination of TRAF3 induced by △RIG-I overexpression was significantly inhibited by the ED of NS1. Collectively, our results suggest that TAPE is a key modulator in the RIG-I pathway in response to IAV infection and that IAV NS1 protein displays the ability to block IFN-b production in a RNA binding-independent manner. Our studies demonstrate the importance of TAPE in antiviral innate immunity against IAV infection and reveal a novel mechanism by which NS1 targets TRAF3 to block innate immune activation.

    1. Introduction I. Pathology of Influenza A virus......................7 II. The life-cycle of the Influenza A virus............7 III. Innate recognition of influenza A virus...........8 IV. TAPE (TBK1-associated protein in endo-lysosome) in the endosomal TLR and cytosolic RLR pathways...............9 V. Interactions between IAV NS1 and the innate immune system.................................................10 2. Materials and methods I. Cell, Viruses, and Reagents........................12 II. Plasmids .........................................12 III. Transfection.....................................12 IV. RNA Interference..................................13 V. Luciferase assay...................................13 VI. Cytokine quantification in cell supernatants......13 VII. Coimmunoprecipitation and Western Blot Analysis..14 VIII. RNA isolation and Reverse transcription polymerase chain reaction (RT-PCR)...............................15 IX. Real-time PCR.....................................15 X. Plaque assay.......................................15 XI. RNA pulldown......................................16 3. Results I. TAPE is essential for type I interferon production in a human lung epithelial cell line A549 upon IAV infection...17 II. TAPE knockout MEFs are impaired type I interferon production upon IAV infection.............................17 III. Effects of NS1 wt and mutants on RIG-I-mediated IFN-b activation by poly(I:C) or 5’-triphosphate dsRNA.........18 IV. The RNA binding domain of LAV NS1 is involved in recognizing 5’-triphosphate moiety of LAV viral RNA......18 V. The effecter domain of LAV NS1 blocks TRAF3 activation in an RNA binding independent manner......................19 4. Discussion.............................................21 5. References.............................................25 6. Figures and figure legends ............................30 7. Appendix...............................................40 I. TAPE knockdown abolishes the RIG-I pathway in response to influenza virus infection..............................40 II. Down regulation of IAV vRNA expression in TAPE deficiency cells at late infection time points............41 III. The amino acids sequence alignment of NS1 protein from pandemic IAV strain.......................................42

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