流行性感冒A型病毒中包含有八段負股RNA基因組，其中第八段RNA基因會經由轉錄合成出mRNA，進而轉譯出第一型非結構性蛋白non-structural protein 1，而第八段RNA基因所產生的mRNA也會經由選擇性剪切形成另一切割形式的mRNA，製造另一個非結構性蛋白質 non-structural protein 2 (NS2)又稱為Nuclear export protein (NEP)。這些蛋白主要會影響病毒複製和傳播，所以在病毒毒性扮演重要角色。而今已知NS1為多功能調控性蛋白，在病毒生活史上較廣為所知，例如它可促進病毒RNA 複製，並可抑制宿主細胞第一型干擾素 (Type I Interferon)，然而目前研究僅知道NEP蛋白在參與病毒RNP (viral ribonucleoprotein)之核質運送。其可能扮演的角色及有交互作用的細胞蛋白並不清楚，為了進一步研究NEP在病毒中和宿主細胞中所參與的機制，及與其有交互作用的細胞蛋白有整體性的瞭解，我們利用串聯親和標籤蛋白純化技術及蛋白質體學尋找這些蛋白，並發現近流感A型病毒中NEP會和18個可能有交互作用的細胞蛋白。這些宿主細胞蛋白依功能可分為：細胞訊息核酸蛋白複合物之傳移、II型核酸聚合酶之轉錄調控、JAK磷酸酶之訊息傳導、及ATP之平衡調控相關。其中前二大類之功能性與已知的流感病毒 RNA之複製及運送非常相關，後二大類則可能與細胞命運，細胞激素調控或助長病毒複製滋長相關。為了印證NEP蛋白質體學中NEP 和 ATP合成酶交互作用，首先我們以免疫共沉澱法及免疫螢光染色先確認NEP 和 ATP合成酶會結合在一起。NEP和粒線體ATP合成酶交互作用也令我們好奇是否影響細胞免疫功能或造成細胞壓力而產生凋亡。另外在前人研究中發現有嗜菌體或病毒可能會產生與ATP合成酶相似結構的蛋白，這些蛋白與病毒核糖核酸結合後會將遺傳物質運送進入新病毒顆粒中，對於病毒繁衍非常重要。在此我們報導NEP可和粒線體中的ATP合成酶 F1 聚合體中 α 及 β次單元體產生交互作用，我們認為他們的功用主要是在幫助病毒繁衍並且會降低細胞內的ATP 濃度，但卻不影響粒線體膜電位的缺失，也不會造成細胞ROS的增加與細胞凋亡。我們證明了NEP和ATP合成酶的結合不只對細胞中ATP濃度造成影響，我們的結果中也發現NEP蛋白可以抑制宿主細胞第一型干擾素，並且調降細胞中ATP能量產生，而這也讓我們知道NEP在幫助A型流行性感冒病毒的毒性、繁衍及傳播上也扮演著重要角色。

The 8th genome segment of Influenza A Viruses (IAV) directs the synthesis of a collinear mRNA encoding for the non-structural protein 1 (NS1) and a spliced mRNA encoding for the nuclear export protein (NEP). While NS1 is largely known as a multi-regulatory factor in facilitating viral RNA replication and antagonizing host Inteferon response, little has been reported regarding the insight contribution of NEP and its cellular partners upon IAV replication. To gain a systemic view of cellular proteins participated in NEP-mediated activities, a Tandem Affinity Tag Purification based proteomic approach was employed and has identified 18 candidates with functional relevance in RNP trafficking, Pol-II transcriptional control, Janus kinase (JAK) signaling, or mitochondria ATP regulation. The first two categories match the predicted role of NEP in vRNA replication and nucleo-cytoplasmic transport of viral RNPs, while the last two categories may accord with novel activities of NEP in cell fates or cytokine regulation for viral multiplication. We report here that NEP interacts with mitochondria ATPsynthase F1 α/β subunits by co-immunoprecipitation assay and a confocal imaging analysis. In addition, NEP downregulated cellular ATP level, but did not affect mitochondria membrane potential or cause cellular ROS and apoptosis. As IAV, unlike many RNA viruses, lack of self-equipped motors similar to the α subunit of F1 to package their genomes, whether NEP hijacks ATP5synthase for vRNP packaging is under investigation. In addition, NEP antagonized type-1 IFN response, whether this links to the down regulated ATP level or the interference of JAK signaling pathway require further investigation. Collectively, our results reveal NEP as a multi-regulatory factor to influence ATP homeostasis and facilitate IAV egression and induced pathogenesis.

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