2012-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/655713摘要：腸病毒 71 型感染可以說是後小兒麻痺時代，最容易造成兒童中樞神經系統感染與傷害的病毒。然而，至今醫界對於腸病毒71 型的分子致病機轉依然了解有限。我們先前的研究結果發現，腸病毒71 型2B 藉由與Mcl-1 的交互作用，增進了Mcl-1在細胞內的穩定度，進一步抑制了細胞的凋亡，更進一步確保了病毒複製的完成。在本計劃的第一年中，我們已經進一步證實我們的假說，腸病毒的2B 蛋白確實可以調控其他具有BH3 domain 的Bcl-2 家族蛋白，包括Bcl-2、Bcl-2A1、Bik、Noxa 等等。這些結果，大幅增進了我們對於腸病毒71 型如何調控細胞凋亡的了解。除了 Mcl-1 等蛋白，我們之前利用yeast two hybrid system 針對腸病毒71 型蛋白的交互作用對象做篩選時，同時也找到其他幾種可以與2B 蛋白交互作用的細胞蛋白，包括腫瘤壞死因子接受體家族的成員9 與18 (tumor necrosis factor receptor superfamily(TNFRSF) members: TNFRSF9 and TNFRSF18)以及甲型干擾素誘發蛋白27(interferonalpha-inducible protein 27, IFI27)。這些都與T 細胞活化的調控有關。本計畫的第一年也已經證實這些交互作用確實存在。我們利用co-immunoprecipitation 的方法證明細胞內大量表現的腸病毒71 型2B 蛋白可以與大量表現的TNFRSF9, TNFRSF18 與IFI27 直接交互作用。我們也利用共軛焦顯微鏡觀察到這些蛋白確實會跟腸病毒71 型2B 蛋白出現在同樣的細胞內位置。本計畫的第二與第三年將進一步探討腸病毒 71 型2B 蛋白與這些分子交互作用的生物意義。這部分亦屬前所未有的開創性研究，也頗具臨床意義，因為腸病毒71 型感染重症病患常常出現嚴重的全身性發炎反應，其臨床表現並不能單純用病毒侵犯與破壞來解釋。腸病毒71 型感染是否會過度激化T 細胞，導致免疫系統失調是一個可能性。了解腸病毒71 型2B 蛋白與這些T 細胞活化路徑相關的接受體的關係，有可能有重要的新發現，也可能提供治療嚴重腸病毒71 型感染新的新思維。<br> Abstract: In the post-polio era, enterovirus 71 (EV71) is the most important etiology of CNSinfections resulting in mortalities or severe neurological deficits in children. However, themolecular pathogenesis of EV71 remained not fully understood.Our earlier studies have shown 2B protein of EV71 regulates expression levels of Mcl-1by direct interactions. Normally, Mcl-1 is tightly regulated by degradation through theubiquitin-proteasome pathway. We have proved that EV71 2B competitively inhibits ubiquitinE3 ligase of Mcl-1. In the presence of EV71 2B, ubiquitination of Mcl-1 is inhibited due tothe blocking of E3 liagase (MULE) docking site (Bcl-2 homologue domain 3) by EV71 2B.As a result, Mcl-1 stability is increased and cellular apoptosis is suppressed. Using EV71infectious DNA clones, we also confirmed that EV71 whose ability to interact with Mcl-1 waslost by mutation is defective in replication. In the first year of the current study, we havefurther confirmed our hypothesis that EV71 directly interacts Bcl-2 members harboringBH3 domain, including Bcl-2, Bcl-2 A1, BiK, and Noxa.On the other hand, in the yeast two-hybrid screening, we also identified several othercellular factors as EV71 2B-interacting proteins. These included tumor necrosis factorreceptor superfamily (TNFRSF) members: TNFRSF9 and TNFRSF18. In addition, interferonalpha-inducible protein 27 (IFI27) was also picked up by the screening. These molecules areinvolved in signaling of T cell activations. Our first year work also confirmed directinteractions between EV71 2B and TNFRSF9, TNFRSF18 and IFI27. We haveconfirmed direct interaction by co-immunoprecipitation experiments and confocalmicroscopic imaging.These proteins are involved in the regulation of host T cell activation is of specialinterests because severe EV71 infections trigger severe systemic inflammatory responsesclinically. The clinical presentation cannot be fully explained by direct viral invasions only.Over activation or dysregulation of T cell activities may also play important roles. EV71possibly execute its modulation on host cytokine signaling through 2B/TNFRSF interactions.Understanding of these molecular pathways will provide basis not only for basic virologicstudies but also studies of therapeutic options in the future.