日本腦炎病毒非結構蛋白NS5阻擾第一型干擾素訊息傳遞及其與細胞因子之交互作用

Japanese Encephalitis Virus NS5 interferes IFN-ß Signaling and its interactions with cellular factors

鄭美秀(MEI-HSIU CHENG)

指導教授 ： 張清堯 ； 林振文

亞洲大學/健康學院/生物科技學系碩士班/碩士(2008年)

摘要

中文摘要
日本腦炎病毒（Japanese encephalitis virus，JEV）是黃質屬病毒的成員之一，其基因體可以轉譯成三個結構蛋白（核蛋白、前膜蛋白、套膜蛋白）及七個非結構蛋白（NS1、NS2a、NS2b、NS3、NS4a、NS4b、NS5）為一含有單股正性的RNA病毒，受感染後主要會入侵神經系統，引起急性腦膜炎並使腦、腦膜和脊髓受損。日本腦炎病毒NS5蛋白所主導產生的RNA聚合酶是病毒基因體複製的關鍵酵素，亦有研究顯示除了影響病毒複製的能力。NS5蛋白已被證實可以抑制第一型干擾素的訊息傳遞。本篇研究目的在於探討NS5蛋白與宿主細胞的蛋白質產生交互作用及其作用與阻斷干擾素訊息傳遞的相關性。以 phage display篩選Brain cDNA library中與NS5交互作用的細胞蛋白。熱休克蛋白70(Heat shock protein 70)被篩選為與NS5交互作用的細胞蛋白之一。利用免疫共同沉澱-西方墨點法發現NS5 和Hsp70蛋白質會有交互作用；並且以共軛焦顯微鏡觀察發現NS5可抑制干擾素處理細胞之STAT1進入細胞核，但是NS5及Hsp70共同存在之下，干擾素處理細胞之STAT1則可進入核內。進一步，以冷光報導基因顯示NS5對干擾素處理細胞之ISRE驅動子冷光報導基因活性具有抑制作用，而與Hsp70交互作用後NS5對干擾素處理細胞之ISRE驅動子冷光報導基因活性的抑制則下降。最後，以二維電泳等蛋白質體技術分析對照細胞與NS5表現細胞對干擾素處理後的蛋白體差異。經軟體比對後發現5個差異性超過2及7個低於0.5的蛋白質點，如HSP60、tubulin beta chain、 calreticulin 、EDAR-associated death domain、Triose-phosphate isomerase、CDK5、Fascin、cyclophilin A、Enolase 1、glyceraldehyde 3-phosphate dehydrogenase、ACTB protein等。因此推測宿主蛋白質Hsp70和日本腦炎病毒NS5蛋白的交互作用，可能與阻擾干擾素訊息傳遞有關，而干擾素處理後的蛋白體差異可幫助推論NS5阻擾干擾素訊息傳遞路徑的佐證。本論文研究成果可利未來進一步研究日本腦炎病毒阻擾干擾素的機制及其治療。

關鍵字

日本腦炎 ； 非結構蛋白 ； 干擾素

並列摘要

Abstract
Japanese encephalitis virus (JEV), a member of Flavivirus, contains a single-stranded positive-sense RNA genome. JEV genome encodes for 3 structural proteins (C, prM, and E) and 7 nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). The non-structural protein 5 (NS5) of Japanese encephalitis virus encodes RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral RNA genome. JEV NS5 protein has been demonstrated the inhibitory effect on the interferon (IFN)-α/β response by blocking Jak-Stat singling pathway. In this study, we intend to investigate the interaction of JEV NS5 protein with host cellular proteins on affecting the IFN-α/β signaling. Heat shock protein 70 (HSP70) was identified as one of the NS5-interacting proteins from the biopanning of a phage displayed human brain cDNA library with bacterial recombinant NS5 protein. Interaction between JEV NS5 and HSP70 in the TE671 human medulloblastoma cells was verified by coimmunoprecipation and confocal imaging. Importantly, interferon stimulating response element (ISRE)-luciferase reporter assay showed that the both expression of JEV NS5 and HSP70, but not the single expression of JEV NS5 had a significant response to IFN-stimulated Jak-Stat signaling in TE671 cells. In addition, confocal imaging indicated IFN-activated STAT1 nuclear translocation was found in the TE671 cells co-expressing JEV NS5 and HSP70, but not in the NS5-expressing cells. Furthermore, the combination of two-dimensional difference gel electrophoresis and mass spectrometry identified up- and down-regulated proteins in response to blocking IFN-stimulated Jak-Stat signaling by JEV NS5 protein. The identified proteins were HSP60、tubulin beta chain, calreticulin, EDAR-associated death domain, triose-phosphate isomerase, CDK5, fascin, cyclophilin A, enolase 1, glyceraldehyde 3-phosphate dehydrogenase and ACTB protein. Therefore, the results demonstrated the interaction of JEV NS5 with HSP70 was involved in blocking IFN-stimulated Jak-Stat signaling. The difference in the IFN-stimulated proteome profiling between mock cells and NS5-expressing cells could be useful for the system-based elucidation of the JEV NS5 role in the IFN antagonist function. The study could be helpful for the investigation of JEV-induced innate immune escape and the clinical therapy against the JEV infection.

並列關鍵字

Japanese Encephalitis Virus ； NS5 ； interferon

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