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    Please use this identifier to cite or link to this item: http://asiair.asia.edu.tw/ir/handle/310904400/2508

    Title: Japanese Encephalitis Virus NS5 interferes IFN-? Signaling and its interactions with cellular factors
    Authors: MEI-HSIU CHENG
    Contributors: Department of Biotechnology
    Keywords: Japanese Encephalitis Virus;NS5;interferon
    Date: 2008
    Issue Date: 2009-11-06 14:11:16 (UTC+8)
    Publisher: Asia University
    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.
    Appears in Collections:[生物科技學系] 博碩士論文

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