訊息傳遞途徑調控牛流行熱病毒進入細胞及誘發自噬體有利於病毒複製

Signal transduction pathways mediating bovine ephemeral fever virus entry and BEFV-induced autophagosome that is beneficial for virus replication

吳銘宏(Ming-Hung Wu)

指導教授 ： 劉宏仁 ； 施玟玲

國立屏東科技大學/農學院/生物科技研究所/碩士(2011年)

https://doi.org/10.6346/NPUST.2011.00134

摘要

牛流行熱病毒 (Bovine ephemeral fever virus； BEFV) 為子彈型病毒科 (rhabdoviridae)成員之一，感染後會導致牛隻急性高燒、呼吸困難及降低泌乳量等，而影響經濟效益。本實驗室已證實牛流行熱病毒經由clathrin-mediated endocytosis 及dynamin-dependent 並需要endosome 之酸化及microtubule 之運送進入宿主細胞，但其活化機制及訊息傳遞路徑尚未證實。本研究首次發現BEFV感染細胞5分鐘誘發Src及JNK磷酸化後而增加COX-2、clathrin 及dynamin-2 之表現量。以CSK (C-terminal Src kinase) 及藥物進行抑制試驗進一步證實牛流行熱病毒藉由引發Src-JNK/SAPK-COX-2 調控路徑使clathrin 及dynamin-2 表現量增加以利病毒進入細胞。此外，以dominant negative Rab5抑制early endosome 形成，結果明顯抑制病毒複製。在另一方面，先前我們已證實利用rapamycin 抑制mTOR (mammalian target of rapamycin) 活性會增強牛流行熱病毒之複製。細胞自噬指細胞對抗外在環境或飢餓時為維持自體衡定的一種降解蛋白質過程。本研究發現牛流行熱病毒感染細胞會增強細胞自噬小體 (autophagosome) 上的細胞微管相關蛋白輕鏈3-II (microtubule-associated protein light chain 3-II, LC3-II) 表現量。利用3-MA (3-methylademine) 及LC3b shRNA 抑制細胞自噬會降低病毒蛋白合成及力價，而以rapamycin 抑制mTOR 活性或營養匱乏使細胞自噬活化亦增強病毒複製。為了證實牛流行熱病毒活化Src、JNK/SAPK 及COX-2與細胞自噬有關，經證實病毒感染後使用Src、JNK/SAPK 及COX-2 抑制劑或感染過度表現Bcl-2 細胞皆抑制LC3-II 之表現量及病毒複製。我們的結果顯示牛流行熱病毒引發Src-JNK/SAPK-COX-2 訊息傳遞路徑，可於病毒感染早期促進病毒進入細胞及於病毒感染中期誘導細胞自噬小體產生以增強利於自身複製。

關鍵字

牛流行熱病毒 ； clathrin-mediated endocytosis ； dynamin-2 ； 細胞自噬 ； 自噬小體 ； 微管相關蛋白輕鏈3 ； Bcl-2

並列摘要

Bovine ephemeral fever virus (BEFV) is a member of the rhabdoviridae family. Cattle infected by BEFV has signs and syndromes including acute fever, dyspnea, and decreased milk yield. We have confirmed that clathrin-mediated endocytosis, dynamin-dependent, endosome acidification and microtubule-dependent transport are required for the cell entry of BEFV. However, the related activation mechanisms and signaling pathways remain unknown. In this study, our results showed that, for the first time, BEFV causes phosphorylations of Src and JNK/SAPK and increases the expressions of COX-2, clathrin and dynamin-2 at 5 or 9 min postinfection. The CSK (C-terminal Src kinase) and pharmacological inhibition tests further confirm that BEFV induces the expressions of clathrin and dynamin-2 via Src-JNK/SAPK-COX-2 pathway to facilitate the cell entry. The blocking in the early endosome formation by dominant negative Rab5 results in repression of BEFV replication. On the other hand, we have demonstrated previously that inhibition of mTOR (mammalian target of rapamycin) by rapamycin enhances replication of BEFV. Autophagy is a protein degradation process by which cells keep homeostasis under stress conditions or during starvation. In this study, we uncover that BEFV increases the level of LC3-II (microtubule-associated protein light chain 3-II) , an autophagosome marker protein. Treated cells with 3-MA and LC3b shRNA which inhibit autophagy formation decreased the production of viral protein and progeny virus titer. When autophagy was induced by either rapamycin or starvation, the viral replication was remarkably increased. To confirm that activation of Src, JNK/SAPK and COX-2 by BEFV is linked to autophagy, the LC3 II expression and viral replication are both suppressed by treating pharmacological inhibitions of Src, JNK/SAPK, COX-2 after BEFV infection and over-expression of Bcl-2 in cells. The results suggest that BEFV can induce Src-JNK/SAPK-COX-2 signaling pathway to promote the cell entry in the early stagy of viral infection and to promote the formation of autophagosome in the middle stagy of viral infection that enhances the viral multiplication.

並列關鍵字

BEFV ； clathrin-mediated endocytosis ； dynamin-2 ； autophagy ； autophagosome ； LC3 ； Bcl-2

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