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Title: | 魚類神經壞死症病毒與細胞自噬的交互作用機制 Interaction between betanodavirus and autophagy |
Authors: | Ren-Xiang Wang 王任翔 |
Advisor: | 齊肖琪(Shau-Chi Chi) |
Keyword: | 魚類神經壞死症病毒,粒線體膜電位,自噬體,ATP,自噬體抑制劑, nervous necrosis virus,mitochondrial membrane potential,autophagy,ATP,autophagy inhibitors, |
Publication Year : | 2009 |
Degree: | 碩士 |
Abstract: | 神經壞死症病毒(NNV)屬於單股正意RNA病毒, NNV感染細胞後會造成粒線體膜電位的流失。自噬體(autophagosome)是細胞在饑餓狀態時形成的雙層膜囊狀構造,可將長效型蛋白質及胞器分解產生胺基酸及ATP,以調控細胞內的能量。本篇研究首次在魚類細胞中發現NNV與細胞自噬之間交互影響的機制。細胞轉殖pEGFP-LC3質體後再感染NNV,可在細胞內觀察到具螢光的細胞自噬體。運用luciferase氧化ATP所放出的冷光量作定量分析,發現細胞在病毒感染後12小時內,ATP會逐漸下降;當病毒感染濃度越高,ATP量則下降越多。用cycloheximide抑制病毒蛋白質的轉譯,ATP量就恢復到未感染時狀態,因此NNV蛋白質的轉譯會造成受病毒感染細胞ATP量的下降。另外,當細胞被高濃度NNV感染時,會出現大量的自噬體,當NNV蛋白轉譯被CHX抑制時,自噬體數量就明顯減少,因此NNV蛋白轉譯會誘發自噬體數量增加。以饑餓法使細胞內ATP量先大幅降低再感染NNV,結果饑餓組細胞內的NNV蛋白量比正常細胞感染NNV組的少,但自噬體數量卻相對較多,因此推論,NNV感染細胞所誘發的自噬作用主要是因NNV造成ATP下降所引起,而不需經由NNV蛋白質過多造成的ER stress所誘發。當自噬體的功能被抑制時,受感染細胞的病毒產量則明顯下降,因此推論,自噬作用有助於病毒的複製,但免疫螢光染色結果顯示, NNV並不會在細胞自噬體膜上形成複製複合體。細胞感染病毒後,再用三種藥劑分別去抑制細胞自噬作用,結果藥物處理組細胞內的ATP量比未加藥劑組低,病毒力價亦較低。當飢餓細胞的自噬作用被藥物抑制時,細胞內的ATP量大幅減少,感染NNV後所產生的病毒量也比未加抑制劑組少,因此推測,病毒感染所誘發的自噬作用,有助於細胞回收蛋白質及胞器來產生ATP,供應病毒複製之用。 Nervous necrosis virus (NNV) is a positive single-stranded RNA virus. NNV infection was reported to induce the loss of mitochondria membrane potential. Autophagy is a unique membrane traffic system for engulfing long-lived proteins and organelles to maintain homeostasis of cellular energy during starvation. This research first reported the interaction between NNV and autophagy in fish cells. After transfection of pEGFP-LC3 plasmids, fluorescence of autophagosomes was observed in NNV-infected cells. Quantitative analysis of ATP by chemoiluminescence, ATP was found to decrease gradually after NNV infection. When viral protein translation was inhibited by cycloheximide (CHX) treatment, the level of ATP restored to the original level of non-infected cells, indicating that NNV replication could decrease ATP of infected cell. By the way, the level of autophagy could be induced by NNV infection, but it was down-regulated when viral protein translation was inhibited by CHX treatment. When the cells were pre-starved to reduce the level of ATP and then infected with NNV, the level of autophagy was higher but the level of viral protein was lower than that in the NNV-infected cells without pre-starvation. Therefore, it is suggested that the up-regulation of autophagosomes in NNV-infected cells was much related with the loss of ATP and less related with ER stress. When NNV-infected cells were separately treated with three autophagy inhibitors, the levels of ATP and the levels of progeny viral titers were lower than that of untreated NNV-infected cells. All the above data suggested that NNV-induced autophagy was helpful to product the cellular ATP and provide the resources for viral replication. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43726 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 動物學研究所 |
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