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  • 學位論文

家禽里奧病毒調控細胞凋亡與細胞週期

Apoptosis and Cell Cycle Regulation by Avian Reovirus

指導教授 : 劉宏仁

摘要


本研究探討家禽里奧病毒 (avian reovirus , ARV) 與家禽里奧病毒之非結構性蛋白p17對細胞的影響。家禽里奧病毒會經由病毒的附著性蛋白σC造成初級雞胚胎纖維母細胞 (primary chicken embryonic fibroblast)及其它細胞凋亡,而該病毒的非結構蛋白p17則會造成細胞生長遲緩。然而,家禽里奧病毒誘導細胞凋亡和細胞生長遲緩的詳細機制,目前仍有待釐清。 本研究首次証明,家禽里奧病毒誘發細胞凋亡是透過p53和線粒體途徑。另外,本研究也首次証明p17會透過 p53依賴性途徑造成細胞週期停滯在G2/M時期以及宿主細胞蛋白轉譯終止。我們也提供了第一手的證據証明在家禽里奧病毒感染細胞時會造成p53和Bax表現增加。不僅如此,Bax還會從細胞質移動到線粒體並造成線粒體的細胞色素C和Smac/DIABLO釋放到細胞質。而病毒感染細胞時也會造成Caspases-9 and -3活化。有趣的是,家禽里奧病毒對表現有人類bcl-2的BHK-21穩定細胞株的感染和傳播力會顯著減少。這代表著病毒誘發BHK-21細胞凋亡時,p53和線粒體傳導的途徑拌演著重要的角色。雙標記法實驗証明,大多數抗原表達細胞並非凋亡,而是有些凋亡但非抗原表達細胞恰巧位於的抗原表達細胞附近。本實驗也証明家禽里奧病毒感染和轉染病毒蛋白p17到細胞所造的細胞週期停滯,會造成G2/M時期的蛋白質如ATM, p53, p21cip1/waf1, Cdc2, cyclin B1, Chk1, Chk2, 和Cdc25C的磷酸化增加,意謂著p17會透過活化ATM/p53/p21cip1/w路徑而造成細胞週期停滯在G2/M時期。而細胞週期停滯在G2 /M時期卻又會增加病毒的複製。 另外,實驗發現在p17 轉染的細胞可發現轉譯啟始因子eIF2與轉譯延伸因子eEF2磷酸化的增加而真核轉譯啟始因子包括eIF4E, eIF4B, and eIF4G 以及 4E-BP1 and Mnk-1的磷酸化則會減少,代表著p17是透過抑制轉譯啟始因子和轉譯延伸因子而造成宿主細胞蛋白轉譯之終止。用雷帕黴素 (rapamycin)抑制mTOR活性則會導致4E-BP1, eIF4B, and eIF4G磷酸化降低,及增加 eEF2 磷酸化但卻不影響病毒複製,這代表著病毒的複製並不依賴cap-dependent轉譯。綜合以上的結果,本研究証明家禽里奧病毒之p17蛋白會誘導細胞週期停滯在G2/M時期並且造成宿主細胞蛋白轉譯終止,此將有利於病毒的複製。

並列摘要


The biological effects of avian reovirus (ARV) and ARV nonstructural protein p17 were studied by infection and transfection systems in vitro. ARV is known to cause apoptosis in primary chicken embryonic fibroblast and several cell lines through the attachment protein σC and cell growth retardation through non-structural protein p17. However the actual mechanisms through which ARV induces apoptosis and cell growth retardation were still completely unknown. This study has demonstrated for the first time that ARV-induced apoptosis is through p53 and mitochondria pathways. It has also demonstrated for the first time that p17 causes G2/M cell cycle arrest and host cellular translation shutoff through p53 dependent pathways. This study provides the first evidence of upregulation of p53 and Bax during ARV infection. Bax upregulation was accompanied by its translocation from the cytosol to mitochondria that caused the release of cytochrome c and Smac/DIABLO from mitochondria to the cytosol. Caspases-9 and -3 were also activated. Stable expression of human bcl-2 in BHK-21 cells reduced the level of infection and its spread in BHK-21 cells. This suggested that p53 and the mitochondria-mediated pathway play an important role in ARV-induced apoptosis in BHK-21 cells. Dual-labeling assay revealed that the majority of antigen-expressing cells were not apoptotic, although, some apoptotic but non-antigen-expressing cells were frequently located in the vicinity of antigen-expressing cells. It is also demonstrated that ARV infection and ARV p17 transfection caused G2/M cellcycle arrest. The G2/M arrest was accompanied by up-regulation and phosphorylation of the G2/M phase proteins ATM, p53, p21cip1/waf1, Cdc2, cyclin B1, Chk1, Chk2, and Cdc25C suggesting that p17 induces a G2/M cell cycle arrest through activation of the ATM/p53/p21cip1/waf1/Cdc2/cylin B1 and ATM/Chk1/Chk2/Cdc25C pathways. The G2/M cell cycle arrest resulted in increased virus replication. Evidence demonstrating that p17 protein is responsible for ARV-induced host cellular protein translation shutoff is also provided. Increased phosphorylation levels of the eukaryotic translation elongation factor 2 (eEF2) and initiation factor eIF2α and reduced phosphorylation levels of the eukaryotic translation initiation factors eIF4E, eIF4B, and eIF4G as well as 4E-BP1 and Mnk-1 in p17-transfected cells demonstrated that ARV p17 suppresses translation initiation factors and translation elongation factors to induce host cellular protein translation shutoff. Inhibition of mTOR by rapamycin resulted in a decrease in the levels of phosphorylated 4E-BP1, eIF4B, and eIF4G and an increase in the levels eEF2 but did not affect ARV replication, suggesting that ARV replication was not hindered by inhibition of cap-dependent translation. Taken together our data indicate that ARV p17-induced G2/M arrest and host cellular translation shutoff resulted in increased ARV replication.

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