家禽里奧病毒進入細胞途徑之探討

Characterization of routes of avian reovirus cell entry

王英傑(Ying-Cheh Wang)

指導教授 ： 劉宏仁

國立屏東科技大學/農學院/生物科技研究所/碩士(2010年)

摘要

家禽里奧病毒 (Avian reovirus , ARV) 是常見感染家禽的病毒，會造成雞隻產生關節炎、慢性呼吸道疾病及營養吸收不良的症狀，造成經濟上的損失。已有許多的研究探討 ARV 的致病機轉，然而，關於 ARV 透過何種方式與機制進入宿主細胞，目前所知仍有限。本研究探討 ARV 進入 Vero 與 DF-1 細胞之途徑與機轉。以 MβCD 及 nystatin 去除細胞膜 lipid rafts 之膽固醇，可有效抑制病毒的感染。額外再加入膽固醇則能夠恢復病毒的感染。感染病毒後再去除膽固醇並不會影響病毒的感染，證實膽固醇可影響 ARV 進入細胞。使用 chlorpromazine 去抑制 clathrin-mediated endocytosis，則不會影響病毒感染細胞。 再者，使用 dynamin GTPase 的抑制劑 dynasore 也能抑制病毒的感染，證實 ARV 進入細胞是 dynamin-dependent。此外，以 NSC23766 、rottlerin 及 SB202190 分別抑制抑制 Rac 1、PKC δ與 MAPK p38 活性，皆能抑制病毒複製，證實 ARV 進入宿主細胞會活化 Rac 1、PKC δ 與 MAPK p38。先以 Rac 1 活性分析證實，PKC δ 與 MAPK p38 皆會活化 Rac 1；接著，以 rottlerin 抑制 PKC δ 活性時，不會影響 MAPK p38，而 SB202190 抑制 MAPK p38 活性時，亦不會影響 PKC δ，證實 ARV 早期進入宿主細胞皆會活化 MAPK p38 與 PKC δ，並且 MAPK p38 與 PKC δ 皆會活化下游 Rac 1。另外，以共軛焦顯微鏡證實，ARV 進入宿主細胞是caveolin-dependent。以chloroquine, bafilomycin A1, 或 ammonium chloride 等藥物也都會抑制 ARV 的感染 ， 證實 endosome 的酸化是病毒進入過程中的必要步驟。最後，使用 norcodazole 打斷 microtubule 可以有效抑制病毒的感染，但用 cytochalasin D 破壞 actin filament 則未抑制病毒感染細胞。本研究首次證實 ARV 進入宿主細胞需經由 caveolin 依賴之路徑 (caveolin-dependent)，dynamin 的幫助 (dynamin-dependent)， Rac 1 、 PKC δ 、 MAPK p38 的活化 (Rac 1、PKC δ and MAPK p38 activation)，endosome 的酸化 (endosome acidification)，及藉由 microtubule 的運送 (microtubule-dependnt transport)。

關鍵字

家禽里奧病毒 ； lipid rafts ； 病毒進入 ； Rac 1 ； PKC δ ； MAPK p38

並列摘要

Avian reovirus (ARV) causes important poultry diseases of considerable economic importance to the poultry industry. Several studies have addressed the mechanism of how ARV causes cell damages, however, little is known about the exact mechanisms of ARV cell entry and subsequent intracellular trafficking. Cellular mechanisms of ARV entry in vero and DF-1 cells were investigated in this study. Our results revealed that infectivity was exquisitely sensitive to methyl-β-cyclodextrin (MβCD) and nystatin, which disrupt lipid rafts by removing cholesterol. These agents inhibited ARV infection in a dose-dependent manner. The addition of exogenous cholesterol to cholesterol-depleted cells reversed this effect. Interestingly, cholesterol depletion postinfection did not affect ARV replication, suggesting that the effect occurs at the level of ARV entry. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not have any effect on ARV infection. Moreover, cells pretreated with dynasore, a dynamin GTPase inhibitor that prevents the scission of dynamin-dependent endocytic vesicles, can block ARV infection in a dose-dependent manner. Besides, Rac 1 inhibitor (NSC23766)、PKC δ inhibitor (rottlerin) and MAPK p38 inhibitor (SB202190) also significantly reduce ARV replication, respectively. On the other hand, ARV induced Rac 1, PKC δ and MAPK p38 activation that critical for virus entry. By Rac 1 activiation assay kit, rottlerin reduced PKC δ phosphorylation but had no effects on MAPK p38 phosphorylation. Treatment with SB202190 to suppress MAPK p38 phosphorylation had no effect on PKC δ phosphorylation. It was discovered that there were not only enhanced MAPK p38 and PKC δ phosphorylation that but also induced downstream to Rac 1 after ARV entry at early stage of viral entry. In addition, the confocal assay was used to demonstrate that ARV entry into cells via caveolin-dependent pathway. Reagents including chloroquine, bafilomycin A1, or ammonium chloride inhibited ARV infection, indicating that an acidic endosomal pH is required for ARV entry. Finally, ARV infection was impaired when the microtubule assembly was disrupted rather than actin filament assembly by norcodazole and cytochalasin D, respectively. Taken together, these findings suggest that ARV cell entry is through caveolin-dependent, dynamin-dependent, Rac 1 activiation, PKC δ activiation, MAPK p38 activiation, endosome acidification, and microtubule-dependnt pathway.

並列關鍵字

avian reovirus ； lipid rafts ； virus entry ； Rac 1 ； PKC δ ； MAPK p38

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