- 學位論文
藉RNA干擾與細胞訊息傳遞來探討家禽里奧病毒之複製
Regulation of Avian Reovirus Replication by RNA Interference and Cell Signaling
摘要
在演化的過程中，宿主與病毒均試著對彼此佔有優勢。為了避免感染，宿主細胞必須具備偵測及消滅入侵病原的能力，其中，雙股RNA相關之免疫反應與凋亡乃是細胞辨識及限制病毒增生的重要機制之一。身為絕對依賴宿主細胞的病原，病毒經常透過改變細胞功能以增加自身之生存優勢，進而躲避免疫反應或擷取所需資源。家禽里奧病毒由於帶有容易誘發免疫反應之雙股RNA基因體，為了生存，此病毒極可能具備能力以拮抗與雙股RNA相關免疫反應，如RNA干擾。然而以人工誘導之RNA干擾仍有效地減低家禽里奧病毒數個基因之表現。值得注意的是病毒自身並未在感染初期引發出可觀之RNA干擾反應，表示家禽里奧病毒可能藉由避免其雙股RNA受到偵測而使免疫反應不啟動。不同於在RNA干擾上被動的躲避，家禽里奧病毒感染細胞後可改變細胞訊息傳遞，至少涉及PI3K-Akt及AMPK這兩條路徑。AMPK活化有助家禽里奧病毒誘導MAPK p38磷酸化，從而維持病毒之複製。此外，AMPK亦參與細胞蛋白質合成，包含轉譯起始與接續之調控。目前雖然已知諸多病毒藉活化PI3K-Akt路徑來減緩細胞凋亡並延長感染時間，但異於大多數病毒的是，在Vero細胞中以兩種常用之抑制劑抑制PI3K並無法減少家禽里奧病毒增殖，由於PI3K-Akt路徑亦可能為細胞啟動免疫反應的媒介，AMPK可能有助於減緩細胞製造免疫相關訊息，進而方便家禽里奧病毒之增殖，而我們的結果則顯示家禽里奧病毒確實可顯著抑制AMPK下游，與蛋白質轉譯相關之因子，其中包含轉譯延長因子eEF2。除了家禽里奧病毒，牛流行熱病毒由於屬於倚賴Akt來複製之子彈病毒科，因此也被用以作為控制組，以瞭解在Vero細胞中，抑制PI3K-Akt路徑對病毒增殖之影響。令人意外的，抗PI3K的化合物無法有效減少卻相反地促進了牛流行熱病毒的增殖，此結果提高了以該類化合物來控制病毒感染的不確定性。
關鍵字
干擾性核醣核酸並列摘要
In the evolution competition, hosts and viruses evolve to owerpower each other. To prevent infection, host cells have to detect and diminish invasive pathogens. Among multiple mechanisms, dsRNA-mediated immune response and apoptosis play important roles by which cells recognize and restrict progression of several viral infections. To survive, viruses undoubtly have ability to dominate several cell functions thereby getting replication superiority. Avian reovirus (ARV) which has double-stranded RNA genome, an easy target to host immune responses, as proposed has potential to counteract RNA interference. However, application of RNA interference successfully reduced expression of three ARV genes as well as progeny titer. Since ARV did not spontaneously induce notable silencing effect on its own genes during early infection, the main strategies how ARV evades from double-stranded RNA-mediated immune responses may be concealing its own structured RNA from detection. In contrast to its passivity in case of RNA interference, ARV infection regulates cell signaling at least including PI3K-Akt and AMPK pathway. Activation of AMPK facilitates phosphorylation of MAPK p38 which supports replication of ARV. Activated AMPK may also contribute to ARV infection simultaneously via down-regulating cellular protein synthesis, thereby increasing viral transcripts competebility and delaying production of immune signals since PI3K-Akt signaling is a potential mediator of cell immune response though several viruses rely on PI3K-Akt signaling for preventing premature apoptosis. Our results as predicted show that several translation-related factors downstream of AMPK, including a translation elongation factor eEF2, are obviously inhibited after ARV infection. As a control, inhibition of PI3K by two frequently used chemicals surprisingly did not down-regulate replication of bovine ephemeral fever virus (BEFV), which in theory critically relies on Akt for efficient replication. The results raise the concern about unpredictability of using such chemicals on controlling viral infection.