臺灣博碩士論文加值系統

RIG-I受體 (RLR) 訊息的活化促使第一型干擾素的反應發生而去對抗流行性感冒病毒的感染。RIG-I和粒線體上的調控蛋白MAVS的過量表現也會促使自噬作用的發生來維持粒線體的恆定。本實驗室先前的研究發現在RIG-I訊息的傳遞下會造成A/Puerto Rico/34/8/H1N1 (PR8) 病毒株的PB1-F2降解，因此我們探討RIG-I訊息路徑的活化下是否會造成其他流感病毒蛋白的降解，我們發現PB1、PA、 HA以及NP的表現量會受到RIG-I的影響而輕微減少，而病毒蛋白PB1-F2、M2以及NS2則較明顯的減少。比較不同流感病毒蛋白受到RIG-I過量表現的影響程度後，我們選擇M2作為我們的研究降解機制的對象。M2在流感病毒的生活史當中主要參與在病毒從核內體釋放到細胞核中；除此之外，M2會藉由阻擋自噬體和溶酶體融合進而幫助病毒逃避病毒感染所引起的自噬作用。
為了瞭解RIG-I如何調節M2病毒蛋白，在RIG-I訊息強化下，我們使用不同抑制劑處理細胞後，觀察M2蛋白表現量。我們發現M2的表現量可以被蛋白酶體抑制劑lactacystin以及三個自噬相關的抑制劑，包含3-MA、Bafilomycin-A1及Leupeptin而救回來，推論蛋白酶體路徑和溶酶體路徑可能都參與M2的降解。在自噬作用與溶酶體路徑研究中，我們更進一步發現在細胞處於自噬標記LC3的過度表現或是CCCP處理誘發自噬作用的情況下，也會促進M2降解，證明自噬作用的確參與在M2的降解當中。而在蛋白酶體路徑的研究過程中，我們探討參與在RIG-I受體訊息路徑中的泛素連接酶是否也參與在M2的降解當中。我們發現在RIG-I訊息的活化狀態下，RNF125、RNF135以及Parkin這些泛素連接酶會更進一步增強M2的降解，故推論泛素連接體可能在RIG-I調節的M2衰退機制中也扮演了一個角色，然而詳細的蛋白酶體降解機制仍然需要更加探討。

RIG-I like receptors (RLR) signaling activation induces type I interferon response to defend against influenza virus infection. RIG-I and MAVS overexpression also induces autophagy to maintain mitochondrial homeostasis. In our previous studies, we observed that activation of RIG-I signaling led to PB1-F2 derived from A/Puerto Rico/34/8/H1N1 (PR8) degradation. As a result, we investigated whether activation of RIG-I signaling induces other influenza viral proteins degradation. We found that PR8 PB1, PA, HA and NP expression level decreased slightly when N-RIG, an active form of RIG-I, was overexpressed, while PR8 PB1-F2, M2 and NS2 viral proteins reduced more significantly. After comparing the expression level of different influenza viral proteins under N-RIG overexpression, we chose viral protein M2 as our model to study the degradation mechanisms. M2 is responsible for virus releasing to nucleus and is known to help virus to evade infection-induced autophagy by blocking autophagosome fusion with lysosome, thus M2 plays an important role in the life cycle of influenza virus.
To investigate how RIG-I mediated M2 degradation, cells were treated with different pharmacological inhibitors in the presence of RLR signaling activation. We observed that M2 expression level could be rescued by a proteasome inhibitor, lactacystin, and three autophagy-related inhibitors, 3-MA, Bafilomycin-A1 and Leupeptin, indicating that M2 might be degraded via both proteasomel and lysosomal pathways. In the study of autophagy and lysosomal pathways, we further observed that M2 could be degraded when cells were transfected with plasmids expressing EGFP-LC3 or treated with CCCP to induce autophagy. It showed that autophagy and lysosomal pathway indeed participated in RIG-I mediated M2 degradation. In the study of proteasome degradation pathway, we also investigated whether E3 ligases involved in RLR signaling led to M2 degradation. We found that several E3 ligases, such as RNF125, RNF135 and Parkin, could further enhance M2 degradation in the presence of RIG-I signaling, suggesting that E3 ligases might also play a role in RIG-I mediated M2 degradation. However, the detailed mechanisms remain to be deduced.

Index
致謝 ........................................... i
摘要 ........................................... iii
Abstract ....................................... v
Index .......................................... vii
List of figures ................................ viii
Chapter 1 Introduction ......................... 1
1.1 Influenza A virus .......................... 1
1.2 Influenza A virus M2 ....................... 3
1.3 RLR signaling .............................. 5
1.4 Protein degradation ........................ 8
Chapter 2 Objective ............................ 11
Chapter 3 Materials and Methods ................ 12
3.1 Materials .................................. 12
3.2 Methods .................................... 17
Chapter 4 Results .............................. 23
Chapter 5 Conclusion ........................... 31
Chapter 6 Discussion ........................... 34
Chapter 7 Figures .............................. 39
References ..................................... 52

List of figures
Figure 1.RIG-I also induced other influenza viral proteins degradation other than PB1-F2 ...........39
Figure 2.The effect of RLR signaling on M2 .......40
Figure 3.NS1 rescued RIG-I induced influenza M2 degradation ......................................42
Figure 4.Screening for the potential degradation pathways of M2 ............................................43
Figure 5.The effect of Bafilomycin A1 and Leupeptin on M2................................................45
Figure 6.M2 degraded by autophagy induction ......47
Figure 7. Activation of RIG-I or MAVS could induce autophagy ........................................49
Figure 8.Several E3 ligases participated in RLR signaling and M2 degradation ...............................51

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