臺灣博碩士論文加值系統

干擾素引起、刺激基因活化，在對抗病毒感染的過程中扮演很重要的角色。RNA病毒引起RLR、MAVS、TBK-1訊息下游的第一型干擾素和第三型干擾基因表現，差別在第一型干擾素幾乎對所有細胞都能起作用，而第三型干擾素只在特定黏膜表面有作用。MAVS在細胞內存在的位置，會決定細胞生成哪種干擾素。粒腺體上的MAVS能刺激第一型干擾素生成，而過氧化物酶體(peroxisome)上的MAVS則會引發第三型干擾素生成。在我們先前的研究發現，GEF-H1在MAVS引起第一型干擾素生成中扮演重要的角色，但仍不清楚GEF-H1是否對第三型干擾素的產生造成影響。為了探討此問題，我利用poly(I:C)刺激老鼠，GEF-H1缺陷老鼠不論是在腸道表皮細胞表現的第三型干擾素和在lamina propria細胞表現的第一型干擾素都相較野生型老鼠偏低。接著我探討這現象是否也會出現在病毒引起干擾素反應。我使用第三型里奧病毒(reovirus T3D)感染老鼠，發現GEF-H1缺陷老鼠對病毒的感受性較高，但不論第一型和第三型干擾素的表現皆未上升。為了找尋GEF-H1在第三型干擾素的詳細分子機制，我使用第三型干擾素的螢光酵素啟動子分析，發現GEF-H1提高了MAVS的第三型干擾素表現量。更進一步研究，接續的利用不同功能突變的GEF-1去分析哪些功能區是對這調節現象有影響，結果顯示GEF-H1的鳥糞嘌玲交換能力對這調節是很重要。總和以上結果，說明GEF-H1除了參與第一型干擾素生成，也在第三干擾素的生成扮演重要的角色。

Induction of interferons (IFNs) and activation of interferon-stimulated genes (ISGs) are critical to innate immunity against viral infection. Almost all cells respond to type I IFNs whereas type III IFN responses are primarily found at the mucosal surfaces. Like type I IFNs, type III IFNs expression induced by RNA virus infection is downstream of RIG-I-like receptors (RLR), MAVS and TBK1. However, the subcellular localization of MAVS determines which IFN species is produced. IFN-λ production is favored when MAVS is located at the peroxisome whereas IFN-β expression is preferred when MAVS is localized to the mitochondria. Our previous works have identified that GEF-H1, a central component of innate immunity, is required for MAVS-mediated type I IFNs expression in response to RNA virus infection.We therefore hypothesize that GEF-H1 could also mediate MAVS-mediated type III IFN production. To characterize the role of GEF-H1 in induction of type III IFN in vivo, WT or GEF-H1-deficient mice were stimulated with poly (I:C). Type III IFN expression of epithelial cells and type I IFN expression of lamina propria cellswere both reduced in GEF-H1-deficient mice after poly(I:C) stimulation. This result showed GEF-H1 is invovled in MAVS-mediated type III IFN expression. To identify the role of GEF-H1 in virus-induced type III IFN expression, WT and GEF-H1-deficient mice were infected with reovirus. The viral replication was measured by plaque assay, and then this result showed GEF-H1-deficient mice were more susceptible to reovirus. However, both type I IFN and type III IFN expressions seemed to be interfered by reovirus. To assess the molecular mechanism of GEF-H1 in induction of IFNL1 promoter activation, I utilized luciferase reporter assays in HEK293T cells in the absence or presence of GEF-H1. Expression of GEF-H1 significantly enhanced wild-type MAVS-induced activation of the IFNL1 promoter. To find out which function domain of GEF-H1 is critical for type III IFN expression, two different GEF-H1 mutant expressing plasmids were used for IFNL1 promoter luciferase assays. The result showed that the GEF function of GEF-H1 is critical for MAVS-mediated IFNL1 luciferase activation. Our dataprovide insight that GEF-H1 not only involves in type I IFNs expression but also takes part in type III IFN expression.

致謝 i
摘要 ii
Abstract iii
Contents v
List of Figure vii
Chapter 1 Introduction 1
1.1 Antiviral response in gastrointestinal tract 1
1.1.1 Reoviridae 1
1.1.2 Mammalian orthoreovirus 1
1.1.3 Reovirus replication cycle 2
1.1.4 Nucleic acid-sensing receptors 2
1.1.5 RLR–MAVS-mediated signal transduction pathway 3
1.1.6 Type I IFN response 4
1.1.7 Transcription regulators of Type I IFN expression 5
1.1.8 Type III IFN response 6
1.1.9 The transcription regulator of type III IFN 6
1.1.10 Antiviral Response of Type III IFN 7
1.2 Rho-related small GTPase 8
1.2.1 Guanine nucleotide exchange factor (GEF) of Rho GTPase 8
1.2.2 GEF-H1 with Rho-independent function 9
Chapter 2 Materials and Methods 11
2.1 Mice 11
2.2 Cell 11
2.3 Virus 11
2.4 Plasmids 12
2.5 Plaque assay 13
2.6 In vivo treatment 14
2.7 Isolation of intestine epithelial cells and lamina propria leukocytes 14
2.8 Gene expression assay 15
Chapter 3 Result 17
3.1 GEF-H1 was required in type III IFN expression via dsRNA-RLR pathway in vivo. 17
3.2 GEF-H1-deficient mice were more susceptible to reovirus infection 17
3.3 GEF-H1 enhancing MAVS-mediated IFNL1 luciferase activity was not affected by repressor-binding region. 18
3.4 GEF-activity of GEF-H1 is required for enhancing MAVS-mediated IFNL1 luciferase activity. 18
3.5 GFP in N-terminal of MAVS interfered the GEF-H1 enhancing effect. 19
Chapter 4 Discussion 21
References 24
Table 46
Table 1. Primers used for RT-qPCR 46

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