探討第三型干擾素生成的分子機制

Abstract

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.