Louber Jade, Kowalinski Eva, Bloyet Louis-Marie, Brunel Joanna, Cusack Stephen, Gerlier Denis
Centre International de Recherche en Infectiologie, INSERM, U1111, CNRS, UMR5308, Université Lyon 1, ENS Lyon, Lyon, France.
European Molecular Biology Laboratory, Grenoble Outstation, Grenoble Cedex 9, France; Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI 3265, Grenoble Cedex 9, France.
PLoS One. 2014 Sep 26;9(9):e108770. doi: 10.1371/journal.pone.0108770. eCollection 2014.
Effective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN) response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments in vitro upon activation, RIG-I is believed to oligomerize after RNA binding in order to transduce a signal. Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales (Ebola, rabies and measles viruses) leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5'ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA), a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient.
宿主对病毒的有效防御依赖于通过诱导I型干扰素（IFN）反应来快速触发天然免疫。为此，微生物相关分子模式由特定的受体检测。其中，RIG-I样受体RIG-I和MDA5在感知细胞质中的病毒RNA后激活IFN基因表达。虽然MDA5在体外激活后形成长丝，但据信RIG-I在与RNA结合后会发生寡聚化以转导信号。在这里，我们表明，模拟单股负链RNA病毒目（埃博拉病毒、狂犬病病毒和麻疹病毒）前导序列的合成RNA与纯化的RIG-I在体外结合不会诱导RIG-I寡聚化。此外，在缺乏内源性功能性RIG-I样受体的细胞中，在用62聚体5'三磷酸双链RNA或聚肌苷酸:聚胞苷酸（一种双链RNA类似物）或麻疹病毒感染激活外源性Flag-RIG-I后，抗Flag免疫沉淀和用Flag肽特异性洗脱表明RIG-I为单体形式。因此，当使用基于高斯荧光素酶的蛋白质互补分析（PCA）（一种更灵敏的细胞内分析方法）时，在RNA刺激后未检测到RIG-I寡聚化。我们的数据表明，RIG-I自我寡聚化以进行信号转导的需求要么是不必要的，要么是非常短暂的。
