Single-molecule and super-resolution studies of RIG-I pathways

我们建议用单分子研究抗病毒先天免疫反应的机制。 体外荧光技术和细胞的超分辨率成像。RIG-I(维甲酸诱导基因-L) 最近，相关的受体被确定为病毒rna的初始传感器，向下游发出信号。 分子。RIG-I具有一个中心的DExD/H RNA解旋酶结构域。N端具有串行卡 (caspase激活和募集结构域)与线粒体抗病毒信号蛋白相互作用 (MAV)，并被TRIM25 E3连接酶泛素化。它还有一个C-末端调控结构域(RD)， 感觉5‘三磷酸，这是病毒RNA的主要标志。Rig-I也识别双绞线 (DS)作为病毒特征的RNA。RIG-I是一种依赖RNA的ATPase，它的ATPase活性与RIG-I密切相关 以及它的信号传递功能。然而，其ATPase活性的作用仍然是一个谜。使用蛋白质诱导 与项目2合作在单分子水平上的荧光增强(PIPE)。我们 发现RIG-I在短dsRNA(20-50bp)上快速且重复地易位。我们还展示了它的 运动被卡片减慢，而5‘三磷酸的存在则加速运动。与 之前的研究表明ATPase活性和RIG-I信号之间存在很强的相关性，这一数据 表明ATP驱动的RNA转位对RIG-I信号转导是必不可少的。在项目3中，我们将解决 许多悬而未决的问题，如RIG-I如何区分病毒RNA和类似的 宿主核糖核酸分子，RIG-L的ATPase活性的作用是什么，RIG-L的功能是如何受核调控的 酸的组成，它与其他蛋白质的相互作用，以及L的转录后和翻译后 修改会影响其功能。具体目标有三个： 在目标1中，我们将研究RIG-I样受体的RNA转位活性。 在目标2中，我们将研究rig-i的负载和寡聚，以及它在病毒上的构象变化。 RNA识别。 在目标3中，我们将使用live研究RIG-I及其伙伴(如MAV和病毒RNA)的细胞定位 细胞成像和超分辨率成像。此外，RIG-I与小牛的相互作用将在 重组体系中的单分子水平。

We propose to investigate the mechanisms of the anti-viral innate immune response using single molecule fluorescence techniques in vitro and super-resolution imaging of cells. RIG-I (retinoic acid inducible gene-l) and related receptors were recently identified as the initial sensors for viral RNA that signal downstream molecules. RIG-I has a central DExD/H RNA helicase domain. The N-terminus possesses tandem CARDs (caspase activation and recruitment domains) that interact with a mitochondrial antiviral signaling protein (MAVS) and are ubiquitinated by TRIM25 E3 ligase. It also has a C-terminal regulatory domain (RD) that senses 5' triphosphate which is the primary signature of viral RNA. RIG-I also recognizes double stranded (ds) RNA as a viral signature. RIG-I is an RNA-dependent ATPase and its ATPase activity closely correlate with its signaling function. However, the role of its ATPase activity has remained a mystery. Using proteininduced fluorescence enhancement (PIPE) at the single molecule level in collaboration with Project 2. we found that RIG-I translocates rapidly and repeatedly on short dsRNA (20-50 bp). We also showed that its movement is slowed down by CARDs and accelerated by the presence of 5' triphosphate. Combined with previous studies that show a strong correlation between ATPase activity and RIG-I signaling, this data indicates that ATP-powered RNA translocation is essential for RIG-I signaling. In Project 3, we will address many of the outstanding questions such as how RIG-I discriminates between viral RNA and similar-looking host RNA molecules, what the role of RIG-l's ATPase activity is, how RIG-l's function is regulated by nucleic acid composition, what its interactions with other proteins are and how RIG-l's post-transcriptional and posttranslational modifications affect its function. There are three specific aims: In Aim 1, we will investigate the RNA translocation activities of RIG-I like receptors. In Aim 2, we will investigate RIG-I loading and oligomerization, and its conformational changes upon viral RNA recognition. In Aim 3, we will investigate cellular location of RIG-I and its partners such as MAVS and viral RNA using live cell imaging and super-resolution imaging. In addition, RIG-I interaction with MAVS will be studied at the single molecule level in a reconstituted system.