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

本研究拟利用单分子抗体研究抗病毒天然免疫应答的机制 体外荧光技术和细胞的超分辨率成像。视黄酸诱导基因-1 和相关的受体最近被确定为病毒RNA的初始传感器， 分子。RIG-I具有中心DExD/H RNA解旋酶结构域。N-末端具有串联CARD （半胱天冬酶激活和募集结构域）与线粒体抗病毒信号蛋白相互作用 （MAVS）并被TRIM 25 E3连接酶泛素化。它还具有C-末端调节结构域（RD）， 有义5'三磷酸，其是病毒RNA的主要特征。RIG-I还识别双链 (ds)RNA作为病毒的标志。RIG-I是一种RNA依赖的ATP酶，其ATP酶活性与RNA依赖的ATP酶活性密切相关。 它的信号功能。然而，其ATP酶活性的作用仍然是一个谜。使用蛋白诱导 荧光增强（PIPE）在单分子水平上与项目2合作。我们 发现RIG-I在短dsRNA（20-50 bp）上快速且重复地易位。我们还表明， CARD使运动减慢，而5 ′-三磷酸的存在使运动加速。结合 先前的研究表明ATP酶活性和RIG-I信号传导之间存在很强的相关性， 表明ATP驱动的RNA易位对于RIG-I信号传导是必需的。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和相似的外观， RIG-1的ATP酶活性的作用是什么，RIG-1的功能是如何被核酸调控的， 酸的组成，它与其他蛋白质的相互作用是什么，以及RIG-1的转录后和翻译后是如何表达的。 修改会影响其功能。有三个具体目标： 在目的1中，我们将研究RIG-I样受体的RNA转运活性。 在目标2中，我们将研究RIG-I的负载和寡聚化，以及病毒感染后其构象的变化， RNA识别 在目标3中，我们将使用活的细胞定位研究RIG-I及其伙伴如MAVS和病毒RNA。 细胞成像和超分辨率成像。此外，RIG-I与MAVS的相互作用将在 在重构系统中的单分子水平。

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.