Mechanistic analysis of a posttranslationally modified innate antiviral effector

描述(申请人提供)：流感和其他新出现的病毒代表着世界范围内的主要健康问题，目前预防或治疗疾病的战略是不够的。I型干扰素是一种细胞因子，通过转录上调数百个基因在细胞中诱导抗病毒状态。这些基因中的一些编码具有直接抗病毒活性的蛋白质，尽管只有一小部分这些蛋白质被机械地表征。在这些蛋白中，干扰素诱导的跨膜蛋白3(IFITM3)最近被我们和其他人证明对所有被测试的流感病毒亚型以及其他一些病毒家族具有广泛的抗病毒活性。此外，这种蛋白质在干扰素效应器中是独一无二的，因为它似乎通过阻止病毒进入或融合而不是抑制病毒复制来发挥作用。使用化学记者，我们已经证明了IFITM3是翻译后棕榈酰化的，这种脂质修饰调节了它的抗病毒活性。此外，我们的初步数据表明，IFITM3也是泛素化的。这项拟议研究的总体目标是通过表征IFITM3的作用机制及其翻译后修饰对细胞的调控，增加对先天抗病毒免疫的了解。在K99阶段，我们将开发生化和显微镜分析来测试棕榈酰化控制IFITM3的适当运输的假设，使其能够与病毒颗粒相互作用并防止病毒进入。有了这些知识，我们将在R00阶段寻找负责IFITM3棕榈酰化的酶，并研究病毒感染期间棕榈酰化酶的全球转录调节。此外，我们将应用K99阶段开发的分析方法来了解泛素化在IFITM3生物学中的作用及其与棕榈酰化的相互作用。分析IFITM3的作用机制以及通过一组独特的翻译后修饰来控制这一活性，可能会为利用I型干扰素的力量对抗病毒病原体提供必要的见解。 公共卫生相关性(由申请人提供)：干扰素诱导的跨膜蛋白3(IFITM3)已被证明对许多病毒病原体具有广泛的抑制活性。由于目前的治疗方法和疫苗不足以对抗病毒疾病，我们试图通过分析IFITM3的转运和与病毒颗粒的相互作用来了解IFITM3的抗病毒作用机制。

DESCRIPTION (provided by applicant): Influenza and other emerging viruses represent major health concerns worldwide, and current strategies for prevention or treatment of disease are insufficient. Type I interferon is a cytokine that induces an antiviral state in cells by transcriptionally upregulating hundreds of genes. Some of these genes encode proteins with direct antiviral activity, though only a small number of these proteins have been mechanistically characterized. Among these proteins, the interferon-inducible transmembrane protein 3 (IFITM3) has been recently shown by us and others to have broad antiviral activity against all subtypes of influenza virus tested as well as a number of other virus families. Furthermore, this protein is unique among interferon effectors in that it appears to act by preventing entry or fusion of viruses rather than inhibiting viral replication. Using chemical reporters, we have shown that IFITM3 is post-translationally palmitoylated and this lipid modification regulates its antiviral activity. Furthermore, our preliminary data indicate that IFITM3 is also ubiquitinated. The overall goal of the proposed research is to increase understanding of innate antiviral immunity by characterizing the mechanism of action of IFITM3 and its cellular regulation by post-translational modifications. In the K99 phase, we will develop biochemical and microscopy assays to test the hypothesis that palmitoylation controls proper trafficking of IFITM3 allowing it to interact with viral particles and prevent viral entry. With this knowledge in hand we will then seek in the R00 phase to identify enzymes responsible for IFITM3 palmitoylation and look at the global transcriptional regulation of palmitoylating enzymes during viral infections. Furthermore, we will apply assays developed in the K99 phase to understand the role of ubiquitination in IFITM3 biology and its interplay with palmitoylation. Analyzing the mechanism of action of IFITM3 and the control of this activity by a unique set of post-translational modifications may provide insights necessary for harnessing the power of type I interferon for combating viral pathogens. PUBLIC HEALTH RELEVANCE (provided by applicant): Interferon-inducible transmembrane protein 3 (IFITM3) has been shown to possess broadly inhibitory activity against a number of viral pathogens. As current therapies and vaccines are insufficient for combating viral disease, we seek to understand the mechanism of antiviral action of IFITM3 by analyzing its trafficking and interactions with viral particles.