TMEM41B: a pan-flavivirus and pan-coronavirus host factor with antiviral potential

项目摘要 节肢动物传播的黄病毒和寄生虫传播的冠状病毒有可能引起严重的 流行病和大流行病。预防和应对病毒爆发的一种策略是开发药物， 病毒完成其生命周期所需目标宿主因子。通过一系列CRISPR/Cas9基因破坏 筛选，我们确定了跨膜蛋白41 B（TMEM 41 B）和密切相关的液泡膜 蛋白质1（VMP 1）是泛黄病毒和泛冠状病毒的关键宿主因子。这两种蛋白质都是高度保守的 在自噬中发挥作用的脂质乱序酶。我们目前的模型是来自黄病毒科和 冠状病毒科家族劫持TMEM 41 B和VMP 1，因为它们能够重塑ER膜并诱导 膜曲率，以建立膜保护的病毒RNA复制细胞器。 我们这项提议的总体目标是了解这两种蛋白质在机制层面上如何支持 黄病毒和冠状病毒感染。我们以前的工作表明，TMEM 41 B是必需的后进入步骤 在病毒RNA复制时或之前。在目标1中，我们将询问病毒生命周期的早期事件，包括主要 WT、TMEM 41 B和VMP 1敲除中的翻译、多蛋白加工和复制细胞器形成 (KO)细胞，以确定黄病毒和冠状病毒的生命周期进展多远，在缺乏任何一种蛋白质。 我们之前的研究表明，缺乏TMEM 41 B和VMP 1，诱导增强的先天免疫反应， 黄病毒感染后。我们假设这两种蛋白质都被募集到病毒RNA复制的位点， 在它们不存在的情况下，RNA复制启动并产生病毒双链RNA（dsRNA）。然而，在这方面， 如果没有适当的复制，细胞器dsRNA就会暴露并触发先天免疫应答。可选择地， 考虑到TMEM 41 B和VMP 1的脂质乱序酶活性和在脂质体内平衡中的功能，它们的缺失可能 诱导ER应激，其触发未折叠蛋白质应答（UPR），该未折叠蛋白质应答与dsRNA协同作用可引起 增强先天免疫反应在目标2中，我们将在缺乏以下任一项的双KO细胞中测试病毒感染： 除了对病原体传感、IFN信号传导和UPR激活至关重要的基因之外，蛋白质也是如此。我们将 进一步进行RNAseq实验以研究干细胞和干细胞衍生的细胞中TMEM 41 B的缺乏。 在病毒复制存在和不存在的情况下代表不同组织谱系的原代样细胞。 最后，在目标3中，将使用一组表型和机制测定来表征天然存在的 我们以前发现TMEM 41 B中的SNP影响黄病毒复制，一些报道的VMP 1缺失， 功能突变体我们将进一步采取深度突变扫描的方法， TMEM 41 B和VMP 1，并确定是否有任何结构域或氨基酸是它们的细胞分化所需的。 和前病毒功能。这种功能性表征将鉴定可以在基因组中详细研究的突变体。 机制分析，并可以确定两种蛋白质中的氨基酸或界面，可以靶向预防 对细胞生物学破坏最小的病毒感染。

Project Summary Arthropod-borne flaviviruses and respiratory-transmitted coronaviruses have the potential to cause severe epidemics and pandemics. One strategy to prepare for and respond to viral outbreaks is to develop drugs that target host factors viruses require to complete their lifecycles. Through a series of CRISPR/Cas9 gene disruption screens, we identified transmembrane protein 41B (TMEM41B) and the closely related vacuole membrane protein 1 (VMP1) as critical pan-flavivirus and pan-coronavirus host factors. Both proteins are highly conserved lipid scramblases with roles in autophagy. Our current model is that viruses from both the Flavivirdae and Coronaviridae families hijack TMEM41B and VMP1 for their ability to remodel ER membranes and induce membrane curvature to establish membrane-protected viral RNA replication organelles. Our overall goal for this proposal is to understand how, on a mechanistic level, both proteins support flavivirus and coronavirus infection. Our previous work indicates that TMEM41B is required at a post-entry step at or prior to viral RNA replication. In Aim 1, we will interrogate early events of the virus lifecycle including primary translation, polyprotein processing, and replication organelle formation in WT, TMEM41B and VMP1 knockout (KO) cells to determine how far the flavivirus and coronavirus lifecycles progress in the absence of either protein. We previously showed that lack of TMEM41B and VMP1, induces a heightened innate immune response upon flavivirus infection. We hypothesize that both proteins are recruited to sites of viral RNA replication, and that in their absence, RNA replication initiates and viral double stranded RNA (dsRNA) is produced. However, without a proper replication organelle dsRNA is exposed and triggers an innate immune response. Alternatively, given TMEM41B’s and VMP1’s lipid scramblase activity and function in lipid homeostasis, their absence may induce ER stress, which triggers an unfolded protein response (UPR) that in synergy with dsRNA may cause a heightened innate immune response. In Aim 2, we will test virus infection in double KO cells that lack either protein in addition to genes that are essential for pathogen sensing, IFN signaling, and UPR activation. We will further conduct RNAseq experiments to investigate lack of TMEM41B in stem cells and stem cell-derived primary-like cells representing different tissue lineages in the absence and presence of viral replication. Lastly, in Aim 3, will use a panel of phenotypic and mechanistic assays to characterize naturally occurring SNPs in TMEM41B that we previously found to impact flavivirus replication, and several reported VMP1 loss-of- function mutants. We will further take a deep mutational scanning approach to comprehensively characterize TMEM41B and VMP1 and determine if any domains or amino acids are differentially required for their cellular and proviral functions. This functional characterization will identify mutants that can be studied in detail in mechanistic assays and may identify amino acids or interfaces in both proteins that can be targeted to prevent virus infection with minimal disruption to cellular biology.