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呼吸道传播的冠状病毒有可能导致严重的 流行病和流行病。准备和应对病毒爆发的一种策略是开发能够 病毒完成其生命周期所需的目标宿主因子。通过一系列CRISPR/Cas9基因破坏 经过筛选，我们鉴定了跨膜蛋白41B(TMEM41B)和与之密切相关的液泡膜 蛋白1(VMP1)是泛黄病毒和泛冠状病毒的关键宿主因子。这两种蛋白质都高度保守 在自噬中起作用的是脂类杂乱物。我们目前的模型是来自黄病毒科和黄病毒科的病毒 冠状病毒科家族劫持TMEM41B和VMP1是因为它们有能力重塑ER膜并诱导 膜弯曲建立膜保护的病毒RNA复制细胞器。 我们这个提议的总体目标是从机制层面上理解这两种蛋白质是如何支持 黄病毒和冠状病毒感染。我们之前的工作表明，TMEM41B在进入后步骤中是必需的 在病毒RNA复制之时或之前。在目标1中，我们将询问病毒生命周期的早期事件，包括主要事件 WT、TMEM41B和VMP1基因敲除中的翻译、多蛋白加工和复制细胞器的形成 (KO)细胞，以确定在没有这两种蛋白质的情况下黄病毒和冠状病毒的生命周期进展到什么程度。 我们之前的研究表明，缺乏TMEM41B和VMP1会导致先天免疫反应增强 一旦感染了黄病毒。我们假设这两种蛋白质都被招募到病毒RNA复制的位置，并且 在没有它们的情况下，RNA复制启动，产生病毒双链RNA(DsRNA)。然而， 如果没有适当的复制细胞器，dsRNA就会暴露并触发先天免疫反应。或者， 鉴于TMEM41B和VMP1的S脂质扰乱酶的活性和在脂质动态平衡中的作用，它们的缺失可能 诱导内质网应激，触发未折叠蛋白反应(UPR)，与dsRNA协同可能导致 增强了先天免疫反应。在目标2中，我们将测试病毒在双KO细胞中的感染情况，这些细胞既没有 除了病原体感应、干扰素信号和UPR激活所必需的基因外，还包括蛋白质。我们会 进一步进行RNAseq实验以调查干细胞和干细胞来源中缺乏TMEM41B 在没有病毒复制和存在病毒复制的情况下，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械分析来表征自然发生的情况 我们之前发现的影响黄病毒复制的TMEM41B中的SNP，以及报告的几个VMP1丢失。 功能突变体。我们将进一步采取深度突变扫描的方式来全面表征 TMEM41B和VMP1，并确定它们的细胞是否需要任何结构域或氨基酸 和前驱功能。这一功能特征将确定可以在 机械分析，并可能识别两种蛋白质中的氨基酸或界面，这些氨基酸或界面可以被靶向阻止 病毒感染，对细胞生物学的破坏最小。

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.