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

TMEM41B：具有抗病毒潜力的泛黄病毒和泛冠状病毒宿主因子

• 批准号: 10587597
• 负责人: Charles M Rice
• 金额: $ 46.09万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-19 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10587597
• 关键词: 2019-nCoV; Address; Amino Acids; Arbovirus Infections; Autophagocytosis; Biological Assay; CRISPR/Cas technology; Cell Lineage; Cells; Cellular biology; Coronavirus; Coronavirus Infections; Disease Outbreaks; Double-Stranded RNA; Drug Targeting; Enzymes; Epidemic; Event; Family; Flavivirus; Flavivirus Infections; Genes; Genome; Goals; Health; Homeostasis; Human; Infection; Innate Immune Response; Integral Membrane Protein; Integration Host Factors; Interferons; Investigation; Knock-out; Knowledge; Lead; Lipid Mobilization; Lipids; Manuscripts; Membrane; Membrane Proteins; Modeling; Molecular; Morbidity - disease rate; Mus; Mutate; Natural Immunity; Organelles; Pathway interactions; Pattern recognition receptor; Pharmaceutical Preparations; Phenotype; Play; Polyproteins; Population; Proteins; Publishing; RNA; RNA Viruses; RNA replication; Reporting; Resistance; Role; Series; Signal Transduction; Single Nucleotide Polymorphism; Site; Structure; Testing; Tissues; Translations; Vacuole; Viral; Viral Genome; Virus; Virus Diseases; Virus Replication; Work; arthropod-borne; combat; conditional knockout; druggable target; embryonic stem cell; endoplasmic reticulum stress; experimental study; genetic approach; genome-wide; human pluripotent stem cell; immune activation; ineffective therapies; insight; lipid metabolism; loss of function; mortality; mutant; mutation screening; pandemic disease; pathogen; prevent; recruit; respiratory; response; stem cells; synergism; therapeutic target; transcriptome sequencing; viral RNA

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.

项目摘要 节肢动物传播的黄病毒和呼吸传播的冠状病毒有可能引起严重的 情节和大流行。准备和应对病毒爆发的一种策略是开发药物 目标宿主因素病毒需要完成其生命周期。通过一系列CRISPR/CAS9基因破坏 屏幕，我们确定了跨膜蛋白41b（TMEM41B）和密切相关的胶水膜 蛋白质1（VMP1）作为关键的泛氟病毒和泛氧化病毒宿主因子。两种蛋白质都是高度保守的 脂质串联酶在自噬中扮演角色。我们目前的模型是来自Flavivirdae和 Coronaviridae家族劫持了TMEM41B和VMP1的重塑和诱导的能力 膜曲率建立受膜保护的病毒RNA复制细胞器。 该提案的总体目标是了解两种蛋白质的支持 黄病毒和冠状病毒感染。我们以前的工作表明在进入后步骤中需要TMEM41B 在病毒RNA复制处或之前。在AIM 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敲除中的翻译，多蛋白处理和复制细胞器形成 （KO）细胞确定在没有任何一种蛋白质的情况下，黄病毒和冠状病毒生命周期的发展。 我们先前表明缺乏TMEM41B和VMP1，引起了先天免疫增长 在黄病毒感染后。我们假设两种蛋白质均被募集到病毒RNA复制的位置，并且 在不存在的情况下，RNA复制启动并产生病毒双链RNA（DSRNA）。然而， 没有适当的复制细胞器dsRNA暴露并触发先天的免疫响应。或者， 鉴于TMEM41B和VMP1的脂质刺激性活性和脂质稳态的功能，他们的缺席可能 诱导ER应力，这会触发与DSRNA协同作用的展开蛋白反应（UPR）可能导致A 先天免疫增强。在AIM 2中，我们将测试缺乏两者的双KO细胞中的病毒感染 蛋白质除了对病原体传感，IFN信号传导和UPR激活必不可少的基因之外。我们将 进一步进行RNASEQ实验，以研究干细胞和干细胞衍生的TMEM41B缺乏 在不存在和存在病毒复制的情况下，代表不同组织谱系的一级样细胞。 最后，在AIM 3中，将使用一系列表型和机械测定面板来表征自然发生的表征 我们以前发现TMEM41B中的SNP会影响黄病毒的复制，有几个报道了VMP1 - 功能突变体。我们将进一步采用深层的突变扫描方法来全面地表征 TMEM41B和VMP1，并确定其细胞是否需要差异 和临时功能。这种功能表征将识别可以详细研究的突变体 机械测定法，可以鉴定两种蛋白质中的氨基酸或界面，以防止 病毒感染对细胞生物学的干扰最小。