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

基本信息

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
项目类别：
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

项目摘要

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.

项目概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准备和应对病毒爆发的一项策略是开发能够预防流行病的药物。病毒需要通过一系列 CRISPR/Cas9 基因破坏来完成其生命周期。通过筛选，我们鉴定出了跨膜蛋白 41B (TMEM41B) 和密切相关的真空膜蛋白 1 (VMP1) 作为关键的泛黄病毒和泛冠状病毒宿主因子，这两种蛋白都是高度保守的。我们目前的模型是来自黄病毒科和黄病毒科的病毒。冠状病毒科劫持 TMEM41B 和 VMP1，因为它们具有重塑 ER 膜并诱导膜曲率以建立膜保护的病毒RNA复制细胞器。我们该提案的总体目标是了解如何在机制层面上支持蛋白质我们之前的工作表明，在输入后步骤中需要 TMEM41B。在病毒 RNA 复制时或之前，我们将探究病毒生命周期的早期事件，包括初级事件。 WT、TMEM41B 和 VMP1 敲除中的翻译、多蛋白加工和复制细胞器形成 (KO) 细胞来确定黄病毒和冠状病毒在没有任何一种蛋白质的情况下生命周期的进展程度。我们之前表明，缺乏 TMEM41B 和 VMP1 会诱导香水先天免疫反应我们勇敢地承认这两种蛋白质都被招募到病毒 RNA 复制位点，并且如果没有它们，RNA 复制就会启动并产生病毒双链 RNA (dsRNA)。没有适当的复制细胞器 dsRNA 就会暴露并触发先天免疫反应。鉴于 TMEM41B 和 VMP1 的脂质扰乱酶活性和脂质稳态功能，它们的缺失可能会影响脂质稳态。诱导 ER 应激，从而触发未折叠蛋白反应 (UPR)，与 dsRNA 协同作用可能会导致在目标 2 中，我们将测试缺乏其中任何一个的双 KO 细胞中的病毒感染。除了对病原体感应、IFN 信号传导和 UPR 激活至关重要的基因之外，我们还会提供蛋白质。进一步进行RNAseq实验来研究干细胞和干细胞来源中TMEM41B的缺乏在病毒复制不存在和存在的情况下，代表不同组织谱系的原代细胞。最后，在目标 3 中，将使用一组表型和机制测定来表征自然发生的我们之前发现 TMEM41B 中的 SNP 会影响黄病毒复制，并且有几个报道称 VMP1 丢失- 我们将进一步采用深度突变扫描方法来全面表征。 TMEM41B 和 VMP1 并确定其细胞是否需要不同的结构域或氨基酸和原病毒功能。这种功能表征将鉴定可以详细研究的突变体。机械分析，可以识别两种蛋白质中的氨基酸或界面，可以有针对性地预防病毒感染对细胞生物学的破坏最小。