Host Genes Critical for Flavivirus Infection

对黄病毒感染至关重要的宿主基因

• 批准号: 10293600
• 负责人: Jan E Carette
• 金额: $ 39.13万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-11-14 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10293600
• 关键词: Antiviral Agents; Antiviral Therapy; Binding; Binding Proteins; Biochemical Genetics; Cell physiology; Cells; Cellular biology; Complex; Congenital Abnormality; Culicidae; Data; Dengue; Dengue Infection; Dengue Virus; Dependence; Disease; Disease Outbreaks; Distant; Endoplasmic Reticulum; Flaviviridae; Flavivirus; Flavivirus Infections; Genes; Genetic Screening; Goals; Hepatitis C virus; Human; Immunoprecipitation; Individual; Infection; Integration Host Factors; Life Cycle Stages; Link; Medical; Membrane; Messenger RNA; Methods; Molecular; Multiprotein Complexes; Nonstructural Protein; Pharmacology; Play; Process; Protein Binding Domain; Protein Isoforms; Protein-Protein Interaction Map; Proteins; Public Health; RNA; RNA Viruses; RNA replication; RNA-Binding Proteins; Ribosomes; Role; Testing; Therapeutic; Translations; Untranslated RNA; Viral; Viral Proteins; Virus; Virus Diseases; Virus Replication; Widespread Disease; ZIKA; Zika Virus; base; crosslink; genetic approach; genome-wide; glycosylation; human disease; innovation; insight; member; mosquito-borne; protein complex; recruit; therapeutic target; unborn child; viral RNA

PROJECT SUMMARY The mosquito-borne flaviviruses have (re-)emerged causing widespread disease with dengue virus and Zika virus as important members. In recent Zika virus outbreaks, it became clear that the virus can cause severe congenital abnormalities in unborn children, posing an immediate threat to public health. Like other positive- stranded RNA viruses, flaviviruses extensively rearrange endoplasmic reticulum (ER) membranes to create a favorable niche for RNA replication. The ER-membrane serves as physical support for the coordinated accumulation of viral and cellular components required for efficient replication. However, little is known about the molecular mechanisms by which viral proteins assemble functional RNA replication complexes and, more specifically, the role of host proteins in this process. Through genome-scale genetic screens, we have discovered that ER-membrane multiprotein complexes, including the oligosaccharyltransferase complex (OST), have critical roles in flavivirus infection. We pinpointed the role of the OST complex to viral RNA replication but unexpectedly found that the enzymatic activity of OST in N-linked glycosylation is dispensable. We demonstrated that the OST complex binds to several nonstructural proteins and is present in complexes associated with viral RNA. Our preliminary data suggest that flaviviruses have evolved to coordinate the assembly of functional RNA replication complexes at the ER membrane through specific interactions between the host proteins and viral proteins/RNA. Two integrated specific aims are proposed to understand the mechanisms by which flaviviruses exploit host factors to establish productive infection. In aim 1, we will use biochemical and genetic approaches to precisely map the protein-protein interactions between individual viral proteins and the cellular factors with a focus on the OST complex. Given the strong dependence on the OST complex, these interaction interfaces may serve as a basis for pharmacological disruption. In aim 2, we will take an RNA-centric approach to systematically uncover cellular proteins that form viral RNA-protein complexes in dengue and Zika infected cells. We will use innovative methods including ChIRP-MS and CLIP- seq, that will provide a detailed and comprehensive overview of the proteins, and even the individual protein domains that bind to the viral RNA. These studies will fundamentally advance our understanding of the molecular mechanisms by which different flaviviruses have subverted ER functions to promote their infectious cycles, and may help to develop therapeutic targets for host-based antivirals.

项目摘要 蚊子传播的黄病毒已经（重新）出现，导致登革热病毒和寨卡病毒的广泛传播 病毒作为重要成员。在最近的寨卡病毒疫情中，很明显，该病毒可导致严重的 胎儿先天畸形，对公共卫生构成直接威胁。像其他积极的- 黄病毒广泛地重排内质网（ER）膜，以产生一种 有利于RNA复制的生态位。ER膜作为协调的细胞的物理支持， 有效复制所需的病毒和细胞成分的积累。然而，人们对 病毒蛋白组装功能性RNA复制复合物的分子机制，以及更多 特别是宿主蛋白在这一过程中的作用。通过基因组规模的遗传筛选， 发现ER-膜多蛋白复合物，包括寡糖基转移酶复合物， (OST)，在黄病毒感染中起关键作用。我们确定了OST复合物对病毒RNA的作用， 复制，但出乎意料地发现OST在N-连接糖基化中的酶活性降低。 我们证明了OST复合物与几种非结构蛋白结合，并以复合物的形式存在 与病毒RNA有关。我们的初步数据表明，黄病毒已经进化到协调 在内质网膜上通过特异性相互作用组装功能性RNA复制复合物， 宿主蛋白和病毒蛋白/RNA。提出了两个综合的具体目标，以了解 黄病毒利用宿主因子建立生产性感染的机制。在目标1中，我们将使用 生物化学和遗传学方法来精确绘制单个病毒之间的蛋白质-蛋白质相互作用， 蛋白质和细胞因子，重点是OST复合物。鉴于对OST的强烈依赖 复杂，这些相互作用界面可以作为药理学破坏的基础。在目标2中，我们 采用以RNA为中心方法系统地揭示形成病毒RNA蛋白的细胞蛋白 登革热和寨卡病毒感染的细胞中的复合物。我们将使用创新的方法，包括ChIRP-MS和CLIP- seq，这将提供一个详细和全面的概述蛋白质，甚至个别蛋白质 与病毒RNA结合的结构域。这些研究将从根本上推进我们对 不同黄病毒破坏ER功能以促进其感染性的分子机制 周期，并可能有助于开发基于宿主的抗病毒药物的治疗靶点。