Host Genes Critical for Flavivirus Infection

对黄病毒感染至关重要的宿主基因

• 批准号: 10054984
• 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/10054984
• 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; Structure; 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.

项目总结 蚊媒的黄病毒重新出现，导致登革热病毒和寨卡病毒的广泛传播。 病毒为重要成员。在最近的寨卡病毒爆发中，很明显，这种病毒可以造成严重的 未出生儿童的先天异常，对公共健康构成直接威胁。就像其他积极因素一样- 链RNA病毒，黄病毒广泛重排内质网(ER)膜，创建一个 对RNA复制有利的生态位。ER膜作为协调的物质支撑 有效复制所需的病毒和细胞成分的积累。然而，人们对此知之甚少。 病毒蛋白组装功能性RNA复制复合体等的分子机制 具体地说，宿主蛋白在这一过程中的作用。通过基因组规模的基因筛查，我们有 发现ER膜多蛋白复合体，包括寡糖转移酶复合体 (Ost)，在黄病毒感染中起关键作用。我们确定了OST复合体对病毒RNA的作用 但却意外地发现，OST在N-连接糖基化中的酶活性是可有可无的。 我们证明了OST复合体与几种非结构蛋白结合，并存在于复合体中 与病毒核糖核酸有关。我们的初步数据表明，黄病毒已经进化成协调 通过特定的相互作用在内质网膜上组装功能性RNA复制复合体 宿主蛋白和病毒蛋白/RNA。提出了两个综合的具体目标来理解 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 精确定位病毒个体间蛋白质相互作用的生物化学和遗传学方法 蛋白质和细胞因子，重点是OST复合体。鉴于对OST的强烈依赖 这些相互作用界面很复杂，可以作为药理干扰的基础。在目标2中，我们将 采取以RNA为中心的方法，系统地发现形成病毒RNA蛋白的细胞蛋白质 登革热和寨卡病毒感染细胞中的复合体。我们将使用包括Chirp-MS和CLIP-MS在内的创新方法- SEQ，这将提供对蛋白质的详细和全面的概述，甚至是单个蛋白质 与病毒RNA结合的区域。这些研究将从根本上促进我们对 不同黄病毒颠覆ER功能促进其感染性的分子机制 这可能有助于开发以宿主为基础的抗病毒药物的治疗靶点。