Host Genes Critical for Flavivirus Infection

对黄病毒感染至关重要的宿主基因

基本信息

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
项目类别：
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

项目摘要

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.

项目概要蚊媒黄病毒（重新）出现，导致登革热病毒和寨卡病毒广泛传播疾病病毒作为重要成员。在最近的寨卡病毒爆发中，很明显该病毒可以造成严重的后果未出生儿童的先天畸形，对公共健康构成直接威胁。和其他积极的一样—— 链状 RNA 病毒、黄病毒广泛地重新排列内质网 (ER) 膜以形成 RNA复制的有利生态位。 ER 膜作为协调的物理支持有效复制所需的病毒和细胞成分的积累。然而，人们对此知之甚少病毒蛋白组装功能性 RNA 复制复合物的分子机制等等具体来说，宿主蛋白在此过程中的作用。通过基因组规模的遗传筛选，我们已经发现内质网膜多蛋白复合物，包括寡糖转移酶复合物 (OST)，在黄病毒感染中起关键作用。我们查明了 OST 复合物对病毒 RNA 的作用但意外地发现OST在N联糖基化中的酶活性是可有可无的。我们证明 OST 复合物与多种非结构蛋白结合并存在于复合物中与病毒RNA有关。我们的初步数据表明黄病毒已经进化到协调通过特定的相互作用，在 ER 膜上组装功能性 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，我们将使用生物化学和遗传学方法可精确绘制个体病毒之间的蛋白质-蛋白质相互作用蛋白质和细胞因子，重点关注 OST 复合物。鉴于对 OST 的强烈依赖虽然复杂，但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，我们将采用以 RNA 为中心的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细胞中的复合物。我们将使用包括 ChIRP-MS 和 CLIP 在内的创新方法 seq，这将提供蛋白质甚至单个蛋白质的详细而全面的概述与病毒 RNA 结合的结构域。这些研究将从根本上增进我们对不同黄病毒破坏内质网功能以促进其传染性的分子机制循环，并可能有助于开发基于宿主的抗病毒药物的治疗靶点。