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：
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
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/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

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.

项目摘要蚊子传播的黄病毒具有（重新），引起了登革热病毒和Zika的广泛疾病病毒是重要成员。在最近的寨卡病毒暴发中，很明显该病毒会引起严重未出生儿童的先天性异常，对公共卫生构成直接威胁。像其他积极的滞留的RNA病毒，黄病毒广泛重新排列内质网（ER）膜以创建一个 RNA复制的有利利基。 ER膜是对协调的物理支持有效复制所需的病毒和细胞成分的积累。但是，对病毒蛋白组装功能性RNA复制复合物的分子机制，更多具体而言，宿主蛋白在此过程中的作用。通过基因组规模的遗传筛选，我们有发现ER - 膜多蛋白复合物，包括寡糖胆囊酶复合酶复合酶复合物（OST），在黄病毒感染中具有关键作用。我们指出了OST复合物到病毒RNA的作用复制但出乎意料地发现，N-连接糖基化中OST的酶活性是可分配的。我们证明了OST复合物与几种非结构蛋白结合，并且存在于复合物中与病毒RNA相关。我们的初步数据表明，黄病毒已经进化以协调通过在ER膜上通过特定相互作用的功能RNA复制复合物组装宿主蛋白和病毒蛋白/RNA。提出了两个集成的特定目标，以了解黄病毒利用宿主因素建立生产性感染的机制。在AIM 1中，我们将使用精确绘制单个病毒之间蛋白质 - 蛋白质相互作用的生化和遗传方法蛋白质和细胞因子侧重于OST复合物。鉴于对OST的强烈依赖复杂，这些相互作用界面可能是药理破坏的基础。在AIM 2中，我们将采用以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和剪辑在内的创新方法 SEQ，它将提供蛋白质甚至单个蛋白质的详细概述结合病毒RNA的结构域。这些研究将从根本上提高我们对分子机制通过不同的黄病毒具有颠覆ER功能以促进其传染性周期，可能有助于开发基于宿主的抗病毒药的治疗靶标。