Defining the functional interface between the ER and flaviviruses

定义 ER 和黄病毒之间的功能界面

基本信息

批准号：
10180892
负责人：
Sara Cherry
金额：
$ 61.39万
依托单位：
UNIVERSITY OF PENNSYLVANIA
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：
2018-06-20 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10180892
关键词：
Affect Americas Antiviral Agents Biochemical Biology CRISPR/Cas technology Cells Cellular Stress Cellular biology Complement Complex Cytoplasm Dengue Dengue Infection Development Diamond Endoplasmic Reticulum Endosomes Flavivirus Flavivirus Infections Generations Genes Genetic Genetic Screening Genome Glycoproteins Goals Grant Health Human Immunology Infection Insecta Integration Host Factors Japanese Encephalitis Laboratories Life Cycle Stages Mass Spectrum Analysis Membrane Molecular Neural Cell Adhesion Molecule L1 Pathway interactions Peptide Signal Sequences Polyproteins Process Production Protein Glycosylation Protein Secretion Proteins RNA RNA Viruses RNA interference screen RNA replication Role Shapes Site Stress Structural Protein Therapeutic Toxic effect Translating Translations Untranslated RNA Vaccines Vector-transmitted infectious disease Viral Viral Pathogenesis Viral Proteins Viral Structural Proteins Virion Virus Virus Diseases Virus-like particle West Nile virus Yellow Fever Yellow fever virus ZIKA ZIKV infection Zika Virus clinical development combat emerging pathogen genome-wide immunogenicity in vivo innovation lipid biosynthesis lipid metabolism multicatalytic endopeptidase complex novel therapeutics polypeptide protein complex protein folding response signal peptidase small molecule ubiquitin ligase virology

项目摘要

Flaviviruses are a genus of related positive-stranded enveloped RNA viruses that significantly impact human health, including dengue (DENV), Zika (ZIKV). West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. The discovery of host factors critical for viral infection reveals new aspects of cell biology, intricate virus-host relationships, and potential targets for antiviral therapeutics. After entering cells and fusing in the acidified endosome, the flavivirus RNA genome penetrates into the cytoplasm and is then translated into a polyprotein and processed at the endoplasmic reticulum (ER). In addition to utilizing many functions of the ER for protein production, flaviviruses extensively remodel ER membranes to create a niche for RNA- dependent RNA replication. The ER also is the site for flavivirus assembly, which enables the production and secretion of new infectious viruses. Thus, the ER serves as a central point for orchestrating many of the essential steps in the flavivirus infection life cycle. Despite this, little is known about the host factors and molecular mechanisms at the ER that are required for optimal translation and processing of the viral proteins or for the assembly of the replication niche. We recently have performed several genetic screens to identify important components in this process. Our genome-wide RNAi screen with WNV in insect cells validated 18 genes associated with ER biology that promote infection. Our CRISPR/Cas9 gene-editing screen in human cells with WNV also identified 12 ER-associated genes. These screens converged on ER-resident proteins as being critical for WNV infection and included genes associated with ER-translocation and signal peptide processing, ER-associated degradation (ERAD), protein glycosylation, protein folding and lipid metabolism. Indeed, infection of WNV, ZIKV, JEV, DENV, and YFV all required specific subunit components of the host signal peptidase complex (SPCS) for processing of the viral polyprotein, the production of viral glycoproteins and thus generation of nascent virions. The objective of this proposal is to define the molecular mechanisms by which flaviviruses use specific ER-associated host proteins to promote viral translation, polyprotein processing, RNA replication, and/or assembly. Aim 1 will define the mechanism by which the ER translocon promotes polyprotein translation and processing while Aim 2 will dissect the role of ER-associated decay (ERAD) in promoting flavivirus replication. Our long-term goal is to determine the mechanisms by which flaviviruses exploit the ER for their replication, as this will reveal both fundamental aspects of virology as well as new avenues for antiviral therapeutics.

黄病毒是一类相关的正链有包膜 RNA 病毒，其显着影响人类健康，包括登革热（DENV）、寨卡病毒（ZIKV）。西尼罗河 (WNV)、日语脑炎（JEV）和黄热病（YFV）病毒。发现对病毒至关重要的宿主因子感染揭示了细胞生物学的新方面、复杂的病毒-宿主关系以及潜在的抗病毒治疗的靶点。进入细胞并与酸化的内体融合后，黄病毒RNA基因组渗透到细胞质中，然后翻译成多蛋白并在内质网（ER）处加工。除了利用 ER 的许多功能之外黄病毒广泛重塑内质网膜，为 RNA 创造一个利基依赖RNA复制。内质网也是黄病毒组装的场所，这使得新的传染性病毒的产生和分泌。因此，ER 充当了中心点协调黄病毒感染生命周期中的许多重要步骤。尽管如此，还是少有了解内质网优化所需的宿主因素和分子机制病毒蛋白的翻译和加工或复制生态位的组装。我们最近进行了几次基因筛选，以确定该过程中的重要组成部分。我们在昆虫细胞中使用 WNV 进行全基因组 RNAi 筛选，验证了 18 个与 ER 相关的基因促进感染的生物学。我们在人类细胞中对 WNV 进行 CRISPR/Cas9 基因编辑筛选还鉴定了 12 个 ER 相关基因。这些筛选集中于 ER 驻留蛋白，如对于西尼罗河病毒感染至关重要，包括与内质网易位和信号相关的基因肽加工、ER 相关降解 (ERAD)、蛋白质糖基化、蛋白质折叠和脂质代谢。事实上，西尼罗河病毒、寨卡病毒、乙脑病毒、登革热病毒和黄热病毒的感染都需要特定的用于处理病毒的宿主信号肽酶复合物（SPCS）的亚基成分多蛋白，病毒糖蛋白的产生，从而产生新生病毒颗粒。这该提案的目的是定义黄病毒使用特定的分子机制 ER 相关宿主蛋白可促进病毒翻译、多蛋白加工、RNA 复制、和/或组装。目标 1 将定义 ER 易位子促进的机制多蛋白翻译和加工，而目标 2 将剖析 ER 相关衰变的作用（ERAD）促进黄病毒复制。我们的长期目标是通过以下方式确定机制：哪些黄病毒利用 ER 进行复制，因为这将揭示两个基本方面病毒学以及抗病毒治疗的新途径。