Identifying and inhibiting the SARS-CoV-2 packaging mechanism

识别和抑制 SARS-CoV-2 包装机制

基本信息

批准号：
10204705
负责人：
JENNIFER A DOUDNA
金额：
$ 51.98万
依托单位：
J. DAVID GLADSTONE INSTITUTES
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-15 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10204705
关键词：
2019-nCoV Affinity Chromatography Antiviral Agents Binding Binding Proteins Biochemical Bioinformatics Biological Assay COVID-19 assay COVID-19 treatment CRISPR/Cas technology Capsid Cell Culture Techniques Cells Chiroptera Combined Modality Therapy Complement Coronavirus Coronavirus Infections Data Detection Development Disease Disease Outbreaks Drug Targeting Drug resistance Enterovirus 71 Foundations Future Gene Delivery Genes Genome Goals Green Fluorescent Proteins HIV Infection Influenza Knock-out Laboratories Libraries Life Cycle Stages Location Mass Spectrum Analysis Measures Mediating Membrane Proteins Methods Middle East Respiratory Syndrome Coronavirus Molecular Murine hepatitis virus Mutation Nucleocapsid Nucleocapsid Proteins Open Reading Frames Population Process Production Proteins Proteomics Publishing RNA RNA Biochemistry RNA Viruses Regimen Reporter Research Role SARS coronavirus SARS-CoV-2 inhibitor Signal Transduction Structural Protein Structure Technology Test Result Testing Therapeutic Transcript Transfection Viral Viral Genome Viral Packaging Viral Pathogenesis Viral Proteins Viral Structural Proteins Virion Virus Virus Diseases Virus-like particle Work Zoonoses base betacoronavirus coronavirus treatment drug development drug discovery effective therapy env Gene Products experience high throughput screening inhibitor/antagonist insight laboratory experience lead candidate prevent screening small molecule small molecule inhibitor stem therapeutic target viral RNA viral detection viral resistance

项目摘要

Project Summary We aim to determine the molecular basis for SARS-CoV-2 viral packaging and to develop a screening strategy to identify inhibitors of this key step in the coronavirus infection cycle. Selective packaging of the viral genome, over more abundant transcripts, involves specific interactions between an RNA packaging signal, viral structural proteins and possibly other factors. Inhibition of this process would block formation of infectious virions and thereby contribute to a therapeutic regimen that would prevent or treat infection. Building on our laboratory’s extensive expertise in RNA biochemistry and virus-like particle research, we propose to determine the functional SARS-CoV-2 packaging signal and to develop a robust small molecule-based assay for SARS-CoV-2 packaging inhibition. To determine the components of the SARS-CoV-2 packaging mechanism, we will generate virus-like particles (VLPs) that contain the structural proteins of the virus but not the viral genome. The absence of the genome renders these VLPs non-infectious and therefore safe to work with. Methods for generating these VLPs derive from published research with other coronaviruses as well as our own lab’s experience working with influenza and HIV VLPs. SARS-CoV-2 VLPs will be produced by co-expressing the viral spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. RNA molecules containing the packaging signal can be packaged into these VLPs and delivered into receiver cells, providing an assay for packaging signal detection. In parallel, this approach will be used to establish a screening assay to identify viral packaging inhibitors. These two aims are independent, yet the results of each workstream will inform both the fundamental and applied aspects of the project. Our long-term objective is to develop a small molecule inhibitor of SARS-CoV-2 viral packaging. This approach has the following advantages: 1) we will naturally detect nucleocapsid inhibitors, which can be potent antiviral drugs as shown for HIV and other viruses due to strict constraints on nucleocapsid function; 2) our approach targets a step in the viral infection cycle that is not currently the focus of major therapeutic discovery efforts, enhancing the opportunity to find a new and/or complementary antiviral strategy; and 3) our screening approach does not require live virus and can be executed safely in most high-throughput screening facilities. The research proposed here will enable the development of new antiviral strategies for treating coronaviruses. SARS-CoV-2 is the third betacoronavirus to trigger a zoonotic outbreak in the last 18 years and estimates suggest that ~5000 related viruses are circulating within bat populations around the world. Our proposal targets a critical yet relatively understudied step of the coronavirus life cycle that is a promising target of selective small- molecule inhibition. The results of this work will inform and enable other viral inhibition efforts and provide a basis for future high-throughput drug discovery initiatives.

项目摘要我们旨在确定SARS-COV-2病毒包装的分子基础，并制定筛查策略确定冠状病毒感染周期中此关键步骤的抑制剂。病毒基因组的选择性包装，在更丰富的转录本上，涉及RNA包装信号，病毒结构之间的特定相互作用蛋白质，可能是其他因素。抑制此过程将阻止传染病的形成和从而有助于一种可以预防或治疗感染的治疗方案。建立我们的实验室 RNA生物化学和类似病毒样粒子研究方面的广泛专业知识，我们建议确定功能 SARS-COV-2包装信号，并开发用于SARS-COV-2包装的强大的基于小分子的测定抑制。为了确定SARS-COV-2包装机制的成分，我们将生成类似病毒的颗粒（VLP）包含病毒的结构蛋白，而不是病毒基因组。缺乏基因组使这些VLP无感染，因此可以安全地使用。生成这些VLP的方法从与其他冠状病毒的发表研究以及我们自己的实验室的经验与Attractza合作和HIV VLP。 SARS-COV-2 VLP将通过共表达病毒尖峰（S），包膜（E），膜（M）和核素（N）蛋白。可以包装包装包装信号的RNA分子进入这些VLP并输送到接收器中，提供了包装信号检测的测定法。并联，该方法将用于建立筛选分析以识别病毒包装抑制剂。这两个目标是独立的，但是每种工作流的结果都会告知项目。我们的长期目标是开发SARS-COV-2病毒包装的小分子抑制剂。这种方法具有以下优点：1）我们将自然检测到核Ocapsid抑制剂，这可能是有效的抗病毒由于严格限制了对核素功能的限制，如HIV和其他病毒所示的药物； 2）我们的方法针对病毒感染周期的一步，目前并不是主要的治疗发现工作的重点增强机会寻找新的和/或互补的抗病毒策略； 3）我们的筛选方法不需要活病毒，并且可以在大多数高通量筛查设施中安全执行。此处提出的研究将使您能够制定新的抗病毒病毒策略。 SARS-COV-2是在过去18年中引发人畜共患爆发的第三次Betacoronavirus和估计表明在世界各地的蝙蝠种群中，约有5000个相关病毒正在流通。我们的建议目标冠状病毒生命周期的一个关键但相对了解的步骤，是选择性小的靶向目标分子抑制。这项工作的结果将为其他病毒抑制作用提供信息，并提供基础对于未来的高通量药物发现计划。