Identifying and inhibiting the SARS-CoV-2 packaging mechanism

项目摘要 我们的目标是确定SARS-CoV-2病毒包装的分子基础，并制定筛选策略 以确定冠状病毒感染周期中这一关键步骤的抑制剂。病毒基因组的选择性包装， 在更丰富的转录，涉及RNA包装信号，病毒结构之间的特定相互作用， 蛋白质和其他可能的因素。抑制这一过程将阻断感染性病毒体的形成， 从而有助于预防或治疗感染的治疗方案。基于我们实验室的 在RNA生物化学和病毒样颗粒研究的广泛专业知识，我们建议确定功能 SARS-CoV-2包装信号，并开发用于SARS-CoV-2包装的稳健的基于小分子的检测方法 抑制作用 为了确定SARS-CoV-2包装机制的组成部分，我们将生成病毒样颗粒 VLP是指含有病毒结构蛋白但不含病毒基因组的VLP。基因组的缺失 使这些VLP不具有传染性，因此可以安全使用。用于产生这些VLP的方法衍生 从已发表的关于其他冠状病毒的研究以及我们自己的实验室处理流感的经验来看， 和艾滋病毒VLP。SARS-CoV-2 VLP将通过共表达病毒刺突（S），包膜（E）， 膜（M）和核衣壳（N）蛋白。含有包装信号的RNA分子可以被包装 进入这些VLP并递送到接收器细胞中，提供用于包装信号检测的测定。同时， 该方法将用于建立筛选测定以鉴定病毒包装抑制剂。这两个目标 是独立的，但每个工作流的结果将通知的基本和应用方面， 项目 我们的长期目标是开发一种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）我们将自然地检测核衣壳抑制剂，其可以是有效的抗病毒剂 由于对核衣壳功能的严格限制，HIV和其他病毒的药物; 2）我们的方法 靶向病毒感染周期中的一个步骤，该步骤目前不是主要治疗发现努力的焦点， 增加寻找新的和/或互补的抗病毒策略的机会;以及3）我们的筛查方法 不需要活病毒，并且可以在大多数高通量筛选设施中安全地执行。 这里提出的研究将能够开发新的抗病毒策略来治疗冠状病毒。 SARS-CoV-2是过去18年来第三种引发人畜共患病爆发的β冠状病毒， 这表明大约5000种相关病毒在世界各地的蝙蝠种群中传播。我们的建议目标 这是冠状病毒生命周期中一个关键但研究相对不足的步骤，是选择性小分子- 分子抑制这项工作的结果将为其他病毒抑制工作提供信息和支持，并提供基础 用于未来的高通量药物发现计划。

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.