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开发筛选策略 以确定冠状病毒感染周期中这一关键步骤的抑制物。病毒基因组的选择性包装， 更丰富的转录本，涉及RNA包装信号、病毒结构之间的特定相互作用 蛋白质，可能还有其他因素。抑制这一过程将阻止传染性病毒粒子的形成和 从而为预防或治疗感染的治疗方案做出贡献。在我们实验室的基础上 在RNA生物化学和病毒样颗粒研究方面拥有丰富的专业知识，我们建议确定 SARS-CoV-2包装信号及基于小分子的SARS-2包装检测方法的建立 抑制力。 为了确定SARS-CoV-2包装机制的组件，我们将生成病毒样颗粒 (VLP)包含病毒的结构蛋白，但不包含病毒基因组。基因组的缺失 使这些VLP不具传染性，因此可以安全使用。用于生成这些VLP的方法派生 来自已发表的对其他冠状病毒的研究以及我们自己实验室处理流感的经验 和艾滋病毒VLP。SARS-CoV-2 VLP将通过共表达病毒尖峰(S)、包膜(E)、 膜蛋白(M)和核衣壳蛋白(N)。含有包装信号的RNA分子可以被包装 进入这些VLP并输送到接收器细胞，为包装信号检测提供了一种检测方法。同时， 这种方法将被用来建立一种筛选试验来鉴定病毒包装抑制剂。这两个目标 是独立的，但是每个工作流的结果都将通知 项目。 我们的长期目标是开发一种针对SARS-CoV-2病毒包装的小分子抑制剂。这种方法 有以下优点：1)我们会自然地检测到核衣壳抑制物，它可以有效地抗病毒 由于对核衣壳功能的严格限制，用于HIV和其他病毒的药物；2)我们的方法 目标是病毒感染周期中目前不是主要治疗发现努力重点的一步， 增加寻找新的和/或补充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，可以在大多数高通量筛查设施中安全执行。 这里提出的研究将使开发治疗冠状病毒的新的抗病毒策略成为可能。 SARS-CoV-2是过去18年来引发人畜共患疫情的第三种贝塔冠状病毒，据估计 表明大约5000种相关病毒正在世界各地的蝙蝠种群中传播。我们的提案针对的是 冠状病毒生命周期中一个关键但相对研究较少的步骤，是选择性小病毒的一个有希望的目标。 分子抑制。这项工作的结果将为其他病毒抑制努力提供参考和支持，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.