Identifying and inhibiting the SARS-CoV-2 packaging mechanism

项目概要 我们的目标是确定 SARS-CoV-2 病毒包装的分子基础并制定筛查策略 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， 超过更丰富的转录本，涉及RNA包装信号、病毒结构之间的特定相互作用 蛋白质和可能的其他因素。抑制这一过程将阻止传染性病毒粒子的形成 从而有助于预防或治疗感染的治疗方案。建立在我们实验室的基础上 凭借在 RNA 生物化学和病毒样颗粒研究方面的广泛专业知识，我们建议确定功能 SARS-CoV-2 包装信号并开发一种强大的基于小分子的 SARS-CoV-2 包装检测方法 抑制。 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）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 不具有传染性，因此可以安全使用。生成这些 VLP 的方法衍生 来自已发表的其他冠状病毒研究以及我们自己实验室处理流感的经验 和 HIV 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。可包装含有包装信号的RNA分子 进入这些 VLP 并递送到受体细胞中，从而提供包装信号检测的分析方法。同时， 该方法将用于建立筛选试验来识别病毒包装抑制剂。这两个目标 是独立的，但每个工作流的结果将告知该工作的基本和应用方面 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。这种做法 具有以下优点：1）我们会自然地检测到核衣壳抑制剂，它可以是有效的抗病毒药物 由于对核衣壳功能的严格限制，针对 HIV 和其他病毒的药物； 2）我们的方法 针对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工作的重点， 增加寻找新的和/或补充的抗病毒策略的机会； 3）我们的筛选方法 不需要活病毒，可以在大多数高通量筛选设施中安全执行。 这里提出的研究将有助于开发治疗冠状病毒的新抗病毒策略。 SARS-CoV-2 是过去 18 年来第三种引发人畜共患疫情的 β 冠状病毒，估计 表明约有 5000 种相关病毒在世界各地的蝙蝠种群中传播。我们的提案目标 冠状病毒生命周期的一个关键但相对而言研究不足的步骤，是选择性小分子的有希望的目标 分子抑制。这项工作的结果将为其他病毒抑制工作提供信息并使其成为可能，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.