RAPID: the role of nsp3, nsp4 and nsp6 of SARS-CoV-2 in double membrane vesicle formation

SARS-CoV-2是一种冠状病毒，会导致呼吸道疾病新冠肺炎，是目前席卷世界的全球大流行的原因。与所有病毒一样，SARS-CoV-2依赖于受感染细胞的许多成分来驱动病毒复制。冠状病毒复制所依赖的所有真核细胞中的一个成分是与细胞不同区域分开的脂膜。冠状病毒劫持宿主细胞蛋白进行病毒复制，以及一些宿主细胞膜，重组它们以产生一种独特的膜结构，支持病毒RNA的生产。然而，目前还不清楚冠状病毒是如何完成宿主细胞膜的这种重组的。这种知识上的差距限制了对SARS-CoV-2如何在宿主细胞中复制的理解，从而限制了可能有助于减缓其复制的治疗方法的发展。因此，为了深入了解冠状病毒重组宿主细胞膜的机制，本项目将研究这种重组事件所需的三种冠状病毒蛋白(nsp3、nsp4和nsp6)。作为一个更广泛的影响，项目工作人员将与当地的初中生和高中生一起讨论SARS-CoV-2不同组成部分的结构，以及这些病毒蛋白的结构如何帮助科学家理解病毒的机制。所有正义RNA病毒都重组宿主细胞膜，以支持病毒RNA复制所需的复制和转录复合体的组装。冠状病毒，包括SARS和MERS，会产生一系列相互连接的双膜小泡，这些小泡似乎是从内质网膜产生的。这些囊泡的形成需要病毒编码的三种完整的膜非结构蛋白nsp3、nsp4和nsp6。然而，这三种NSP蛋白共同作用产生双膜囊泡的分子机制尚不清楚。为了深入了解这些机制，本项目将研究SARS-CoV-2的NSP3、NSP4和NSP6的结构和功能。具体地说，这项工作将1)确定与NSP3、NSP4和NSP6相互作用的宿主细胞蛋白；2)确定这三种病毒蛋白的原子级结构。为了实现这一点，这些蛋白质将在人肺细胞系中表达，并通过免疫沉淀和质谱仪筛选结合伙伴。这些蛋白质的结构将使用X射线结晶学和电子显微镜相结合的方法进行研究。了解NSP蛋白质的结构及其相互作用将为这种病毒如何利用其宿主细胞，导致毁灭性的混乱提供重要的洞察。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

SARS-CoV-2 is a coronavirus that leads to the respiratory disease COVID-19 and is the cause of a global pandemic currently gripping the world. SARS-CoV-2, like all viruses, relies on many components of the infected cell to drive viral replication. One component of all eukaryotic cells that coronaviruses rely on for their replication are lipid membranes that separate from different regions of a cell. Coronaviruses hijack host cell proteins for viral replication, as well as some of the host cell membranes, restructuring them to generate a unique membrane architecture that supports the production of viral RNA. However, it is currently unclear how coronaviruses accomplish this restructuring of host the cell membrane. This gap in knowledge has limited the understanding of how SARS-CoV-2 replicates in host cells and thus limited the development of therapeutics that might be useful to slow its replication. Therefore, to gain insight into the mechanisms by which coronaviruses restructure host cell membranes, this Project will investigate three coronavirus proteins (nsp3, nsp4 and nsp6) that are required for this restructuring event. As a Broader Impact, Project staff will engage with local middle and high school students to discuss the structures of different of SARS-CoV-2 components and how the structures of these viral proteins inform scientists’ understanding of viral mechanisms. All positive sense RNA viruses restructure host cell membranes to support the assembly of the replication and transcription complexes required for viral RNA replication. Coronaviruses, including SARS and MERS, generate a series of interconnected double membrane vesicles that appear to be generated from endoplasmic reticulum membranes. The formation of these vesicles require three integral membrane nonstructural proteins, nsp3, nsp4 and nsp6, that are encoded by the virus. However, the molecular mechanisms by which these three nsp proteins work together to generate double membrane vesicles is unknown. To gain insight into these mechanisms, this Project will study the structure and function of nsp3, nsp4 and nsp6 from SARS-CoV-2. Specifically, this work will 1 ) identify the host cell proteins that interact with nsp3, nsp4 and nsp6; and 2) determine the atomic level structures of these three viral proteins. To accomplish this, these proteins will be expressed in human lung cell lines and screened for binding partners by immunoprecipitation and mass spectrometry. The structure of these proteins will be investigated using a combination of X-ray crystallography and electron microscopy. Knowledge of the structure of the nsp proteins and their interactions will provide important insight into how this virus takes advantage of its host cell, resulting in a devastating pandemic.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.