RAPID: Interaction of the Coronavirus M protein with a Myosin V motor protein

COVID-19冠状病毒是一个世纪以来对全球健康最严重和最危险的威胁之一。目前的COVID-19疫情是由SARS-CoV-2病毒引起的，并且由于我们目前无法抑制病毒在细胞内的组装而加剧，这影响了冠状病毒在人类患者中的传播。SARS- cov -2是包括SARS、MERS和小鼠肝炎病毒（MHV）在内的相关冠状病毒家族的一员。这些是包裹在脂质膜中的RNA病毒，脂质膜中嵌入了许多不同的蛋白质。然而，总的来说，人们对病毒蛋白如何在人体细胞内包装在一起以组装冠状病毒颗粒知之甚少。这种知识的缺乏阻碍了针对病毒颗粒在人体细胞内组装的策略的发展。然而，病毒编码的一种膜蛋白，由于其膜结合而被称为M蛋白，是所有其他冠状病毒蛋白组装的起始种子。最近发现，MHV版本M蛋白的尾部可以与一种用于在人体细胞内运输的运动蛋白相互作用。由于这类冠状病毒的m蛋白尾部序列非常保守，因此本项目旨在确定这种相互作用是否可在SARS-CoV-2和其他相关冠状病毒之间推广。如果这种相互作用存在于SARS-CoV-2中，那么该项目将确定与肌凝蛋白马达相互作用的M蛋白部分。这将使未来的研究能够评估可以中断这种相互作用的药物，这可能会改变SARS-CoV-2和其他冠状病毒组装和传播的能力。作为更广泛的影响，该项目将为抗击Covid-19大流行提供急需的信息。人们对冠状病毒粒子在人体细胞中组装所需的过程知之甚少。包括SARS- cov -2、SARS、MERS和MHV在内的β -冠状病毒家族都利用类似的蛋白质汇编，包括一种被称为M蛋白的膜糖蛋白。M蛋白是其他冠状病毒蛋白组装成成熟病毒颗粒的中心。该项目旨在扩展最近的发现，即MHV的M蛋白与肌球蛋白Vb （MYO5B）的特定26个氨基酸选择性剪接外显子（称为外显子d）相互作用。这是首次发现与M蛋白相互作用的关键细胞内运输蛋白。该项目旨在确定这种相互作用是否保留在其他β -冠状病毒家族成员（包括SARS-CoV-2病毒）的M蛋白中。该项目还将确定M蛋白与MYO5B外显子D之间相互作用的分子基础。本项目将首先利用酵母2杂交法评估SARS- cov -2 （COVID-19）、SARS和MERS等相关冠状病毒的内尾结合情况。其次，将利用冠状病毒尾部的随机突变来确定与MYO5B外显子d相互作用的共同氨基酸基序。第三，将在HeLa细胞中表达野生型和突变型冠状病毒蛋白，并评估阻断与M蛋白上MYO5B相互作用的突变对内质网、高尔基体和质膜运输的影响。如果与sars - cov - 2m蛋白验证，与MYO5b外显子D相互作用的位点可以作为破坏冠状病毒粒子组装的靶点。该RAPID项目有望为未来开发破坏宿主细胞中病毒粒子组装的分子策略提供见解，这可能导致治疗Covid-19的新疗法。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The COVID-19 coronavirus represents one of the most acute and dangerous threat to global health in a century. The present COVID-19 outbreak is caused by the SARS-CoV-2 virus and is exacerbated by our present inability to inhibit the assembly of virus inside cells, which affects the spread of coronavirus within human patients. SARS-Cov-2 is a member of a family of related coronaviruses that includes SARS, MERS, and Mouse Hepatitis Virus (MHV). These are RNA viruses wrapped in a lipid membrane, which has a number of different proteins embedded in it. In general however, relatively little is known about how viral proteins are packaged together inside human cells to assemble coronavirus particles. This lack of knowledge impedes the development of strategies that could target the assembly of virus particles inside human cells. However, one membrane protein encoded by the virus, designated the M protein because of its membrane association, serves as the starting seed for the assembly of all the other coronavirus proteins. It has recently been discovered that the tail of the MHV version of the M protein can interact with a motor protein used for trafficking inside human cells. Because the sequence of the M-protein tails in this class of coronaviruses is very conserved, this project seeks to determine if this interaction is generalizable among SARS-CoV-2, and other related coronaviruses. If this interaction is present in SARS-CoV-2, then this project will identify the parts of the M protein that interact with the myosin motor. This would allow future research to evaluate drugs that can interrupt this interaction, which may alter the ability of the SARS-CoV-2 and other coronaviruses to assemble and spread. As a Broader Impact, the Project will provide critically needed information for fight the Covid-19 pandemic. Relatively little is known about the processes required for the assembly of coronavirus virions in human cells. The family of beta-coronaviruses, including SARS-CoV-2, SARS, MERS and MHV all utilize a similar compendium of proteins, including a membrane glycoprotein protein, termed the M protein. M protein serves as the nidus for assembly of other coronavirus proteins into the mature virus particle. This project seeks to expand on the recent finding that the M-protein of MHV interacts with a specific 26 amino acid alternatively spliced exon of Myosin Vb (MYO5B), termed exon D. This observation is the first to identify a key intracellular trafficking protein that interacts with the M protein. This project seeks to identify whether this interaction is preserved in the M proteins of other beta-coronavirus family members, including the SARS-CoV-2 virus. The Project will also determine the molecular basis of the interaction between M protein and Exon D of MYO5B. The Project will first utilize yeast 2-hybrid assays to evaluate the binding of the internal tails of SARS-CoV-2 (COVID-19), SARS and MERS and other related coronaviruses. Second, random mutagenesis of the coronavirus tails will be utilized to determine the common amino acid motifs used for interaction with MYO5B Exon D. Third, wild type and mutant coronavirus proteins will be expressed in HeLa cells, and mutations that block interactions with MYO5B on M protein will be evaluated for their effects on trafficking through the endoplasmic reticulum, the Golgi apparatus and to the plasma membrane. If verified with the SARS-CoV-2 M protein, the site of interaction with MYO5b Exon D could be utilized as a target for disruption of the assembly of coronavirus virions. This RAPID Project is expected to provide insights for the future development of molecular strategies to disrupt virion assembly in host cells, which could lead to new therapeutics for the treatment of Covid-19.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.