EAGER: Collaborative Research: Design of Inhibitors for ORF7a and ORF7b Oligomerization in COVID-19

有了这个奖项，化学部的生命过程计划化学和化学，生物工程，环境和运输系统司正在资助布赖恩伯杰博士（弗吉尼亚大学）和Jeffery Klauda博士（马里兰州大学）研究来自COVID 19病毒的两种名为ORF7a和OR7b的蛋白质，这两种蛋白质与病毒对其宿主（例如人类细胞）的危害程度有关。 该研究将重点关注这两种蛋白质如何形成更大的蛋白质复合物，进而影响病毒与感染细胞之间的相互作用，并影响宿主的免疫反应。 该研究为可用于探测病毒繁殖的肽的开发提供了信息。 该研究是基于计算和实验方法相结合的使用。 Berger博士和Klauda博士通过Addgene向科学界免费分发为该项目开发的质粒和相关方案，从而使致力于寻找当前流行病解决方案并将未来爆发的可能性降至最低的全球科学界能够快速使用他们的研究成果。 这项工作将为使用最先进的实验和计算方法研究关键挑战的博士后研究员提供培训。该小组将通过在弗吉尼亚大学和马里兰州大学举行的会议和讲习班以及通过出版物向更广泛的社区传播研究结果。 研究人员还计划通过参与其机构现有的外展计划，告知和教育学生有关病毒传播和预防的可能机制。本研究项目旨在了解ORF7a与BST-2的跨膜和跨膜寡聚化以及ORF7b的同源寡聚化的特异性基础。利用基于E.大肠杆菌AraC蛋白（AraTM和DN-AraTM测定法），研究确定了负责细菌膜中蛋白寡聚化的特定氨基酸残基和结构基序。这些知识为BST-2/ORF7a杂寡聚体和ORF7b同源寡聚体的形成提供了计算模型。 反过来，计算模型用于对跨膜肽序列进行关键的新预测，这些跨膜肽可能影响涉及ORF7a和ORF7b的蛋白质-蛋白质相互作用。这些预测和肽的性质通过合成肽库和使用AraTM、DN-AraTM和哺乳动物的基于细胞的荧光共振能量转移测定来测试。使用基于哺乳动物细胞的BST-2功能和细胞凋亡测定来实现候选序列的验证。这些研究的结果可以为OR7a和ORF7b同源和异源寡聚化提供高分辨率、实验验证的模型，以及可用于探测ORF7a和ORF7b在体内病毒繁殖中的作用的肽序列。该奖项反映了NSF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。

With this award, the Chemistry of Life Processes Program in the Chemistry Division and the Chemical and Biochemical Engineering Program in the Chemical, Bioengineering, Environmental and Transport Systems Division are funding Dr. Bryan Berger (University of Virginia) and Dr. Jeffery Klauda (University of Maryland) to investigate two proteins named ORF7a and OR7b from the COVID19 virus that have been implicated in how harmful the virus is to its host, e.g. the human cells. The research will focus on how these two proteins form larger protein complexes that in turn affect the interactions between the virus and the infected cells and influence the immune response of the host. The research informs the development of peptides that could be used to probe the viral propagation. The research is based on the use of a combination of computational and experimental methods. Dr. Berger and Dr. Klauda distribute to the scientific community free of charge through Addgene the plasmids and associated protocols developed for this project, thus enabling the global scientific community that works on finding a solution to the current pandemic and to minimizing the possibility of future outbreaks to quickly use the outcomes of their research. This work will provide training for post-doctoral fellows working on critical challenges using state-of-the-art experimental and computational methods. The results of the research will be disseminated by the team to the greater community through conferences and workshops at University of Virginia and University of Maryland and through publications. The researchers also plan to inform and educate students on possible mechanisms of virus transmission and prevention by participation in existing outreach programs at their Institutions.This research project seeks to understand the basis of specificity for transmembrane and juxtamembrane oligomerization of ORF7a with BST-2 and for homooligomerization of ORF7b. Using bacterial transcriptional assays for membrane protein dimerization based on the E. coli AraC protein (AraTM and DN-AraTM assays), the researches determine specific amino acid residues and structural motifs responsible for the protein oligomerization in bacterial membranes. This knowledge informs computational models for formation of BST-2/ORF7a heterooligomers and ORF7b homooligomers. In turn, the computational models are used to make critical new predictions of sequences for transmembrane peptides that could influence protein-protein interactions involving ORF7a and ORF7b. These predictions and the properties of the peptides are tested by synthesizing peptide libraries and using AraTM, DN-AraTM, and mammalian, cell-based fluorescence resonance energy transfer assays. Validation of candidate sequences are achieved using mammalian cell-based assays for BST-2 function and apoptosis. The results of these studies could provide high-resolution, experimentally validated models for OR7a and ORF7b homo and heterooligomerization, as well as peptide sequences that can be used to probe the roles of ORF7a and ORF7b in viral propagation in vivo.This grant is being awarded using funds made available by the Coronavirus Aid, Relief, and Economic Security (CARES) Act supplement allocated to MPS and ENG.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.