Host Substrate Profiling of SARS-CoV-2 Virus Protease

项目总结 严重急性呼吸系统综合症冠状病毒2(SARS-CoV-2)是病毒性肺炎爆发的原因。 2019年冠状病毒病(新冠肺炎)造成广泛的发病率和死亡率。主要蛋白酶(MPRO) 由SARS-CoV-2编码的病毒处理病毒多蛋白并促进病毒复制。一旦激活，这一点 酶可以通过裂解宿主蛋白来逃避宿主的先天免疫反应。沙门氏菌的宿主蛋白底物 SARS-CoV-2 MPRO的特征还不是很好。直接检测宿主蛋白的主要挑战 靶标的低丰度和技术局限性与目前方法的特异性和敏感性有关。 因此，这项提议旨在开发一种更具体和更敏感的方法，称为N末端组学，以 发现由MPRO在SARS CoV-2感染期间产生的独特蛋白质片段。具体目标 这一建议的主要内容是：1)开发和验证新的探针，以鉴定由 细胞裂解物中SARS-CoV-2 MPRO的研究和2)SARS-CoV-2 MPRO内源底物的鉴定和定量 在受感染的哺乳动物细胞中。这项拟议研究的理由是，它的成功将促进更大的 了解这种SARS-CoV-2改变哪些宿主途径会导致严重的疾病状况。 此外，如果新冠肺炎的话，Mpro的宿主靶点可以用于开发治疗方案来治疗重症病例。 这项研究的预期结果是，我们的方法将更敏感，更容易适应 宿主底物分析任何致病的蛋白水解酶，它将有助于绘制细胞路径图 被病原体劫持以入侵宿主系统。

PROJECT SUMMARY Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the viral pneumonia outbreak of coronavirus disease 2019 (COVID-19) causing widespread morbidity and mortality. Main protease (Mpro) encoded by SARS-CoV-2 processes the viral polyproteins and facilitates viral replication. Once activated, this enzyme can evade the host innate immune responses by cleaving host proteins. The host protein substrates of SARS-CoV-2 Mpro are not well characterized. The major challenges with the direct detection of host protein targets is their low abundance and technical limitations with the specificity and sensitivity of the current methods. Thus, this proposal is designed to develop a more specific and sensitive approach termed N-terminomics to discover unique protein fragments that are generated by Mpro during SARS CoV-2 infection. The Specific Aims of this proposal are: 1) Development and validation of novel probes to identify protease fragments generated by SARS-CoV-2 Mpro in cell lysate and 2) Identify and quantify the endogenous substrates of SARS-CoV-2 Mpro in infected mammalian cells. The rationale for the proposed research is that its success would facilitate a greater understanding of which host pathways are altered by this SARS-CoV-2 to cause a severe disease condition. Moreover, host targets of MPro can be used to develop treatment regimens to treat severe cases if COVID-19. The expected outcome of this research is that our approach will be more sensitive and readily adaptable for the host substrate profiling of any pathogenic proteases and it will aid in mapping cellular pathways which are hijacked by pathogens to invade the host system.