Exploring the dynamics of nsp1 and RNA interaction in SARS-CoV with undergraduate researchers

项目摘要 病毒宿主关闭蛋白选择性地劫持细胞机器以帮助病毒繁殖。在SARS冠状病毒 在SARS-CoV-1和SARS-CoV-2中，非结构蛋白1（nsp 1）通过以下方式作为宿主关闭因子： 通过核糖体在宿主mRNA上停滞，然后切割，抑制宿主基因表达， 腐烂然而，nsp 1如何选择性地靶向宿主mRNA而不是病毒RNA仍然是一个未解决的难题。 本研究的长期目标是研究nsp 1介导的宿主关闭的步骤，以清楚地了解 它如何特异性地选择宿主mRNA，而不是类似于宿主的加帽和多聚腺苷酸化的病毒RNA mRNA。该项目的基本原理是基于观察到，从 病毒RNA前导序列消除了它逃避宿主关闭的能力。区分病毒的一个关键因素 从宿主mRNA中提取的RNA主要是通过nsp 1和SL 1之间的相互作用实现的。根据我们之前公布的结果 nsp 1与应激颗粒蛋白相关，并将G3 BP 1蛋白从应激颗粒中分离， 建议检查应激颗粒的蛋白质和RNA组成，以确定病毒RNA是否受到保护 由于其特异性定位与nsp 1和病毒RNA之间的相互作用有关，因此不会腐烂。在 目的1，我们建议进行深入的研究nsp 1和SL 1之间的相互作用，将映射 负责结合的序列，表征与SL 1结合的宿主蛋白的蛋白质组学谱，和 分析nsp 1对宿主mRNA翻译和稳定性的影响。在目标2中，我们建议审查 在nsp 1存在下分解应力颗粒，并研究其对选择性裂解和衰变的影响。 宿主mRNA。这项多学科的合作研究将使本科生在一个 与生物化学家，计算机科学家和免疫学家团队的高度变革经验， 探索生物医学科学中的当前和相关主题。总的来说，我们的研究将把nsp 1的能力与 将病毒RNA序列与RNA稳定性和定位结合起来，并使我们能够研究 导致宿主mRNA裂解和衰变。最后，本项目将确定nsp 1的相互作用者，并为 用于设计抗病毒疗法。

Project Summary Viral host shutoff proteins selectively hijack cellular machinery to aid viral propagation. In SARS coronaviruses (SARS-CoV-1 and SARS-CoV-2), nonstructural protein 1 (nsp1) serves as the host shutoff factor by dampening host gene expression through ribosomal stalling on host mRNAs followed by their cleavage and decay. However, how nsp1 selectively targets host mRNAs over the viral RNA remains an unsolved puzzle. The long-term goal of this research is to study the steps of nsp1-mediated host shutoff to clearly understand how it specifically selects host mRNAs but not capped and polyadenylated viral RNA that resembles host mRNAs. The rationale for this project is based on the observation that the deletion of stem-loop 1 (SL1) from the viral RNA leader sequence eliminates its ability to escape host shutoff. A critical factor in differentiating viral RNA from host mRNA lies in the interaction between nsp1 and SL1. Based on our previously published results that nsp1 associates with stress granule proteins and disengages G3BP1 protein from the stress granule, we propose to examine the protein and RNA composition of stress granules to determine if viral RNA is protected from decay because of its specific localization connected to the interaction between nsp1 and viral RNA. In Aim 1, we propose to conduct an in-depth study of the interaction between nsp1 and SL1 that will map the sequence responsible for binding, characterize the proteomic profile of the host proteins bound to SL1, and analyze the effect of nsp1 on host mRNA translation and stability. In Aim 2, we propose to examine the disassembly of stress granules in the presence of nsp1 and study its effect on selective cleavage and decay of host mRNA. This multidisciplinary collaborative research will engage undergraduate students to be trained in a highly transformative experience with a team of biochemists, computer scientists, and immunologists to explore a current and relevant topic in biomedical sciences. Overall, our research will connect nsp1’s ability to bind viral RNA sequence to RNA stability and localization and will allow us to examine the mechanism that leads to host mRNA cleavage and decay. Finally, this project will identify interactors of nsp1 and pave the way for designing anti-viral therapeutics.