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

与本科生研究人员一起探索 SARS-CoV 中 nsp1 和 RNA 相互作用的动态

• 批准号: 10730676
• 负责人: Anita Nag
• 金额: $ 40.65万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-03 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10730676
• 关键词: 2019-nCoV; Amino Acids; Antiviral Agents; Antiviral Therapy; Binding; Binding Proteins; Binding Sites; Biochemical; C-terminal; Career Choice; Cells; Charge; Complex; Computer Analysis; Computer Models; Computers; Coronavirus; Ensure; Future; G3BP1 gene; Gene Expression; Goals; Heat shock proteins; Hepatitis C virus; Immunologist; Knowledge; Life; Maps; Mediating; Mentors; Messenger RNA; Middle East Respiratory Syndrome Coronavirus; Modeling; Mutation; Nonstructural Protein; Pathway interactions; Poly A; Polyadenylation; Process; Protein Biosynthesis; Proteins; Proteomics; Publishing; RNA; RNA Binding; RNA Sequences; RNA Stability; RNA analysis; Research; Research Personnel; Ribosomes; Role; SARS coronavirus; Science; Scientist; Specificity; Structural Models; Structure; Training; Translating; Translational Repression; Translations; Viral; Viral Proteins; cricket paralysis virus; design; experience; flexibility; inhibitor; innovation; mRNA Stability; mRNA Transcript Degradation; mRNA Translation; multidisciplinary; stem; stress granule; undergraduate student; viral RNA; zoonotic coronavirus

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.

项目概要 病毒宿主关闭蛋白选择性地劫持细胞机器以帮助病毒传播。在SARS冠状病毒中 （SARS-CoV-1 和 SARS-CoV-2），非结构蛋白 1 (nsp1) 通过以下方式充当宿主关闭因子 通过宿主 mRNA 上的核糖体停滞，然后裂解和抑制宿主基因表达 衰变。然而，nsp1 如何选择性地靶向宿主 mRNA 而不是病毒 RNA 仍然是一个未解之谜。 本研究的长期目标是研究nsp1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，但不选择类似于宿主的加帽和聚腺苷酸化病毒 RNA mRNA。该项目的基本原理是基于以下观察：茎环 1 (SL1) 的删除 病毒 RNA 前导序列消除了其逃避宿主关闭的能力。区分病毒的关键因素 来自宿主 mRNA 的 RNA 存在于 nsp1 和 SL1 之间的相互作用中。根据我们之前发布的结果 nsp1 与应激颗粒蛋白结合并使 G3BP1 蛋白与应激颗粒分离，我们 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 由于其特定的定位与 nsp1 和病毒 RNA 之间的相互作用有关，因此可以避免腐烂。在 目标 1，我们建议对 nsp1 和 SL1 之间的相互作用进行深入研究，以绘制 负责结合的序列，表征与 SL1 结合的宿主蛋白的蛋白质组谱，以及 分析nsp1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 nsp1 存在下应力颗粒的分解并研究其对选择性裂解和衰变的影响 宿主 mRNA。这项多学科合作研究将让本科生接受培训 生物化学家、计算机科学家和免疫学家团队的高度变革性经验 探索生物医学科学中当前的相关主题。总的来说，我们的研究将把 nsp1 的能力联系起来 将病毒 RNA 序列与 RNA 稳定性和定位结合起来，使我们能够检查病毒 RNA 序列的机制 导致宿主 mRNA 裂解和衰变。最后，该项目将识别 nsp1 的交互者并铺平道路 用于设计抗病毒疗法。