Understanding Communications Between Viral RNAs and Human Proteins

了解病毒 RNA 和人类蛋白质之间的通讯

• 批准号: RGPIN-2022-03391
• 负责人: Patel, Trushar
• 金额: $ 2.91万
• 依托单位: University of Lethbridge
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748480
• 关键词: Understanding; Communications; Between; Viral; RNAs

Background: Globally, viral infections are responsible for millions of deaths and impose a severe financial burden on healthcare systems. Viruses containing RNA as their genetic material have emerged as highly pathogenic and infectious. My interdisciplinary, integrative and innovative research program aims to provide critical insight into the human protein-viral RNA interactions that are of fundamental importance for viral replication. Aims and Approaches: The viral non-coding terminal regions are essential for virus replication. Therefore, over the next 5 years, my lab will investigate the structural features of 5' and 3' non-coding RNAs (Aim 1), identify human proteins that are being recruited by viral non-coding RNAs (Aim 2), and establish the structural basis of host protein - viral RNA complexes (Aim 3). Viral RNAs will be prepared using in vitro RNA transcription, and size exclusion chromatography. Using pull-down and mass-spectrometry assays, human proteins that specifically interact with viral RNAs will be identified. Human proteins will be expressed in E. coli, animal, or insect cells, followed by their purification by means of affinity and size-exclusion chromatography. Microscale thermophoresis will aid the determination of affinity between viral RNAs and human proteins. The viral RNA-human protein complexes will be characterized using light scattering and analytical ultracentrifuge techniques, followed by structural studies using solution X-ray scattering, and cryo-electron microscopy. Impact: The proposed research will highlight mechanisms by which viral RNAs exploit human proteins to aid viral replication. The identification and comparison of the structural features of viral non-coding RNAs could provide insights into how viral non-coding RNAs adapt and evolve, which can affect viral pathogenicity, spread, and resistance and influence agriculture, and biotechnology areas. Ultimately, the detailed insights of structural features of a broad range of RNA viruses can promote the urgently required framework to develop strategies to treat viral diseases by targeting host proteins - viral RNA complexes. HQP: My priority is to work on interdisciplinary research programs involving cutting-edge biochemical, biophysical, -omics, and structural biology techniques to create a high-quality, cross-institutional training environment to stimulate and foster the development of highly skilled research personnel. Such skills will help trainees with their future careers in academia, government agencies, and industries related to agriculture, biomedicine, or biotechnology research, and ultimately improve the quality of life of Canadians.

背景：在全球范围内，病毒感染造成数百万人死亡，并给卫生保健系统造成严重的经济负担。以RNA为遗传物质的病毒具有高致病性和传染性。我的跨学科、综合性和创新性的研究项目旨在为人类蛋白质-病毒RNA相互作用提供关键的见解，这对病毒复制至关重要。目的和方法：病毒非编码末端区是病毒复制的关键。因此，在接下来的5年里，我的实验室将研究5‘和3’非编码RNA的结构特征（Aim 1），鉴定被病毒非编码RNA招募的人类蛋白质（Aim 2），建立宿主蛋白-病毒RNA复合物的结构基础（Aim 3）。病毒RNA将使用体外RNA转录和大小排斥层析制备。使用下拉和质谱分析，将鉴定出与病毒rna特异性相互作用的人类蛋白质。人蛋白将在大肠杆菌、动物或昆虫细胞中表达，然后通过亲和层析和大小排斥层析纯化。微尺度热泳将有助于确定病毒rna与人蛋白之间的亲和力。病毒rna -人蛋白复合物将使用光散射和分析超离心技术进行表征，随后使用溶液x射线散射和冷冻电子显微镜进行结构研究。影响：提出的研究将强调病毒rna利用人类蛋白质来帮助病毒复制的机制。通过对病毒非编码rna结构特征的鉴定和比较，可以深入了解病毒非编码rna如何适应和进化，从而影响病毒的致病性、传播和抗性，并影响农业和生物技术领域。最终，对广泛的RNA病毒结构特征的详细了解可以促进迫切需要的框架，以制定通过靶向宿主蛋白-病毒RNA复合物治疗病毒性疾病的策略。HQP：我的首要任务是从事涉及尖端生化、生物物理、组学和结构生物学技术的跨学科研究项目，创造一个高质量的跨机构培训环境，以刺激和培养高技能研究人员的发展。这些技能将有助于受训人员未来在学术界、政府机构以及与农业、生物医学或生物技术研究相关的行业从事职业，并最终提高加拿大人的生活质量。