Understanding Communications Between Viral RNAs and Human Proteins

在全球范围内，病毒感染导致数百万人死亡，并给医疗保健系统带来严重的财政负担。含有RNA作为其遗传物质的病毒已成为高致病性和传染性的病毒。我的跨学科，综合和创新的研究计划旨在提供关键的洞察人类蛋白质-病毒RNA的相互作用是病毒复制的根本重要性。 目的和方法：病毒非编码区是病毒复制所必需的。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），并建立宿主蛋白质-病毒RNA复合物的结构基础（Aim 3）。将使用体外RNA转录和分子排阻色谱法制备病毒RNA。使用下拉和质谱分析，将鉴定与病毒RNA特异性相互作用的人蛋白质。人蛋白质将在E.大肠杆菌、动物或昆虫细胞，然后通过亲和和尺寸排阻色谱法对其进行纯化。微尺度热泳将有助于确定病毒RNA和人类蛋白质之间的亲和力。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，然后使用溶液X射线散射和冷冻电子显微镜进行结构研究。 影响：拟议的研究将突出病毒RNA利用人类蛋白质来帮助病毒复制的机制。对病毒非编码RNA结构特征的识别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，对各种RNA病毒结构特征的详细了解可以促进迫切需要的框架，以开发通过靶向宿主蛋白-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物化学，生物物理学，组学和结构生物学技术的跨学科研究项目，以创造一个高质量的，跨机构的培训环境，以刺激和促进高技能研究人员的发展。这些技能将帮助学员在学术界，政府机构和与农业，生物医学或生物技术研究相关的行业的未来职业生涯，并最终提高加拿大人的生活质量。

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.