Live-cell imaging of SARS-CoV-2 replication organelle formation and RNA synthesis

SARS-CoV-2 复制细胞器形成和 RNA 合成的活细胞成像

• 批准号: 10644286
• 负责人: Zandrea Ambrose
• 金额: $ 19.58万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10644286
• 关键词: 2019-nCoV; Acute respiratory infection; Biogenesis; Biological; Biological Assay; COVID-19; COVID-19 mortality; COVID-19 prevention; COVID-19 treatment; Cell Line; Cell membrane; Cells; Cellular biology; Cessation of life; Chiroptera; Complex; Confocal Microscopy; Coronavirus; Cytoplasm; Disease; Electron Microscopy; Endoplasmic Reticulum; Evaluation; Future; Genes; Genetic Transcription; Genome; HIV-1; Human; Image; Immune response; Impaired cognition; Individual; Infection; Integration Host Factors; Investigation; Label; Lead; Length; Light; Membrane; Messenger RNA; Methods; Middle East Respiratory Syndrome Coronavirus; Molecular; Molecular Virology; Mutation; Nonstructural Protein; Organelles; Pathway interactions; Peer Review; Peptide Hydrolases; Pneumonia; Polyproteins; Prevention; Process; Proteins; Publications; RNA; RNA chemical synthesis; Replication-Associated Process; Research; Role; SARS coronavirus; SARS-CoV-2 infection; SARS-CoV-2 variant; Symptoms; Time; Translating; Translations; Universities; Vaccines; Variant; Vesicle; Viral; Viral Genome; Virion; Virulent; Virus; Virus Replication; Visualization; airway epithelium; betacoronavirus; cell fixing; genomic RNA; human coronavirus; improved; insight; knock-down; live cell imaging; new therapeutic target; nonsynonymous mutation; novel; novel coronavirus; post SARS-CoV-2 infection; prevent; protein expression; receptor binding; reverse genetics; small molecule; spatiotemporal; superresolution imaging; superresolution microscopy; trafficking; transmission process; ultra high resolution; variants of concern; viral RNA; virology; virus host interaction

SARS-CoV-2 is a novel b-coronavirus identified in 2019 that causes the disease COVID-19, which is responsible for over 6 million deaths since late 2019. While recent virology studies have clarified many aspects of how SARS- CoV-2 infects cells and causes disease, questions remain on the spatio-temporal processes of post-entry replication steps, which may be useful for targeting novel therapies. Using reverse genetics and live cell and super-resolution microscopy of labeled SARS-CoV-2 proteins expressed in cells or during SARS-CoV-2 infection, we propose two aims to gain better understanding of virus-host interactions during infection of human airway cells. We will understand the role of host proteins in the biogenesis of SARS-CoV-2-induced double- membraned vesicles (Aim 1) and visualize the origin and trafficking of SARS-CoV-2 RNA synthesis (Aim 2) for WT virus and variants of concern (e.g., Alpha, Delta, and Omicron). The studies will be performed in real-time in human airway epithelial cell lines and deidentified primary cells. These aims will be performed by fluorescently labeling SARS-CoV-2 proteins and the viral RNA as well as host cell proteins. Small molecules, knock down of host factors, and mutations in the viral genome (including those found in highly circulating variants) will be used to alter these processes and, thus, infectivity to study replication mechanisms. In addition, correlative light- electron microscopy (CLEM) will provide structural information on these replication processes. Improved understanding of SARS-CoV-2 infection may lead to more effective COVID-19 therapies for infected individuals and could prepare us for preventing or treating new coronaviruses that arise in the future.

SARS-CoV-2是2019年发现的一种新型b型冠状病毒，导致COVID-19疾病，自2019年底以来，该疾病已造成600多万人死亡。虽然最近的病毒学研究已经阐明了SARS-CoV-2如何感染细胞并导致疾病的许多方面，但关于进入后复制步骤的时空过程仍然存在问题，这可能对靶向新疗法有用。使用反向遗传学和活细胞和超分辨率显微镜标记的SARS-CoV-2蛋白在细胞中表达或在SARS-CoV-2感染，我们提出了两个目的，以获得更好地了解病毒感染过程中的人体气道细胞的宿主相互作用。我们将了解宿主蛋白在SARS-CoV-2诱导的双膜囊泡的生物发生中的作用（Aim 1），并可视化WT病毒和相关变体（例如，Alpha、Delta和Omicron）。这些研究将在人气道上皮细胞系和去识别原代细胞中实时进行。这些目标将通过荧光标记SARS-CoV-2蛋白和病毒RNA以及宿主细胞蛋白来实现。小分子、宿主因子的敲除和病毒基因组中的突变（包括在高循环变体中发现的那些）将被用来改变这些过程，从而改变感染性以研究复制机制。此外，相关光电子显微镜（CLEM）将提供这些复制过程的结构信息.对SARS-CoV-2感染的进一步了解可能会为感染者带来更有效的COVID-19治疗方法，并为我们预防或治疗未来出现的新冠状病毒做好准备。