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

基本信息

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
项目类别：
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

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

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项目摘要

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年确定的一种新型B-核纳病毒，导致疾病COVID-19，该病毒自2019年底以来造成超过600万人死亡。尽管最近的病毒学研究已经阐明了SARS-COV-2感染细胞并导致疾病的许多方面，但问题仍然存在于续期复制的时期过程中，该步骤可能是针对新的，该步骤的定位，该步骤有用，该步骤有用。使用反向遗传学以及在细胞中或SARS-COV-2感染期间表达的标记的SARS-COV-2蛋白的活细胞和超分辨率显微镜，我们提出了两个旨在更好地了解人类气道细胞期间病毒宿主相互作用的目标。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SARS-COV-2 RNA合成的起源和运输（AIM 2）（AIM 2），用于WT病毒和关注的变体（例如Alpha，Delta和Omicron）。这些研究将在人类气道上皮细胞系和去识别的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执行。小分子，抑制宿主因子以及病毒基因组中的突变（包括在高循环变体中发现的）将用于改变这些过程，从而改变研究复制机制的感染性。另外，相关光学显微镜（CLEM）将提供有关这些复制过程的结构信息。对SARS-COV-2感染的了解得以提高，可能会导致对感染者的更有效的COVID-19 COVID疗法，并可以为我们预防或治疗未来出现的新冠状病毒做好准备。