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Multiplexed imaging of viral protein processing and assembly in live cells

活细胞中病毒蛋白加工和组装的多重成像

基本信息

批准号：
10708987
负责人：
Christopher Davis Snow
金额：
$ 47.96万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-21 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10708987
关键词：
Acceleration Address Affinity Alphavirus Antibodies Binding Biochemistry Biogenesis Biological Process Biology Capsid Capsid Proteins Cells Chimera organism Chimeric Proteins Color Communities Complex Computers Consumption Darkness Data Development Directed Molecular Evolution Engineering Ensure Epitopes Eukaryotic Cell Flavivirus Genetic Materials Genetic Recombination Genome HIV-1 Hand Homo Hypersensitivity Image Imaging Device Imaging technology Immunoglobulin Fragments Infection Investigation Kinetics Label Lead Libraries Life Life Cycle Stages Light Machine Learning Mechanics Membrane Methods Microscopy Modeling Molecular Molecular Biology Molecular Virology Monitor Peptides Photobleaching Polyproteins Positioning Attribute Predisposition Process Protein Dynamics Protein Engineering Protein Fragment Proteins Reagent Recombinant Proteins Refractory Replication-Associated Process Research Research Personnel Ribosomes Scientist Signal Transduction Specificity Technology Testing Time Translations Validation Variant Viral Viral Proteins Virus Virus Diseases Virus Replication Visualization antibody mimetics cost design experimental study in vivo in vivo imaging innovation live cell imaging live cell microscopy molecular dynamics molecular imaging multiplexed imaging next generation non-invasive imaging novel particle prediction algorithm protein structure protein structure prediction rational design real-time images scaffold single molecule spatiotemporal success temporal measurement virology

项目摘要

Imaging the full lifecycle of viral proteins in vivo is essential for understanding the molecular processes underlying viral infection. Live-cell imaging has long been performed using fluorescent protein fusion tags such as GFP. However, these tags can alter the size and function of targeted proteins. Furthermore, slow maturation, degradation, and photobleaching of tags results in the loss of signal, making it difficult to track the early life and ultimate fate of many proteins. Viral polyproteins, in particular, remain refractory to imaging in vivo due to their hypersensitivity to tags and the extensive processing and assembly they undergo during viral biogenesis. The use of linear epitope tags reversibly labeled by genetically encoded live-cell probes can solve many of these issues. Unfortunately, engineering functional probes for live-cell imaging of epitopes has been costly and time-consuming. In the proposed research, we combine expertise in protein engineering, single-molecule microscopy, and biochemistry to refine and accelerate the rational design of orthogonal epitope/probe pairs for highly multiplexed imaging of full viral protein lifecycles in living cells. We demonstrate the power of our strategy in our Preliminary Data by creating novel scFvs that bind the commonly used HA and Flag epitopes with high affinity in a variety of demanding live-cell imaging scenarios. In Aim 1, we will use our tested strategy to develop scFv against additional viral epitope tags and validate their utility in imaging experiments. To identify chimeric scFv that are both soluble and active within the cellular milieu, we will graft known epitope-specific CDR loops onto a unique panel of stable scFv scaffolds. In Aim 2, we will use state-of-the-art machine learning protein modeling and design methods to develop predictive binding models for scFv:viral-epitope complexes, validate a scFv design pipeline, engineer scFv libraries encoding multiple new peptide-binding solutions, and screen using innovative high-throughput, high-content in vivo methods. In Aim 3, we will demonstrate the utility of our newly developed scFv in live-cell imaging experiments by probing several critical aspects of viral biology. Specifically, we will use our engineered scFv to visualize and quantify the translation dynamics of flavivirus transmembrane polyproteins, and to monitor alphavirus particle assembly kinetics. Overall, this project will provide a powerful new pipeline for generating scFv proteins that can track viral proteins in living cells. The reagents we generate will provide the virus molecular biology community with new, versatile imaging tools to better illuminate many important biological processes.

对病毒蛋白在体内的整个生命周期进行成像对于理解其分子机制至关重要