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：
10455219
负责人：
Christopher Davis Snow
金额：
$ 51.77万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-06 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10455219
关键词：
Address Affinity Alphavirus Antibodies Binding Biochemistry Biogenesis Biological Process Biology Capsid Capsid Proteins Cells Chimera organism Chimeric Proteins Color Communities Complex Computers Consumption Crystallization Data Development Directed Molecular Evolution Engineering Ensure Epitopes Eukaryotic Cell Flavivirus Genetic Materials Genetic Recombination Genome HIV-1 Hand Homo Hypersensitivity Image Imaging Device Immunoglobulin Fragments Infection Investigation Kinetics Label Lead Libraries Life Light Machine Learning Mechanics Microscopy Modeling Molecular Molecular Biology Molecular Virology Monitor Peptides Phage Display Photobleaching Polyproteins Positioning Attribute Process Protein Dynamics Protein Engineering Proteins Protocols documentation Reagent Recombinant Proteins Refractory Replication-Associated Process Research Research Personnel Ribosomes Scientist Signal Transduction Specificity Technology Testing Time Translations Validation Variant Viral Viral Proteins Viral Structural Proteins Virus Virus Diseases Virus Replication cost design design and construction experimental study in vivo in vivo imaging live cell imaging live cell microscopy model design molecular dynamics molecular imaging multiplexed imaging next generation non-invasive imaging novel particle prediction algorithm protein structure prediction real-time images scaffold single molecule spatiotemporal success temporal measurement virology

项目摘要

Project Summary 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 computational protein modeling and design to develop novel predictive binding models for scFv:viral- epitope complexes, establish and test protocols for de novo scFv design, engineer large scFv libraries encoding multiple new peptide-binding solutions, and screen using phage display. 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.

项目摘要成像体内病毒蛋白的完整生命周期对于理解分子过程至关重要潜在的病毒感染。长期以来，使用荧光蛋白融合标签进行了活细胞成像作为GFP。但是，这些标签可以改变目标蛋白的大小和功能。此外，慢标签的成熟，降解和光漂白导致信号丢失，因此很难跟踪许多蛋白质的早期生命和最终命运。尤其是病毒多蛋白仍然对成像的耐火性体内由于其对标签的高敏性以及病毒期间经历的广泛处理和组装生物发生。使用基因编码的活细胞探针可逆标记的线性表位标签可以解决其中许多问题。不幸的是，用于表位的活细胞成像的工程功能探针已经昂贵且耗时。在拟议的研究中，我们结合了蛋白质工程专业知识，单一的专业知识分子显微镜和生物化学，以完善和加速正交的合理设计活细胞中全病毒蛋白生命周期的高度多重成像的表位/探针对。我们证明通过创建结合常用的HA和在各种苛刻的活电池成像场景中具有高亲和力的旗帜表位。在AIM 1中，我们将使用我们的测试的策略以开发针对其他病毒表位标签的SCFV并验证其成像的效用实验。为了识别在细胞环境中既可溶性又活跃的嵌合SCFV，我们将移植已知的表位特异性CDR循环循环到稳定的SCFV支架的独特面板上。在AIM 2中，我们将使用最新 ART计算蛋白建模和设计，以开发SCFV的新型预测结合模型：病毒 EPITOPE COPPLEES，建立和测试从头SCFV设计的协议，工程师大型SCFV库编码多个新的肽结合解决方案，并使用噬菌体显示器进行屏幕。在AIM 3中，我们将通过探测几个关键病毒生物学方面。具体来说，我们将使用工程的SCFV可视化和量化翻译黄病毒跨膜多蛋白的动力学，并监测α病毒颗粒组装动力学。总体而言，该项目将为生成可以追踪病毒的SCFV蛋白提供强大的新管道活细胞中的蛋白质。我们生成的试剂将为病毒分子生物学界提供新的，多功能成像工具可以更好地阐明许多重要的生物学过程。