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

• 批准号: 10587280
• 负责人: Christopher Davis Snow
• 金额: $ 53.24万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 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/10587280
• 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; Methods; Microscopy; Modeling; Molecular; Molecular Biology; Molecular Virology; Monitor; Peptides; Photobleaching; Polyproteins; Positioning Attribute; Process; Protein Dynamics; Protein Engineering; 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; antibody mimetics; cost; design; design and construction; experimental study; in vivo; in vivo imaging; innovation; 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; protein structure prediction; rational design; real-time images; scaffold; single molecule; spatiotemporal; success; temporal measurement; virology

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.

在体内对病毒蛋白的全生命周期进行成像对于理解分子是必不可少的。 处理潜在的病毒感染。长期以来，活细胞成像一直使用 荧光蛋白融合标签，如GFP。但是，这些标记可以更改大小和 靶蛋白的功能。此外，缓慢的成熟、降解和光漂白 标签导致信号丢失，使人难以追踪早期生活和最终命运 很多蛋白质。尤其是病毒多蛋白，由于它们的特性，在体内仍然难以进行成像。 对标签及其在病毒期间经历的广泛加工和组装的超敏反应 生物发生学。利用遗传编码的活细胞可逆标记的线性表位标记 探测器可以解决其中的许多问题。不幸的是，活细胞的工程功能探针 对表位进行成像既昂贵又耗时。在拟议的研究中，我们 结合蛋白质工程、单分子显微镜和生物化学的专业知识 完善和加快高复用正交表位/探针对的合理设计 活细胞中完整病毒蛋白生命周期的成像。我们在以下方面展示了我们的战略的力量 我们通过创建绑定常用HA和Flag的新型scFv来获得初步数据 在各种要求严格的活细胞成像场景中具有高亲和力的表位。在目标1中，我们将 使用我们测试的策略针对其他病毒表位标签开发单链抗体，并验证其 在成像实验中的实用性。鉴定既可溶又有活性的嵌合单链抗体 在细胞环境中，我们将把已知的表位特异性CDR环嫁接到一个独特的稳定的 ScFv支架。在目标2中，我们将使用最先进的机器学习蛋白质建模和 设计方法开发单链抗体：病毒-表位复合体的预测结合模型，验证 ScFv设计流水线，设计编码多个新的肽结合解决方案的scFv文库， 并采用创新的高通量、高含量体内筛选方法。在《目标3》中，我们将 通过探测来展示我们新开发的单链抗体在活细胞成像实验中的应用 病毒生物学的几个关键方面。具体地说，我们将使用我们设计的scFv可视化 并量化黄病毒跨膜多蛋白的翻译动力学，并监测 甲型病毒颗粒组装动力学。总体而言，该项目将提供一条强大的新管道 用于产生单链抗体蛋白，可以在活细胞中跟踪病毒蛋白。我们的试剂 GENERATE将为病毒分子生物学社区提供新的、通用的成像工具 更好地阐明了许多重要的生物过程。