Multiplexed imaging of viral protein processing and assembly in live cells

项目摘要 对体内病毒蛋白质的整个生命周期进行成像对于理解分子过程至关重要 潜在的病毒感染活细胞成像长期以来一直使用荧光蛋白融合标签进行， 作为GFP。然而，这些标签可以改变靶蛋白的大小和功能。此外，慢 标签的成熟、降解和光漂白导致信号的丢失，使得难以跟踪标记物。 许多蛋白质的早期生命和最终命运。特别是病毒多聚蛋白， 由于它们对标签的超敏性以及它们在病毒感染期间经历的广泛加工和组装， 生物起源使用由遗传编码的活细胞探针可逆标记的线性表位标签可以解决 许多这些问题。不幸的是，用于表位活细胞成像的工程功能探针已经被 昂贵且耗时。在拟议的研究中，我们联合收割机结合蛋白质工程，单一， 分子显微镜和生物化学，以完善和加速正交合理设计 表位/探针对用于活细胞中完整病毒蛋白生命周期的高度多重成像。我们证明 在我们的初步数据中，我们的策略的力量是通过创建结合常用HA的新型scFv， 在各种苛刻的活细胞成像场景中标记具有高亲和力的表位。在目标1中，我们将使用 开发针对额外病毒表位标签的scFv并验证其在成像中的效用的测试策略 实验为了鉴定在细胞环境中既可溶又有活性的嵌合scFv，我们将嵌合scFv移植到 将已知的表位特异性CDR环连接到一组独特的稳定scFv支架上。在目标2中，我们将使用 最先进的计算蛋白质建模和设计，以开发新的预测结合模型的scFv：病毒- 表位复合物，建立和测试从头scFv设计的方案，工程化大型scFv文库 编码多种新的肽结合溶液，并使用噬菌体展示进行筛选。在目标3中，我们 通过探测几个关键的细胞成像实验，证明了我们新开发的scFv在活细胞成像实验中的效用。 病毒生物学的各个方面。具体地说，我们将使用我们的工程化scFv来可视化和量化翻译。 黄病毒跨膜多蛋白的动力学，并监测甲病毒颗粒组装动力学。 总的来说，这个项目将提供一个强大的新的管道，产生单链抗体蛋白，可以跟踪病毒 活细胞中的蛋白质我们生产的试剂将为病毒分子生物学界提供新的， 多功能成像工具，以更好地阐明许多重要的生物过程。

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.