Multiplexed imaging of viral protein processing and assembly in live cells

项目摘要 在体内对病毒蛋白的全生命周期进行成像对于理解分子过程是必不可少的 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进行，如 作为GFP。然而，这些标签可以改变目标蛋白质的大小和功能。此外，速度慢 标签的成熟、降解和光漂白会导致信号的丢失，使得跟踪标签变得困难 许多蛋白质的早期生命和最终命运。尤其是病毒多蛋白，仍然对成像不敏感。 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 生物发生学。使用由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 许多这样的问题。不幸的是，用于抗原表位活细胞成像的工程功能探针已经被 既昂贵又耗时。在拟议的研究中，我们结合了蛋白质工程方面的专业知识，单一- 分子显微镜，生物化学，提炼和加速合理的正交设计 表位/探针对，用于在活细胞中对完整的病毒蛋白生命周期进行高度多元化的成像。我们展示了 我们的策略在初步数据中的力量是通过创建新的scFv来绑定常用的HA和 标记表位在各种要求苛刻的活细胞成像场景中具有高亲和力。在目标1中，我们将使用我们的 针对额外的病毒表位标签开发单链抗体的测试策略，并验证其在成像中的有效性 实验。为了鉴定在细胞环境中既可溶又有活性的嵌合单链抗体，我们将 已知的表位特异性CDR循环到一组独特的稳定的ScFv支架上。在目标2中，我们将使用状态- 最先进的计算蛋白质建模和设计，以开发新型单链抗体预测结合模型：病毒- 表位复合体，建立和测试从头设计单链抗体的方案，设计大型单链抗体文库 编码多种新的多肽结合溶液，以及使用噬菌体展示的屏幕。在《目标3》中，我们将 通过探索几个关键的关键技术，展示我们新开发的单链抗体在活细胞成像实验中的有效性 病毒生物学的各个方面。具体地说，我们将使用我们设计的scFv可视化和量化翻译 黄病毒跨膜多蛋白的动力学，并监测甲型病毒颗粒组装动力学。 总体而言，该项目将为产生能够追踪病毒的单链抗体蛋白提供一条强大的新管道。 活细胞中的蛋白质。我们生产的试剂将为病毒分子生物界提供新的、 多功能成像工具，可更好地照亮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.