Multiplexed imaging of viral protein processing and assembly in live cells

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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.