# Multiplexed imaging of viral protein processing and assembly in live cells

> **NIH NIH R01** · COLORADO STATE UNIVERSITY · 2023 · $479,592

## Abstract

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.

## Key facts

- **NIH application ID:** 10708987
- **Project number:** 5R01AI168459-02
- **Recipient organization:** COLORADO STATE UNIVERSITY
- **Principal Investigator:** Christopher Davis Snow
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $479,592
- **Award type:** 5
- **Project period:** 2022-09-21 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10708987

## Citation

> US National Institutes of Health, RePORTER application 10708987, Multiplexed imaging of viral protein processing and assembly in live cells (5R01AI168459-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10708987. Licensed CC0.

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