Official websites use .gov

A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS

A lock ( 🔒 ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Need Help

Multiplexed imaging of viral protein processing and assembly in live cells

Award Number: R01AI168459
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/21/2022
PERIOD OF PERFORMANCE END DATE: 07/31/2027

Group Awards By:

View Award Description

Multiplexed imaging of viral protein processing and assembly in live cells - 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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $421,947 )
2026	2026	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	000	5	7/28/2026	NON-COMPETING CONTINUATION	$421,947
														Subtotal = $421,947

Issue Date FY: 2025 ( Subtotal = $422,409 )
2025	2025	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	000	4	8/4/2025	NON-COMPETING CONTINUATION	$422,409
														Subtotal = $422,409

Issue Date FY: 2024 ( Subtotal = $422,255 )
2024	2024	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	000	3	8/1/2024	NON-COMPETING CONTINUATION	$422,255
														Subtotal = $422,255

Issue Date FY: 2023 ( Subtotal = $479,592 )
2023	2023	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	000	2	7/12/2023	NON-COMPETING CONTINUATION	$479,592
														Subtotal = $479,592

Issue Date FY: 2022 ( Subtotal = $532,386 )
2022	2022	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	000	1	9/21/2022	NEW	$532,386
														Subtotal = $532,386

Grand Total All Awards = $2,278,589

Top

All Categories

About

Search

Reports

Data Submission

Award Information

Multiplexed imaging of viral protein processing and assembly in live cells

Award Number: R01AI168459

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/21/2022

PERIOD OF PERFORMANCE END DATE: 07/31/2027

Federal Websites

Department of Health & Human Services

HHS Operating Divisions

HHS Staff Divisions

Download A Document Viewer