A split GFP approach to assay SARS-CoV-2 spike-dependent cell fusion

M Jane Morwitzer; Ying Yi Zheng; Heather Friberg; Jeffrey R Currier

doi:10.1016/j.jviromet.2025.115296

A split GFP approach to assay SARS-CoV-2 spike-dependent cell fusion

J Virol Methods. 2025 Nov 1:340:115296. doi: 10.1016/j.jviromet.2025.115296. Online ahead of print.

Authors

M Jane Morwitzer¹, Ying Yi Zheng¹, Heather Friberg¹, Jeffrey R Currier²

Affiliations

¹ Department of Virus-Host Interactions, Viral Diseases Program, Walter Reed Army Institute of Research, Silver Spring, MD, United States.
² Department of Virus-Host Interactions, Viral Diseases Program, Walter Reed Army Institute of Research, Silver Spring, MD, United States. Electronic address: jeffrey.r.currier.civ@health.mil.

PMID: 41177465
DOI: 10.1016/j.jviromet.2025.115296

Abstract

The SARS-CoV-2 spike (S) protein plays a central role in viral entry through receptor binding and membrane fusion, making it a key target for therapeutic interventions. While existing assays for studying spike-mediated fusion can be complex and lack real-time monitoring, we present a split GFP-based cell fusion assay that provides a straightforward and adaptable platform for evaluating fusion dynamics. This assay utilizes a split GFP system in non-adherent 293-F cells to detect fusion events, allowing for reliable assessment of spike-targeting monoclonal antibodies and fusion inhibitors. Our study demonstrates the effectiveness of this approach in evaluating the inhibitory potential of therapeutics against multiple SARS-CoV-2 variants. The results highlight the assay's ability to distinguish variant-specific fusion characteristics and inhibitor responses, particularly in the context of Omicron's altered entry pathways. By enabling the quantitative, real-time assessment of spike-mediated fusion, this platform provides a valuable tool for therapeutic screening, supporting future efforts to develop antiviral strategies against SARS-CoV-2 and other emerging coronaviruses.

Keywords: ACE2; Cell fusion; Fusion inhibitors; SARS-CoV-2; Spike protein; Split GFP reporter assay; TMPRSS2.