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

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

M Jane Morwitzer  1 , Ying Yi Zheng  1 , Heather Friberg  1 , Jeffrey R Currier  2
Affiliations

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

M Jane Morwitzer et al. J Virol Methods. .

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.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known financial conflicts of interest or personal relationships that may have influenced the work presented in this paper. Disclaimer: This manuscript has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. The investigators have adhered to the policies for protection of human subjects as prescribed in AR 70–25.

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