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J Virol Methods
. 2025 Oct 31:115296.
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
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; SARS-CoV-2; TMPRSS2; cell fusion; fusion inhibitors; spike protein; split GFP reporter assay.
. 2025 Oct 31:115296.
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
- PMID: 41177465
- DOI: 10.1016/j.jviromet.2025.115296
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; SARS-CoV-2; TMPRSS2; cell fusion; fusion inhibitors; spike protein; split GFP reporter assay.