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

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.