Viral RNP Complex Polymerase Activity

To investigate the polymerase activity of viral RNP complexes (PB1, PB2, PA, and NP genes), we additionally constructed a Gaussia luciferase reporter plasmid (pPolI-GLuc)21 (link). The RNP complex gene and the pPolI-GLuc plasmids were transfected into 5 × 10⁴ 293T cells (ATCC, Manassas, VA, USA) using X-tremeGENE (Roche, Basel, Switzerland), and the cells were incubated at 37 °C for 24 h. After treating the cells using BioLux Gaussia Luciferase Assay Kit (New England Biolabs, Ipswich, MA, USA), luciferase activity was measured on a SpectraMax L plate reader (Molecular Devices, Sunnyvale, CA, USA). The cells transfected with empty plasmids were used as background luciferase expression. Luciferase activity was normalized by WST-1 treatment (Roche, Basel, Switzerland)22 (link).