Wst 1 treatment

Viral polymerase activity was analyzed in Vero cells with a Gaussia luciferase mini-replicon assay. The Gaussia luciferase gene was inserted as a reporter between PR8 NA non-coding regions (pPolI-Gluc plasmid). The Vero cells at 70–80% confluency were transfected with the four plasmids expressing viral polymerase complex proteins (PB2, PB1, PA, and NP) and pPolI-Gluc plasmid using TransIT-2020 (Mirus Bio LLC, Madison, WI). At 12 hours post-transfection, luciferase expression was estimated and adjusted by WST-1 treatment (Roche, Germany). For quantitative real-time PCR (qRT-PCR), the Vero cells were inoculated at an MOI of 0.01 with rIETR/PA:E31 or rIETR/PA:K31. The cellular RNA was isolated at the indicated time point, and cDNA was synthesized using Oligo(dT) primer. The expression of NP mRNA was analyzed using NP-specific primer sets. The RNA expression was then normalized using that of GAPDH mRNA, and the relative expression levels in rIETR/PA:E31- and rIETR/PA:K31-infected cells were calculated by the ΔΔC_t method.

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).