Fig. 3. Mutational screen of modified lysines reveals distinct effects on mRNA transcription and vRNA replication.

a Schematic of the polymerase reconstitution assay. Cells are co-transfected with plasmids for PB2, PB1, PA, and NP together with a firefly luciferase-encoding vRNA minigenome under control of the pol-I promoter and a Renilla luciferase under control of the pol-II promoter. FF activity correlates with the activity of the viral polymerase. Renilla activity serves as an internal transfection control. b Generation of recombinant influenza viruses. Cells are co-transfected with 8 bi-directional pHW2000 plasmids encoding the viral genome segments. Viral mRNA and vRNA is generated from pol-II and pol-I promoters, respectively. Illustrations created with biorender. c–e Polymerase reconstitution assay with A/R-substitutions of the modified lysines in PB2 (c), PA (d), and PB1 (e). Relative FF activities are presented as the mean percentage activity using the wild type (WT) polymerase (±SEM), n = 3 independent biological replicates. P values < 0.05 compared to WT from Dunnett’s multiple comparison one-way ANOVA test are indicated. Success (+) or failure (−) to generate recombinant viruses is depicted below. Virus rescue was considered negative if three independent rescue attempts failed. Sites located in functional domains (white), interaction regions with RNA templates (light red), or viral/host proteins (gray) are highlighted. f–h Quantification of FF mRNA and cRNA levels from the polymerase reconstitution assay for mutations in PB2 (f), PA (g), or PB1 (h) using qRT-PCR using segment and RNA species-specific primers. Values are depicted as mean relative to WT (±SEM), n = 3 independent biological replicates. GAPDH served as housekeeping control. P values < 0.05 compared to WT from Dunnett’s multiple comparison two-way ANOVA are indicated. i–k Viral titers after rescue (i) or passaging (j, k), n = 3 independent biological replicates (±SEM). Viral titers were determined 48 h p.i. as plaque forming units (PFU) per ml. l–n Immunofluorescence of WT or mutated versions of PB2 (l), PA individually (m), or in combination with HA-tagged PB1 (n)⁷⁴. 24 h post transfection A549 cells were fixed and analyzed using the indicated antibodies. Cell nuclei were visualized using DAPI. Representative cells from one experiment are shown. Scale bar = 10 µm.