NS1 RNA binding is not required for rescue but CPSF30 interaction domain is essential. 293T cells were transfected with plasmid mixes to express recombinant vRNPs containing influenza RdRP with either WT-NP, del20NLS-NP, or no NP, and FLAG-PA vRNA as template in the presence or absence of NS1 and NS1 mutants as indicated. Total protein was separated by SDS-PAGE and analyzed by Western blotting with anti-FLAG antibody to visualize FLAG-PA products. Anti-NS1 detects NS1. Anti-tubulin detects tubulin and serves as a loading control. Shown is a representative experiment, repeated in triplicate.