Human ifn β 1a

293 cells were seeded in 12-well plates 1 day before transfection for 30–50% confluency at the time of transfection. Transfection was performed using TransIT-LT1 according to the manufacturer's instructions. Plasmids transfected included 250 ng firefly luciferase in a pISRE reporter plasmid (Agilent)47 (link), 50 ng pCAGGS-hrluc for normalization and 1–100 ng (in threefold dilutions) pCAGGS-VP24, with wells receiving <100 ng VP24 balanced by empty vector. Twenty-four hours later, 1,000 IU human IFN-β 1a (PBL Assay Science) were added. Cells were harvested and reporter activity was measured 8 h after IFN application as described for the IFN production assay.

Confluent Calu-3 cells in 96-well plates (~6 × 10⁴ cells/well) were infected with SARS-CoV-2 (2000 pfu per well). At 1 or 24 hpi, we added active or inactive IAV DIPs (DI244 or OP7) at indicated fractions (% v/v) with respect to the cell culture volume of 100 µL. Whenever indicated, we additionally added 0.8 µM ruxolitinib (Cayman Chemical (Ann Arbor, MI, USA), #Cay11609-1) to these wells. Alternatively, remdesivir (MedChem Express (Monmouth Junction, NJ, USAnited States), #HY-104077) or human IFN-β-1A (PBL assay science (Piscataway, NJ, USA), #11415-1) (instead of IAV DIPs) were added at indicated concentrations at 1 hpi. Supernatants were collected at indicated time points for quantification of SARS-CoV-2 titers (plaque assay) and for protein quantification of secreted IFNs using commercially available ELISA kits (see below). In addition, infected cells were lysed using solution RL for subsequent total RNA extraction using the innuPREP RNA Mini Kit 2.0 (Analytik Jena (Jena, Germany), #845-KS-2040050), according to the manufacturer’s instructions, for gene expression analysis via real-time RT-qPCR.