Influenza a pr 8 76 pr8

Influenza A/PR/8/76 (PR8) was purchased as purified allantoic fluid from Charles River Laboratories (Spafas, CT, USA). Plasmids coding for CHIKV-GFP and CHIKV-mCherry were purchased from EVA and obtained from the Andres Merits laboratory, respectively. SINV-GFP was derived from the pTR339 infectious clone. Plasmids were linearized overnight with NotI (Thermo Fisher Scientific # FD0593), then purified on columns (Macherey-Nagel #740609.50). In vitro transcription was performed using Ambion SP6 mMessage mMachine (Thermo Fisher Scientific #AM1340), according to manufacturer's instructions, and RNA was subsequently purified by phenol–chloroform extraction. Ten million BHK cells were electroporated with 10 μg of IVT RNA (1.2 kV, 25 μF, and infinite resistance) and put in culture with complete medium. Virus was harvested 72 h later, and was further passaged once for 24 h on BHK cells, before purification by ultracentrifugation to avoid protein contamination.

Atg5^+/+ cells and Atg5^–/– MEFs were obtained from Christian Munz, University of Zurich, Switzerland. Atg7^–/– MEFs were obtained from Stephen Tait, University of Glasgow. All cell lines in this study were cultivated in complete growth medium, comprised of Dulbecco’s modified Eagle’s medium with high glucose, pyruvate and GlutaMAX and supplemented with non-essential amino-acids, HEPES buffer, penicillin/streptomycin (all reagents, ThermoFisher Scientific) and 10% foetal calf serum (GE Healthcare, A15-502). Shield1 (Clontech, 632188) was added at a final concentration of 1 µM (stock maintained at 1 mM) in growth media. Ethanol, the solvent for Shield1, was used as a control at a 1:1000 dilution in growth media. Treatments were used at the following concentrations: chloroquine (Sigma, C6628), 50 µM; PP242 (Selleck Chemical, S2218), 1 µM; MG132 (Sigma, C2211) 10 µM; recombinant IFN-β, at the indicated concentrations (BioLegend, 581302); and thapsigargin (Sigma, T9033) 3 µg/mL. Blocking IFNAR antibody (BD Pharmingen, 561183) or isotype control (BD Pharmingen, 553447) were used at 10 µg/mL and added to culture media 1 h before infection for the duration of the experiment. Influenza A/PR/8/76 (PR8) and ΔNS1 PR8 were purchased as purified allantoic fluid or purified antigen, respectively, from Charles River Laboratories (Spafas, CT, USA).