2.1. Plasmids

The plasmid pCAGGS-NS1 encoding the full-length NS1 sequence of A/Puerto Rico/8/1934 (PR8), NS1 R38A/K41A, NS1 E96A/E97A, NS1 ∆1-73 (N), or NS1 ∆74-230 (C) was previously described [16,24,25]. Plasmid FLAG-NS1 was obtained by cloing IAV PR8 NS1 amplified by PCR using primers with Nhe I and Xba I sequences at each end (NheI-NS1-FLAG-PR8-F: 5’-GGCCGCTAGCATGGATCCAAACACT GTGTCAA-3’ and XbaI-NS1-FLAG-PR8-R: 5’-GGCCTCTAGATCAAACTTCTG ACCTAATTGT-3’) into p3xFLAG-CMV-14 vector (Sigma-Aldrich, Saint Louis, MO, USA) linearized with corresponding restriction enzymes.

Plasmids expressing HA-tagged wild-type MAVS and deletions of CARD, PRD, or transmembrane domain (TMD) (namely ΔCARD, ΔPRD and ΔTMD) were provided by P.P. To generate plasmids expressing human MAVS and its variants with deletion of TM domain tagged with an HA sequence at the C-terminus, the inserts were respectively amplified by PCR from human A549 cDNA using a primer set (NheI-MAVS-F: 5′-TCGA GCTAGC ATGCCGTTTGCTGAAGACA-3′ with XbaI-MAVS-R: 5′-ACTCTAGACTACCCGGGGGCATAATCTGGCACATCATAAGGGTAGTAGTGCAGACGCCGCCGGTACAG-3’ or XbaI-MAVS-TM-R: 5′-AC TCTAGACTACCCGGGGGCATAATCTGGCACATCATAAGGGTAGTAGTGCAGACGCCGCCGGTACAGAGGTGAGGGCCTGTGGCATGGC-3′). The cDNA of full-length MAVS or ΔTM was digested by Nhe I and Xba I enzymes at 37 °C for 1 h and then ligated with pcDNA3.1 plasmid digested with corresponding enzymes.