a, Venn diagram of influenza virus NS1s bearing a SUMO site in the C-terminal domain (tail). Bottom: amino acidic sequence of the NS1 tail from A/Brevig Mission/1/1918 (H1N1) bearing SUMOylation site and PDZ domain ligand site. 1918 NS1 C-termini is unique (1 out 1341 H1N1 from human isolates). b, Conservation plot of amino acidic sequences of NS1 among different viral isolates and hosts. The color-coding is the difference between the conservation score and average value of the score across the protein. Protein domains of NS1 are shown at the bottom. c, Ectopic expression of SUMO2 and the indicated NS1 (WT NS1 and the SUMO-consensus mutants at positions K70 and K227) in A549 cells. Immunoprecipitation (IP) with anti-Flag antibody, and Western Blotting (WB) analysis with anti-NS1 and anti-SUMO2/3 antibodies are shown. d, IP with anti-NS1 antibody and WB with anti-NS1 and anti-SUMO2/3 antibodies in A549 cells transfected with the indicated NS1s. The tail swapping of H3N2 NS1 was performed using the sequences from the indicated viruses, all of human origins but Hong Kong 1992 and Indonesia 2005 which are avian viruses. e, IP and WB with anti NS1- antibody of whole cellular extract from A549 cells infected with the reassortant NS1 virus bearing the segment 8 encoding for 1918 NS1 (see Figure 2B). NEM: N-Ethylmaleimide, a SUMO peptidase inhibitor