Fig 5.

SUMOylation affects NS1's ability to neutralize IFN production. HEK293FT cells were transfected with (+) or without (−) the indicated mammalian expression constructs, including (i) the T7T7NS1 and T7T7NS1K70AK219A constructs previously described; (ii) Pol II(PB2+PB1+PA+NP), a mix of four RNA polymerase II-driven constructs for the expression of the viral proteins PB2, PB1, PA, and NP, which together form the functional vRdRp; (iii) Pol I(PB2), an RNA polymerase I-driven construct for the production of the PB2 vRNA; (iv) wt-ASL, an expression construct coding for the wild-type form of the artificial SUMO ligase for NS1; and (v) mut-ASL, a mutated form of the artificial SUMO ligase for NS1 containing two amino acid substitutions, R38A and K41A. At 48 h posttransfection, culture supernatants and total cell extracts were collected. The IFN levels present in the supernatants were analyzed by using a bioassay. The total cell extracts were analyzed by SDS-PAGE and immunoblotting using anti-T7 MAb and anti-Ubc9 MAb or anti-SUMO1 MAb and anti-GAPDH MAb as primary antibodies and highly cross-absorbed IRDye 800 CW- and IRDye 680 LT-conjugated anti-mouse and anti-rabbit IgG antibodies as secondary antibodies. The immunoblots were read and analyzed by using an Odyssey CLx infrared imaging system. (A) Graphic representation of the percentage of inhibition of IFN production observed for cells transfected with the indicated constructs. The numbers in brackets indicate the corresponding sample in the gel presented in panel B. (B) Immunoblots of a representative group of transfected cells showing the expression of T7T7NS1 and SUMOylated T7T7NS1 (T7T7NS1^SUMO), the overall SUMOylation profile obtained, and the levels of expression of the ASL. (C) Percentage of SUMOylated NS1 obtained in cells transfected with the indicated combinations of expression constructs. The numbers in brackets indicate the corresponding sample in the gel presented in panel B. (D) Relative quantification of the levels of ASL compared to the amount observed in cells transfected with wt-ASL. In all cases, the quantitative data presented represent the cumulative data obtained from four sets of samples obtained in independent experiments. Error bars indicate standard deviations.