FIG 6.

Interaction of NS1 with MLKL enhances MLKL-mediated NLRP3 inflammasome activation and IL-1β processing and secretion. (A) HEK293T cells were transfected with plasmids expressing NLRP3 inflammasome components and pro-IL-1β along with the indicated plasmids expressing null (empty vector), MLKL, NS1, or NS1(R38A/K41A). The supernatant and cell lysates were collected at 20 h posttransfection and analyzed by Western blotting with the indicated antibodies. (B) HEK293T cells were transfected as described above for panel A, and IL-1β levels in the supernatants of the above-described samples were measured by an ELISA. R, reconstitution (pcDNA-NLRP3 plus pcDNA-ASC plus pCMV-Flag-pro-caspase 1 plus pcDNA-pro-IL-1β). ***, P < 0.001; ****, P < 0.0001. Results are representative of data from three independent experiments performed in duplicates.