Suppression of IFN by nsp11 is via inhibition of RLR-I signaling.

<p>(A) HeLa cells were transfected with vector control or nsp11 and at 24 h post-transfection, stimulated with poly(I:C) (0.5 μg/ml) for 6 h. Cells were harvest and fractionated to nuclear and cytosolic fractions using Nuclear/Cytosol Fractionation Kit (BioVision). Both nuclear and cytosol fractions were electrophoresed by SDS-PAGE, and Western blot was conducted using α-IRF3 PAb, α-Flag MAb, α-PARP PAb, α-HSP90 MAb, and α-β-action MAb. PARP was included as a nuclear protein marker, and HSP90 was included as a cytosol protein marker. Actin was used as a loading control. Flag antibody was used to detect nsp11 expression. (B) HeLa cells were transfected with empty vector, nsp11, or K3779A, followed by stimulation with poly(I:C) (0.5 μg/ml) for 6 h. Cells were lysed and the lysates were subjected to SDS-PAGE and Western blot using rabbit α-pIRF3 PAb and rabbit α-IRF3 PAb. Rat α-FLAG MAb antibody was used to detect WT nsp11 and K3779A mutant expression. (C) HeLa cells were transfected with nsp11, and at 24 h post-transfection treated with 20 ng/ml of TNF-α for 5, 10, or 20 min. Cells were lysed at indicated time points and each lysate was subjected to SDS-PAGE and Western blot using α-nsp11 PAb, α-β-actin MAb, α-phosphor-IκB αPAb, or α-p65 PAb. Flag antibody was used to detect nsp11 expression. (D, E) MARC-145 cells were co-transfected with pIFN-β-luc (0.5 μg), pRL-TK (0.05 μg), and indicated amounts of nsp11or empty vector along with the constitutively active form of RIG-I (0.5 μg) (D) or MAVS (0.5 μg) (E). Cells were lysed at 40 h post-transfection and assayed for luciferase activities. Each set of samples were compared to the value of RIG-I or MAVS alone transfection (*, P<0.05; **, P<0.01 by one-way ANOVA and bonferroni multiple comparison).