miR-144 attenuates the host response to influenza virus by targeting the TRAF6-IRF7 signaling axis
Fig 4
miR-144 negatively regulates IRF7 activity and TRAF6 expression.
(A) Viral load in the BAL of WT or IRF7-/- mice infected with influenza virus was quantified by qRT-PCR after 24 h. n = 3 mice per genotype. (B) Gene expression in WT or IRF7-/- mouse lungs following infection with influenza virus for 24 h was measured by qRT-PCR. Mean expression ±SEM in IRF7-/- relative to wild-type mice is plotted. n = 3 mice per genotype. (C) Luminescence assay of cells expressing firefly luciferase fused to the complete coding sequence plus 3’-UTR of murine Irf7 or 3’-UTR only of Trim30 and miR-144, miR-451, or vector alone. Firefly/Renilla luminescence relative to cells expressing miR-451 for 4 independent experiments performed in triplicate are plotted (means ±SEM). Concordant results were obtained in TC-1 lung epithelial cells. (D) Sequences of the 2 computationally predicted miR-144 target sequences in the Traf6 3’-UTR and the mutant target sequences used for the experiments shown in E. (E) Luciferase assays were performed as in C using firefly luciferase fused to the complete 3’-UTR of murine Traf6 or the intact or mutant miR-144 target sites in the Traf6 3’-UTR shown in D. Means ±SEM for 5 (complete UTR) or 3 (individual miR-144 sites) independent experiments performed in triplicate are shown. *p = 0.014,**p = 0.010. (F) Reduced TRAF6 mRNA expression in influenza virus-infected TC-1 cells expressing miR-144 compared to those expressing vector alone by qRT-PCR. Means ±SEM for 3 independent experiments are shown, *p<0.05. (G) Reduced TRAF6 protein in TC-1 cells expressing miR-144 compared to those expressing miR-451 or vector alone pre- or 6 h post-infection with influenza virus. See S5D Fig for a representative immunoblot. Means ± SEM for 3 independent experiments are graphically displayed. * p<0.05, ** p<0.01.