miR-144 negatively regulates IRF7 activity and TRAF6 expression.

<p>(<i>A</i>) Viral load in the BAL of WT or IRF7^-/- mice infected with influenza virus was quantified by qRT-PCR after 24 h. <i>n</i> = 3 mice per genotype. (<i>B</i>) 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. <i>n</i> = 3 mice per genotype. (C) Luminescence assay of cells expressing firefly luciferase fused to the complete coding sequence plus 3’-UTR of murine <i>Irf7</i> or 3’-UTR only of <i>Trim30</i> 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. (<i>D</i>) Sequences of the 2 computationally predicted miR-144 target sequences in the <i>Traf6</i> 3’-UTR and the mutant target sequences used for the experiments shown in <i>E</i>. (<i>E</i>) Luciferase assays were performed as in <i>C</i> using firefly luciferase fused to the complete 3’-UTR of murine <i>Traf6</i> or the intact or mutant miR-144 target sites in the <i>Traf6</i> 3’-UTR shown in <i>D</i>. 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. (<i>F</i>) 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. (<i>G</i>) 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006305#ppat.1006305.s007" target="_blank">S5D Fig</a> for a representative immunoblot. Means ± SEM for 3 independent experiments are graphically displayed. * p<0.05, ** p<0.01.</p

miR-144 attenuates the host response to influenza virus by targeting the TRAF6-IRF7 signaling axis

<div><p>Antiviral responses must rapidly defend against infection while minimizing inflammatory damage, but the mechanisms that regulate the magnitude of response within an infected cell are not well understood. miRNAs are small non-coding RNAs that suppress protein levels by binding target sequences on their cognate mRNA. Here, we identify miR-144 as a negative regulator of the host antiviral response. Ectopic expression of miR-144 resulted in increased replication of three RNA viruses in primary mouse lung epithelial cells: influenza virus, EMCV, and VSV. We identified the transcriptional network regulated by miR-144 and demonstrate that miR-144 post-transcriptionally suppresses TRAF6 levels. <i>In vivo</i> ablation of miR-144 reduced influenza virus replication in the lung and disease severity. These data suggest that miR-144 reduces the antiviral response by attenuating the TRAF6-IRF7 pathway to alter the cellular antiviral transcriptional landscape.</p></div

Decreased TRAF6 levels are sufficient to impair host control of influenza virus replication.

<p>(<i>A</i>) Western blotting of TRAF6, IRF7, and β-actin was performed using protein lysates from the indicated LET1 cells. Densitometric quantification of 3–4 independent experiments was performed and means ±SEM normalized to actin and relative to control shRNA cells is as follows: TRAF6 Western. Control shRNA, 1; TRAF6 shRNA, 0.43±0.15; miR-144, 0.52±0.12; miR-451, 0.79±0.12. IRF7 Western. Control shRNA, 1; TRAF6 shRNA, 0.47±0.12; miR-144, 0.68±0.09; miR-451, 1.16±0.26. (<i>B</i>) Gene expression by qRT-PCR in LET1 cells stably expressing TRAF6-specific shRNAs and infected with influenza virus for 18 h is shown relative to the level in infected LET1 cells expressing control shRNA. Means ±SEM for 3 independent experiments performed in duplicate are shown. (<i>C</i>) qRT-PCR of influenza viral load in LET1 cells expressing TRAF6-specific or control shRNAs or in the cell-free supernatants 18 h post-infection. Means ± SEM for 3 independent experiments performed in duplicate are shown. MOI = 5; * p<0.05, ** p<0.01. (<i>D</i>) Cells infected as described in <i>C</i> were stained for viral NP protein and analyzed by flow cytometry. Data are representative of 2 independent experiments. (<i>E</i>) Heat map of antiviral gene expression generated from the log₂ ratios of gene expression between the following cells: miR-144^-/-/miR-144^+/+ type I lung epithelial cells, TC-1+miR-144+miR-451/TC-1+vector alone, LET1+miR-144/LET1+vector alone, IRF7null vs WT lungs, LET1+TRAF6 shRNAs/LET1+control shRNA. All cells were infected with PR8 influenza virus for 24 h, gene expression measured by qRT-PCR, and mean intensities for 3 independent samples compared. Red (blue) represents up-(down-) regulation relative to controls. (<i>F</i>) Network model for miR-144 regulation of the TRAF6-IRF7-antiviral network.</p

miR-144 impairs control of viral replication by murine lung epithelial cells.

<p>(<i>A</i>) miR-144/451 KO or WT lung epithelial cells were infected with influenza virus. Mean viral load ±SEM at 24 h was quantified by qRT-PCR of viral M gene and normalized to host EF-1 (<i>n</i> = 3). * p<0.05. ** p<0.01, NS, not significant. (<i>B</i>) TC-1 cells were stably transduced with retrovirus expressing GFP along with miR-451+miR-144, miR-144, miR-451, miR-155, or retroviral vector alone and compared with untransduced (parent) cells. Viral load in influenza-infected cells was quantified as in <i>A</i>. Means ±SEM for 3–7 independent experiments are shown and p-values calculated for miR-144+miR-451 and miR-144-expressing cells relative to each other cell line. * p<0.05. ** p<0.01, NS, not significant. (<i>C</i>) TC-1 cells were infected as described in <i>B</i> and the mean percentage ±SEM of influenza NP⁺ cells quantified by flow cytometry (<i>n</i> = 4–9 independent experiments); p-values were calculated for cells expressing miR-144 alone relative to each other control cell line. (<i>D</i>) Primary mouse lung epithelial type I cells (LET1) were immortalized while being transduced with lentivirus expressing miR-144, miR-451, or vector alone. Viral load in influenza-infected cells (<i>n</i> = 6–9) or released into supernatants (<i>n</i> = 2–7) was measured as described in <i>B</i>, and means ±SEM are shown. (<i>E</i>) Infectious virus in the supernatants of LET1 cells infected with influenza, EMCV, or VSV measured by plaque assay. Means ±SEM are shown for 3 independent experiments performed in duplicate. A similar increase in viral load was observed when LET1 cells expressing miR-144 were compared with cells expressing miR-451. * p<0.05, ** p<0.01, ns = not significant.</p

miR-144 and miR-451 are expressed in lung epithelial cells.

<p>(<i>A</i>) Expression of miR-144 and miR-451 in total lung cells and FACS-purified lung hematopoietic and epithelial cell populations were measured by qRT-PCR and plotted in arbitrary units relative to sno-202 expression. Means ± SEM for 3–8 samples are shown. (<i>B</i>) miR-144/451^null or wild-type littermate mice were infected with influenza virus for 0.5, 1, 3, 7, or 12 d. Influenza virus was quantified by qRT-PCR of viral M gene in lungs (normalized to EF-1) or BAL, or by plaque assay of BAL. Means ± SEM are shown; 0.5 d (<i>n</i> = 8–9), 1 d (<i>n</i> = 3–6), 3 d (<i>n</i> = 4–11), 7 d (<i>n</i> = 5), 12 d (<i>n</i> = 3–6). PFUs were below the detection limit at 12 d (ND, not detected). Similar data was obtained in two independent experiments. (<i>C</i>) Weight-loss was quantified over 12 d as a correlate of morbidity (<i>n</i> = 10–19 mice per time point); reduced weight-loss in KO mice was observed in 5 independent experiments; * p<0.003.</p

miR-144 attenuates the IRF7 transcriptional network.

<p>(<i>A</i>) Heat map depicting microarray analysis of influenza-infected TC-1 cells stably expressing miR-144+miR-451 or vector alone, with genes grouped by functional annotation. The indicated genes were differentially expressed by >2-fold after 24 h relative to infected cells expressing vector alone and clustered by biological function. Mean intensities for 3 independent experiments are shown relative to uninfected control (vector alone) cells. Genes containing IRF7 motifs within 1 kB of the transcriptional start site are indicated by “+”. (<i>B</i>) Expressions of miR-regulated genes in influenza-infected miR-144+miR-451-expressing TC-1 cells (MOI = 5, 24 h) were measured by qRT-PCR, normalized to EF-1, and plotted relative to the levels in vector-only control cells. Means±SEM for 3 independent experiments are shown. (<i>C</i>) qRT-PCR was performed as described in <i>B</i> on primary type I epithelial cells sorted from miR-144/451^null or WT littermates and infected with influenza virus <i>in vitro</i>, <i>n</i> = 3. * p<0.05, ** p<0.01.</p

Global expression profiling of lung tissue isolated from influenza-infected mice.

<p>A) Heatmap depicting 385 genes whose expression is altered in the lung during the first 24 hours following infection with influenza virus (permutation q-value=<0.001 and > 5-fold difference). B) Mean expression of all genes within each cluster (K means with k=5 for 385 genes) grouped by virus strain (X31 – green; PR8 – red; VN – blue). The number of genes in each cluster is indicated. Error bars depict the SEM for all genes in the cluster at each time point.</p

Tnf and Ly6i expression in PR8 or X31 infected LET-1 cells.

<p>LET-1 cells were infected with either PR8 (red) or X31 (green) and relative levels of endogenous (<b>A</b>) Tnf and (<b>B</b>) Ly6i transcripts determined from RNA isolated at specified time points. Error bars represent SEM for n=2.</p

Pathway enrichment analysis.

<p><b>A</b>) Network diagram of TREM1 signaling canonical pathway from IPA. Colored nodes indicate genes with RNA expression measures above background in murine lungs infected with influenza virus. Green indicates higher expression in X31-infection than PR-infection at the indicated time point (>= 2 Fold). Grey color indicates that the difference in expression is < 2 fold. <b>B</b>) Average expression measure of all genes above background annotated as belonging to the “TREM1 signaling” (top) and “Differential Regulation of Cytokine Production in Macrophages and T Helper Cells by IL-17A and IL-17F” (bottom) pathways. PR8=red, VN=blue, X31=green. Error bars represent the SEM.</p

PR8 and X31 replication in LET-1 cells.

<p>A) LET-1 cells were infected with either PR8 (red) or X31 (green) and treated with TNF (100 ng/mL) two hours post infection (dashed lines) or buffer only (solid lines). (B) Percent of live LET-1 cells (derived from WT or IFNAR KO mice) at 24h post infection with or without Tnf added to the media (at 2h). (C) Tnf dose response in PR8 and X31 infected LET-1 cells (n=3). Tnf was added 2h post-infection, viral M gene was measured at 24h post-infection. Error bars represent SEM.</p