Figure 6. Effect of miRNA inhibition on influenza replication. A549 cells were treated with the appropriate miRNA inhibitor (25 nM) for 48 hours, followed by infection with A/WSN/33 (MOI = 0.001). Cellular supernatant was tested for infectious virus production by a modified TCID50 48 hpi. Data is expressed as TCID50/ml and is representative of two independent experiments. doi:10.1371/journal.pone.0037169.g006 Cell pathways associated with the validated host genes affect influenza replication Expanding from the primary screen toward defining host cell pathways involved in influenza virus replication requires assess- ment of the signaling pathways, molecular networks, and biological processes that are linked to the five validated host genes. Dynamic pathway analysis identified three major pathways for the five validated genes, i.e. NF-kB pathway, CRE/CREB signaling pathway, and cellular apoptosis (Figure S3). To verify the host genes in these pathways, A549 cells were transfected with a luciferase assay reporter plasmid governed by the promoter of interest linked to a firefly luciferase gene. The level of pathway activation was determined by luciferase expression, which was normalized to a control plasmid expressing Renilla luciferase to account for transfection efficiency. Pathway analysis suggested that MST1 and PRSS12 genes participate in the CRE/CREB signaling pathway. RNAi of MST1 by siRNA targeting (siMST1) led to a significant (p,0.05) increase in CRE/CREB signaling compared to the siNEG and CRE controls in the presence or absence of influenza virus infection (Figure 3A). RNAi of PRSS12 by siPRSS12 did not affect CRE/CREB activation relative to controls. Interestingly, RNAi of DPP3 using siDPP3 significantly (p,0.05) increased CRE/CREB activation under mock-infected conditions despite DPP3 not being implicated in the CRE/CREB pathway, and silencing of ADAMTS7 significantly (p,0.05) abrogated CRE/CREB activation during infection. Pathway analysis also indicated that ADAMTS7, CPE, and MST1 genes were potentially involved in the NF-kB activation pathway (Figure S3). siADAMTS7 significantly (p,0.01) abrogated NF-kB activa- tion regardless of infection or mock-treatment (Figure 3B). Interestingly, a similar finding was observed for DPP3, a gene not previously linked to the NF-kB signaling pathway by our analysis. siCPE did not affect NF-kB activation under mock conditions, but during infection NF-kB activation levels were significantly (p,0.05) downregulated. siMST1 also had a negative effect on NF-kB activation regardless of infection. Elevated NF-kB Human Genes Required for Influenza Replication levels were also seen in controls receiving the NF-kB plasmid alone but this was not significant compared of siNEG controls. Dynamic pathway analysis predicted DPP3 to be involved in apoptotic pathways (Figure S3), and as a luciferase/Renilla pathway reporter system was unavailable, a human apoptosis array was performed to determine the level of mRNA expression of various pro- and anti-apoptotic host genes in relation to DPP3 expression (Figure 4). After confirming RNAi silencing (.95%) of DPP3 by PCR (data not shown), gene expression was compared in cellular RNA extracted from infected A549 cells treated with either siDPP3 or siNEG (50 nM). While most genes tested did not vary in expression levels relative to siNEG-treated cells, four pro- apoptotic genes were found to be substantially increased when DPP3 was silenced: BCL2L10, TNFSF10, TNFRSF25, and TNFSF8. Of these four genes, TNFSF10 was significantly (p,0.05) increased 6-fold, and TNFSF8 was significantly (p,0.05) increased 5-fold compared to siNEG control treated cells. TNFRSF25 was substantially increased as well. These findings show that inhibition of DPP3 expression initiates higher expression of pro-apoptotic factors, features that may negatively impact influenza virus replication and associated modulation of cellular apoptosis. Silencing of the other four target protease genes did not significantly affect levels of pro- or anti-apoptotic genes compared to cells treated with siNEG as predicted by the Ingenuity pathway analysis, although TNFSF10 was substantially increased when cells were treated with siADAMTS7 (Table S1). miRNAs govern host genes required for influenza replication Pathway analysis of the five validated host genes revealed potential miRNA interaction (Figure S4) with eight miRNAs (miR- 1254, miR-1272, miR-17-5p, miR-17-3p, miR-106B, miR-106B*, miR-124-a, and miR-124*). To determine the role of these miRNAs in regulating ADAMTS7, CPE, DPP3, MST1, and PRSS12, A549 cells were treated with miRNA hairpin inhibitors as previously described [52], or treated with a negative control from C. elegans that shares no homology with known human miRNA sequences, and gene expression levels determined by qPCR. As gene modulation by miRNAs is usually subtle and multi-targeted, the effect of the miRNA inhibitors on host gene mRNA levels was determined 24 hours post-treatment. All 8 miRNA inhibitors were tested for their effect on each of the 5 validated genes; however, only those inhibitors that affected gene expression are shown (Figure 5). At 24 h post-treatment, ADAMTS7 gene expression levels increased 20-fold when miR- 106B was inhibited, and 40-fold when miR-124* was inhibited (Figure 5A). Treatment with siRNA targeting the gene examined was performed as a control. CPE expression was slightly decreased by miRNA inhibitors, as there was no response above 1.0 (Figure 5B). In contrast, inhibition of miR-106B and miR-124* resulted in a .20-fold and .40-fold increase of DPP3 gene expression, respectively (Figure 5C), while miR-1254, miR-1272, and miR-17-3p inhibition caused a decrease of DPP3 expression. MST1 expression was increased to similar levels as DPP3 with the same miRNA inhibitors, while the other miRNA inhibitors resulted in a slight (but significant for miR-17-3p) decrease in expression (Figure 5D). Finally, PRSS12 expression levels were significantly (p,0.05) increased in response to miR-106B inhibi- tion, but a slight decrease was detected by inhibition of miR-1254 (Figure 5E). These results show the same miRNAs can regulate different genes both subtly and robustly. Given the evidence that miR-1254, miR-1272, miR-17-5p, miR-17-3p, miR-106B, miR-106B*, miR-124-a, and miR-124* are involved in governing aspects of ADAMTS7, CPE, DPP3, PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e37169