Human Genes Required for Influenza Replication Figure 2. RNAi of individual host protease genes down- regulates replication of a clinical influenza isolate. A549 cells were reverse transfected with 100 nM of the novel siRNA targeting siADAMTS7, siCPE, siDPP3, siMST1, and siPRSS12. After 48 hours, cells were infected with A/New Caledonia/20/99 at an MOI of 0.1 in the presence of 1 ug/ml TPCK-trypsin. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID50. Data is expressed as TCID50/ml. Data is representative of two independent experiments. (*p,0.05 vs. siNEG). doi:10.1371/journal.pone.0037169.g002 with 100 nM of a single siRNA targeting each of the validated genes was greatly (siCPE, siMST1 and siPRSS12) and significantly (p,0.05; siADAMTS7 and siDPP3) reduced (Figure 1C). Influ- enza virus M gene levels reflected the low level of infectious virus and NP staining observed for A549 cells treated with siRNAs targeting the five validated genes, as well (data not shown). Host genes validated with a clinical influenza virus isolate Since A/WSN/33 was used in the primary and secondary screens because of its ability to grow in the absence of trypsin, a Figure 4. RNAi of DPP3 (siDPP3) inhibits influenza replication by modulation of apoptotic genes. A549 cells were reverse transfected with 50 nM of siDPP3 or siNEG. After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. After 18 hours of infection, cellular RNA was isolated and apoptosis gene expression profiles were determined by array. Gene expression was normalized to GAPDH levels. Silencing DPP3 resulted in upregulated levels of the pro-apoptotic genes BCL2L10, TNFSF10, TNFSF25 and TNFSF8. Data is representative of three independent experiments. * p,0.05. doi:10.1371/journal.pone.0037169.g004 feature facilitating high throughput screening, cross-validation of ADAMTS7, CPE, DPP3, MST1, and PRSS12 genes was performed in A549 cells with A/New Caledonia/20/99. Using the same siRNAs previously used to validate the genes (Figure 1C), A549 cells treated with 100 nM of the siRNAs significantly (p,0.05) reduced A/New Caledonia/20/99 virus replication (Figure 2). The differences in viral titer were at least 1.5 logs lower compared to siNEG-treated cells for all five genes examined. These results show that ADAMTS7, CPE, DPP3, MST1, and PRSS12 contribute to influenza A virus replication, and provide promising disease intervention targets. Figure 3. Analysis of host gene involvement in major cellular pathways. A: A549 cells were reverse cotransfected with CRE/CREB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p,0.05, ** p,0.01 compared to siNEG control (mock), # p,0.05, ## p,0.01 compared to siNEG control (A/WSN/33) B: A549 cells were reversed cotransfected with NF-kB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p,0.05, ** p,0.01 compared to siNEG control (mock), # p,0.05, ## p,0.01 compared to siNEG control (A/ WSN/33). Data is representative of three independent experiments. doi:10.1371/journal.pone.0037169.g003 PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e37169