Table 1. Human protease gene hits. Gene ADAMTS7 Name ADAM metallopeptidase with thrombospondin type 1, motif 7 Accession number siRNA sequences NM_014272 CPE Carboxypeptidase E NM_001873 DPP3 Dipeptidyl-peptidase 3 NM_005700 MST1 Macrophage stimulating 1 (hepatocyte growth factor-like) NM_020998 PRSS12 Neurotrypsin, motopsin NM_003619 *: Refers to the pool of four siRNAs used in the primary screen. #: Refers to the individual siRNA used for the validation step. doi:10.1371/journal.pone.0037169.t001 critical for replication. Pathway analysis revealed that these five genes were linked to cAMP responsive binding element (CREB), NF-kB, and apoptosis pathways, thus we examined and verified host gene regulation of these pathways. As the tempo of host gene expression is governed by miRNAs which have important roles in regulating host genes during viral infection and replication [45,50,51], an additional analysis was performed to identify miRNAs that might potentially regulate the five validated host genes required for influenza virus replication. A library of miRNA antagonists was used to confirm miRNA regulation, and several miRNAs were identified to affect virus replication and host gene regulation, notably miR-1254, miR-106B, miR-106B*, miR-124- a, and miR-124*. This finding shows varied miRNA expression patterns associated with the regulation of various genes involved in global host cell pathways governing inflammation (NF-kB), cAMP/calcium signaling (CRE/CREB), and apoptosis, and adds to our understanding of how miRNAs may govern host gene regulatory networks involved in the response to influenza virus replication. Results Human protease genes required for influenza replication A primary RNAi screen of 481 host protease genes in a human type II respiratory epithelial cell line (A549) identified 24 genes SMARTpool*: GCGAGGACCCGGAGAAGUA GAACGUGGGCUGUGACUUC CAACGAGGACUACUUCAUU GCAGAUACUGUGUGGGUGA Novel siRNA for Validation#: CCAAGGACAUUAUCGACUU SMARTpool*: GAAAGAAGGUGGUCCAAAU GCUUAUACCUGGAAACUAU GGAAUAGACCACGAUGUUA GGAUGCAAGACUUCAAUUA Novel siRNA for Validation#: GAUGAGACGCGGAGUGGUA SMARTpool*: ACACGGUGCUGCUGCGUAA GAGGGAAUCACCACCUAUU GCAGCAAGAUCCGGUCUGU GCUCAGACGUGCAGCUUCU Novel siRNA for Validation#: GAUCCUUCUCUGAGCGUUU SMARTpool*: GUACGGACCUGCAUCAUGA GGAAUGGCCUGGAAGAGAA GCUGUGACCUCUUCCAGAA CGACAACUAUUGCCGGAAU Novel siRNA for Validation#: GACCAAAGGUACGGGUAAU SMARTpool*: GGACUGAGCUGAAUACAUA GAUGAUGGAUGGACUGAUA GAGCAUAACUGUGGCCAUA GGACGAGCCUAUUCAAGAA Novel siRNA for Validation#: GAGCAAGAACCAUGGCUUA important for A/WSN/33 influenza virus replication (Figure S1). The gene hits identified were $1.5s and #21.5s from the mean Z-score. Endpoint validation of the gene hits in the primary screen included influenza nucleoprotein (NP) cell localization determined by immunohistochemistry, determining the level of infectious virus by TCID50 assay, as well as influenza matrix (M) gene copy number determined by qPCR (data not shown). For validation of primary gene hits, a novel siRNA targeting the same gene but at a different seed site (Table 1) was required to produce the same phenotype as observed in the screen. Protein levels of the protease genes were assessed to ensure that RNAi silencing was decreasing target gene expression (Figure S2). After validation, five genes were identified that decreased virus replication when silenced: ADAMTS7, CPE, DPP3, MST1, and PRSS12 (Table 1). Silencing of these protease genes did not cause cytotoxicity in A549 cells compared to the cytotoxicity (siTOX) control. siMEK (an siRNA targeting MAP2K) and siNEG (a negative, non- targeting siRNA control) both displayed low level cytotoxicity possibly due to the importance of MEK in cellular signaling, and the induction of an inflammatory response to siNEG due to off- target effects (Figure 1A). Silencing of the five validated genes resulted in considerably decreased influenza NP staining com- pared to the negative (2) control, and was consistent with low NP staining in cells treated with the positive (+) siRNA control, siMEK (Figure 1B). The level of infectious virus from A549 cells treated Human Genes Required for Influenza Replication Function Role in inflammation of extracellular matrix [56,77,78,79] Role in local opioid network in the lung [57,80,81,82] Role in inflammation and apoptosis [83,84,85] Role in inflammation and response to tissue injury [55] Unknown PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e37169