MST1, and PRSS12 gene expression (Figure 5), the role of these miRNAs in the regulation of influenza virus replication was determined (Figure 6). A549 cells were treated with individual miRNA inhibitors and the cells infected with A/WSN/33 to determine the effect on virus replication (Figure 6). Of the 8 miRNA inhibitors tested, inhibition of miR-106B was associated with a decrease in influenza virus replication, while inhibition of miR-124 resulted in an increase in virus replication with respect to the negative control. Inhibition of the other eight miRNAs had more subtle effects with slight increases or decreases of influenza virus replication. The decrease of virus replication associated with inhibition of miR-106B is likely associated with a decrease of CPE gene expression as RNAi silencing of CPE was associated with low levels of virus replication (Figures 1 and 2). The level of virus replication was confirmed by qPCR M gene levels and was consistent with the findings observed in Figure 5 (data not shown). The results show that some of the miRNAs modulate host genes critical for influenza virus replication, thus it likely that the tempo of host gene expression is differentially regulated in response to influenza virus infection. Further, the results provide evidence that targeting miRNAs may offer an alternative disease intervention approach to control influenza virus replication. Discussion Some human proteases are known to have a direct function in the replication of influenza virus [38,39], but the role of other proteases in the biology of virus replication and host cell pathways they affect are not fully elucidated. To better understand protease gene requirements for influenza virus replication, and discover novel disease intervention targets, we conducted an RNAi screen of 481 genes comprising the human protease genome and validated five genes, ADAMTS7, CPE, DPP3, MST1, and PRSS12 that are required for influenza replication. The primary and secondary RNAi screens were performed with A/WSN/33 influenza virus because of its ability to replicate in the absence of trypsin in a type II respiratory epithelial (A549) cell line. All five genes showed reduced A/WSN/33 titers and some showed a substantial decrease in NP staining to the level where NP was not detected using a NP-specific monoclonal antibody. This may have been due to an inability of the virus to reenter the host cells for subsequent infection as a consequence of protease gene silencing. To confirm the findings for WSN/33 infection, the five validated genes were assessed against a clinical influenza virus strain, i.e. A/ New Caledonia/20/99. RNAi silencing of all five genes resulted in decreased levels of A/New Caledonia/20/99 replication. Al- though previous RNAi host factor screens for influenza virus replication did not identify any of the five protease genes in this study, the same host cell pathways were identified which included the genes validated in this study [27,29,30,31,32]. Specifically, our pathway analysis for this study showed that ADAMTS7, CPE, DPP3, MST1, and PRSS12 were involved in three previously identified host cell pathways, specifically CRE/CREB signaling, NF-kB activation, and apoptosis. It is likely that different pathway interaction is co-opted by the virus at different stages in the virus replication process, but further study is needed to determine the exact process. An additional caveat may be the use of an immortalized cell line to identify gene hits. Clonal cell lines can have misregulated gene expression relative to primary cells, particularly for genes involved in cell growth and differentiation [53]. Use of a primary human cell line to perform this study, e.g. normal human bronchial epithelial (NHBE) cells may be more desirable; however, primary cells often have reduced transfection efficacy and issues with maintenance of a uniform stage of Human Genes Required for Influenza Replication differentiation that makes it difficult to perform high-throughput screening [35]. Therefore, it is possible that some genes discovered may not translate to a primary cell line, so further evaluation is needed to confirm the results of this study. CRE/CREB signaling is an important cellular process that serves a variety of functions. Most notably with respect to influenza infection, CRE/CREB signaling has been shown to activate protein kinase A (PKA) and thus have a role in protein synthesis [54]. In this study, MST1 and PRSS12 were implicated in CRE/ CREB signaling; however, in response to influenza infection, CRE signaling levels in cells treated with siPRSS12 were unchanged compared to both pathway reporter and siNEG controls. However, RNAi of the MST1 gene resulted in significantly higher CRE signaling levels regardless of infection. These findings may suggest that MST1 is involved in some aspect of CRE signaling or the cAMP response, and without MST1, the calcium response element is not activated. ADAMTS7, CPE, and MST1 have been shown to have a role in tissue injury and inflammation [55,56,57], and pathway analysis performed in this study identifying their linkage to NF-kB pathway activation is consistent with these earlier findings. However, there was no significant effect of these genes on the NF-kB pathway relative to controls. However, A549 cells treated with siRNAs targeting ADAMTS7 and DPP3, a gene not predicted to be involved in the NF-kB pathway, resulted in complete abrogation of NF-kB regardless of influenza infection. This finding suggests that NF-kB signaling may involve ADAMTS7 and DPP3. As ADAMTS7 plays a role in maintenance of the extracellular matrix [56] and DPP3 is a metallopeptidase, it is possible that silencing these genes is decreasing the inflammatory response because less damage is being done to basement tissue. ADAMTS7 has also been shown to be upregulated by TNF-a and IL-1b secretion [56], both of which are activated upon influenza infection [43]. DPP3 is involved in apoptosis modulation and is over-expressed in cancerous cells [58]. RNAi of the DPP3 gene was performed to evaluate the effect on apoptotic gene expression. The results showed induction of BCL2L10, TNFSF10, TNFRSF25 and TNFSF8 pro-apoptotic genes. BCL2L10 is a pro-apoptotic gene that interacts with caspase 9 to signal apoptosis [59], and TNFSF8 and TNFRSF25 are involved in the pro-inflammatory response through NF-kB signaling [60,61]. TNFSF10, also known as TRAIL, has been shown to be involved in H5N1 virus-induced apoptosis in human monocyte-derived macrophages [62]. Since DPP3 silencing resulted in a sharp decrease of influenza replication as quantified by TCID50 and NP staining, the findings suggest that reducing DPP3 gene expression causes influenza virus infected cells to initiate apoptosis without interference by the known anti-apoptosis activities of the influenza NS1 protein [3]. It is possible that without the NS1-mediated apoptosis delay, the virus is not able to replicate and bud before the host cell is eliminated. Furthermore, as NF-kB appears to have a role in apoptosis modulation, the effect of DPP3 silencing on NF-kB activation observed in this study is predictable and consistent with earlier findings [63]. Host gene expression is governed by miRNAs [44,64], thus it is important to understand potential miRNA regulation of host genes validated as required for influenza virus replication. The results from the miRNA hairpin inhibitor studies showed that targeting the predicted miRNAs had a considerable and significant outcome on most host gene expression. For example, inhibition of miR- 106B and miR-124* resulted in 20-fold and 40-fold increased ADAMTS7 gene expression levels, respectively, while CPE gene expression was slightly reduced by miRNA inhibitors. As expected, some miRNA inhibitors had differential effects on host genes PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e37169