required for influenza virus replication. For example, inhibition of miR-106B had little effect on CPE gene expression, but dramatically increased DPP3 gene expression. These findings show that expression of host genes required for influenza virus replication can be regulated by multiple miRNAs, and suggests that targeting miRNAs to regulate host gene expression may be a strategy to regulate influenza virus replication. As predicted from the host gene expression studies, miRNA inhibitors also affected influenza virus replication. For example, inhibition of miR-106B, a miRNA known to cause cell cycle arrest when inhibited in a laryngeal cancer model [65], resulted in substantially decreased virus replication. Human papillomavirus oncoproteins E6 and E7 have also been reported to dysregulate miR-106B [66]. Inhibition of miR-124-a resulted in an increase of influenza virus replication relative to the negative control. Although miR-124-a was not found to regulate any of the 5 host protease genes from our screen, it is reported to have a putative target in both swine influenza virus and the 2009 pandemic H1N1 strain [67]. Inhibition of other miRNAs also subtly modulated infectious virus levels, but not to a level significantly different from the NEG control. In this study, inhibition of miR-106B increased ADAMTS7, DPP3, MST1 and PRSSS12 gene expression, but subtly reduced CPE gene expression, an effect that resulted in reduced influenza virus replication, implying the effect of miR- 106B inhibition on CPE may be dominant. Additionally, inhibition of miR-1254 resulted in slightly decreased CPE, DPP3, MST1 and PRSS12 gene expression levels, and this translated to slightly decreased influenza virus replication. Since none of the 5 validated genes were increased by inhibition of miR- 124-a, it is likely miR-124-a affects influenza replication through other genes yet identified. These findings indicate that while miRNAs do modulate host gene expression, they likely modulate influenza replication indirectly and perhaps at different points in the replication pathway. Further study is needed to validate miRNA involvement and mechanism of virus inhibition or assistance. Taken together, these findings suggest novel targets and potentially new therapies for regulating influenza replication and the pathways co-opted during infection. Materials and Methods Cells and virus stocks A549 cells (ATCC CCL-185) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, Logan, UT) containing 5% heat-inactivated FBS (HyClone, Logan, UT). Cells were frozen in 10% DMSO and 90% FBS to create one stock of a single cell passage that was used for the entire screen. A stock of MDCK cells (ATCC CCL-34) was also propagated and stored using the same conditions. A/WSN/33 (H1N1) influenza virus was used for the primary siRNA screen as this virus has the ability to replicate without the need for exogenous trypsin [68]. For the validation studies, A/New Caledonia/20/99 (H1N1) influenza virus was also used. All viruses were propagated in 9-day-old embryonated chicken eggs as previously described [69]. Viruses were titrated in MDCK cells and titers calculated by the method developed by Reed and Muench [70]. Protease library screen A primary screen using four pooled siRNAs to target each gene of the 481 genes in the human protease library (SMARTpool; Dharmacon ThermoFisher, Lafayette, CO) was performed using A/WSN/33 influenza virus and type II human alveolar pneumo- cytes (A549 cells) similar to a method previously described [27]. siRNAs were resuspended in Dharamcon siRNA buffer to a Human Genes Required for Influenza Replication concentration of 1 uM and stored at 280uC until use. The screen was conducted in two steps, a primary screen targeting all 481 human protease genes, followed by a smaller-scale validation screen of the primary hits to confirm the genes identified were essential for influenza virus replication. In all studies, a siRNA targeting the MEK gene (siMEK), a well-characterized human kinase gene important for influenza replication [4,71], was used to control for the transfection efficiency and host gene silencing. A non-targeting siRNA control (siNEG) was also used in all assays. The siRNA SMARTpool library constituents, siMEK, and siNEG transfected A549 cells at .85% efficiency, and a few (,2%) siRNA pools targeting HP genes induced cytotoxicity as deter- mined by adenylate kinase (AK) release in the cell culture supernatant [72,73] and by visual inspection of cell morphology. A549 cells were reverse transfected with the siRNA 48 hours before infection with influenza A/WSN/33 (MOI = 0.001). The amount of infectious virus was measured 48 hpi by titration of A549 cell supernatant on MDCK cells, and the results normalized to siNEG-treated cells. All assays were run in duplicate and the entire screen assay was repeated twice. Both plate-based controls and assay-wide controls, e.g. siMEK, siNEG, and siTOX, were included on each individual assay plate. The results were normalized to the plate median for the SMARTpools. After excluding cytotoxic siRNAs based on detection of AK, primary screen data was subject to analysis. The primary screen, performed as two independent studies, was analyzed using a scaling methodology that sets the non-targeting control siRNA (siNEG) at an arbitrary value of 1.0, and the negative control siTOX at zero. siRNAs targeting host genes were assigned a score based on the distribution of these values. Wells in the primary screen with a percent of differentiation $|1.5| standard deviations from the plate mean in both duplicate assays were considered primary hits. Of the 481 HP genes targeted, twenty-four genes were identified as ‘‘primary hits’’. Hits that caused greater than 20% cytotoxicity in A549 cells were excluded (Figure 1A). Z-scores were computed based on this data for each gene hit and those $1.5s and #21.5s were considered for validation. Reverse transfection Lyophilized siRNAs in 96-well plates were diluted with HBSS (HyClone, Logan, UT) and allowed to incubate for 5 minutes. Dharmafect-1 transfection reagent (Lafayette, CO) and HBSS were added such that each well received 0.004 ml of transfection reagent and 0.096 ml of HBSS. The siRNA/transfection reagent mix was allowed to incubate for 20 minutes at room temperature after which 0.08 ml of 1.56104 A549 cells suspended in DMEM/ 5% FBS was added to each well, and the plate incubated for 48 hours at 37uC in 5% CO2. The final concentration of siRNA for all primary screen transfections was 50 nM. Cytotoxicity and virus infection To determine if siRNA gene silencing was cytotoxic, the cell supernatants from siRNA transfected A549 cells were analyzed for adenylate kinase (AK) using a Toxilight kit (Lonza, Rockland, ME). Results were normalized to a siTOX control, i.e. a siRNA control (Dharmacon) causing complete cell death by 48 hours. siRNA transfected cells with luminescence greater than or equal to 20% of the siTOX control were not considered for further evaluation. A549 cells were subsequently infected with A/WSN/ 33 at an MOI of 0.001 pfu/cell. Cells were incubated for 48 hours at 37uC/5% CO2. PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e37169