Endpoint assays Virus titers in siRNA-treated A549 cells infected with A/WSN/ 33 were determined by modified TCID50 or hemagglutination assay (HA). Briefly, virus infected A549 cell culture supernatants were serially diluted ten-fold, and added to MDCK cells. The MDCK cell plates were incubated for 72 h, followed by an HA using 0.5% chicken red blood cells as previously described [74]. To further identify gene hits the cells were screened for nucleoprotein (NP) by IHC staining and evaluated using high content analysis. For NP staining, the cells were fixed with 3.7% formaldehyde and stained with anti-NP monoclonal antibody (5 ug/ml; H16-L10-4R5) and the antibody staining detected using Alexa Fluor 488 labeled goat anti-mouse IgG (1 ug/ml; Invitro- gen, Carlsbad, CA). Cells were counterstained with DAPI (2 ug/ ml) (Invitrogen, Carlsbad, CA) and visualized by immunofluores- cent microscopy (206, EVOS digital inverted fluorescent micro- scope, Advanced Microscopy Group, Bothell, WA). As an additional hit identification endpoint, real-time qRT-PCR analysis was performed to quantify influenza M gene copy number in the cells as previously described [75]. Briefly, RNA was purified using the RNeasy kit (Qiagen, Valencia, CA), and cDNA was synthesized using a SuperScript First Strand cDNA synthesis kit (Invitrogen, Carlsbad, CA) and appropriate primer/probe set [75] to detect the M gene. PCR was performed using the amplification cycle: 10 minutes at 95uC followed by 40 cycles of 95uC for 30 seconds, 60uC for 1 minute, and 72uC for 30 seconds. M gene copies were normalized to the siNEG control. Validation of gene hits Individual novel siRNAs (Dharmacon) were used to target a different seed site on the same gene for gene hits identified during the primary screen (Table 1). Gene silencing was confirmed by qPCR using SybrGreen (Qiagen, Valencia, CA) to detect the dsDNA product allowing for quantification of gene silencing relative to cells treated with the siNEG control. Primers targeting each individual hit were compared to control GAPDH levels. For phenotype validation, A549 cells were transfected with 100 nM siRNA, incubated 48 h at 37uC in 5% CO2, infected with A/ WSN/33 (MOI = 0.001), and the amount of infectious virus was measured 48 hpi by TCID50 and NP staining assays to validate the screen phenotype previously observed. From the primary gene hits, five genes essential for influenza virus replication were validated using the novel siRNAs. The five genes were also tested using A/New Caledonia/20/99 at an MOI of 0.001 as described above; however infection was done in the presence of TPCK- trypsin (1 ug/ml). Western blot To determine protein levels in A549 cells treated with siRNA specific for the target protease genes, A549 cells were untreated, treated with 50 nM of the siNEG control, or treated with 50 nM of the appropriate siRNA. After 72 hours, cells were lysed in 1% TritonX-100 and proteins from total cell lysate were separated on a 4–20% Tris-HCl precast SDS PAGE gel (BioRad, Hercules, CA) and transferred to a PVDF membrane. Membranes were blocked in 5% BSA/TBS- 0.05% tween and incubated with 1 ug/ml rabbit polyclonal primary antibodies to ADAMTS7, CPE, DPP3, MST1, or PRSS12 (Abcam, Cambridge, MA). A goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (Invitrogen) was used to visualize the proteins. Human Genes Required for Influenza Replication Host cell pathway analysis To survey the spectrum of host cell pathways that may be linked to the validated gene hits, pathway analysis was performed using Ingenuity Pathway Analysis software (Ingenuity Systems, http:// www.ingenuity.com). The results provided network modeling of the validated genes and identified several functional groups (NF- kB, CRE/CREB signaling, apoptosis) linked to protease gene expression, host cell miRNA regulation, and influenza replication. Reporter system assays Reporter plasmids used to confirm pathway were obtained from SABiosciences/Qiagen as a dual luciferase Cignal Reporter Assay Kit. The reporters consisted of a transcription factor of interest linked to a firefly luciferase gene, a positive control plasmid (luciferase linked to a CMV promoter), and a negative control plasmid (luciferase with no promoter). A549 cells were co- transfected with 100 nM of siRNA and 100 ng of the appropriate plasmid (reporter, positive, or negative control plasmids) using either the SureFECT (SABiosciences) or Attractene (Qiagen) transfection reagents. As a transfection control, the plasmid kit also contains a Renilla luciferase plasmid fused to a CMV promoter, and all data was normalized to Renilla luciferase expression. After 24 h of transfection, cells were allowed to rest for one day and then infected with A/WSN/33 (MOI = 0.001) for 24 h. Cell lysate was taken and luciferase expression was measured using a Safire2 Microplate reader (Tecan, Ma¨nnedorf, Switzerland). Apoptosis array A human apoptosis array was obtained from SABiosciences/ Qiagen. A549 cells were transfected with 50 nM of either the appropriate siRNA or siNEG. After 48 h, cells were infected with A/WSN/33 at an MOI of 0.001. RNA was isolated 18 hpi, a time point chosen because it is late enough for apoptosis modulation to become evident at the transcriptional level, but sufficiently early such that cells were not destroyed [76], and then treated with DNase I to remove any genomic DNA. cDNA was synthesized using an RT2 First Strand Kit (SABiosciences) and PCR was performed using RT2 SYBR Green Master Mix (SABiosciences) per the manufacturer protocol. Data was analyzed by calculating 2(2DDCt). Silencing of the protease genes relative to siNEG-treated cells was confirmed for each independent experiment. miRNA studies A library of miRNA hairpin inhibitors synthesized as RNA oligonucleotides with novel secondary structure designed to inhibit the function of endogenous miRNA, and chemically enhanced to improve efficacy and longevity (miRIDIAN, Dharmacon, Lafay- ette, CO) were used to target host cell miRNAs identified as potential regulators of host genes validated as important for influenza virus replication. miRNA mimics were not used since treatment with mimics results in extremely high concentrations of miRNA relative to biological levels. In these studies, A549 cells (1.56104) were transfected with 25 nM of an appropriate miRNA inhibitor using Dharmafect-1 per the manufacturer’s protocol. After 48 h, the cells were infected with A/WSN/33 at an MOI of 0.001 for an additional 24 h or 48 h. Viral replication was assayed by TCID50 and qPCR for M gene as described above. Gene specific primers were also used to quantify changes in host gene expression in cells receiving the miRNA inhibitors using Sybr- Green as described above. PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e37169