Statistics The primary siRNA protease gene screen was performed independently and in duplicate. HA results for each gene were assigned values 0–8 based on the dilution of the HA readout. HA values were normalized to the average of the siNEG control readout per plate. Robust z-scores (Z = xi2x/sx) were used as the normalizing method and calculated for each gene to determine hits based upon the standard deviation where xi is the raw measurement on the ith siRNA, and sx are the mean and the standard deviation, respectively, of all measurements within the plate. Normalization of raw data removes systematic plate-to-plate variation, making measurements comparable across plates. Statis- tical analyses for the cross-strain validation studies, the reporter studies, and miRNA studies were performed using GraphPad Prism software using the Mann-Whitney U test. Supporting Information Figure S1 Calculated standard deviation of the z-scores of the human protease library. The primary screen provided two independent studies and 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 greater than 1.5 standard deviations above the plate mean in both duplicates were considered primary hits. Of the 481 HP genes targeted, 24 were genes were identified as ‘‘primary hits’’. (TIF) Figure S2 siRNA treatment decreases protein levels of the target protease genes. A: A549 cells were untreated (A549), treated with 50 nM of siNEG, or 50 nM of the appropriate siRNA. After 72 hours, cells were lysed in 1% TritonX-100 and protease gene levels were assessed by Western References 1. Kandel R, Hartshorn KL (2001) Prophylaxis and treatment of influenza virus infection. BioDrugs 15: 303–323. 2. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, et al. (2003) Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA 289: 179–186. 3. Hale BG, Randall RE, Ortin J, Jackson D (2008) The multifunctional NS1 protein of influenza A viruses. J Gen Virol 89: 2359–2376. 4. Basler CF (2007) Influenza viruses: basic biology and potential drug targets. Infect Disord Drug Targets 7: 282–293. 5. Betakova T (2007) M2 protein-a proton channel of influenza A virus. Curr Pharm Des 13: 3231–3235. 6. Hsieh HP, Hsu JT (2007) Strategies of development of antiviral agents directed against influenza virus replication. Curr Pharm Des 13: 3531–3542. 7. Jefferson T, Demicheli V, Di Pietrantonj C, Rivetti D (2006) Amantadine and rimantadine for influenza A in adults. Cochrane Database Syst Rev. CD001169 8. Lowen AC, Palese P (2007) Influenza virus transmission: basic science and implications for the use of antiviral drugs during a pandemic. Infect Disord Drug Targets 7: 318–328. 9. Matheson NJ, Harnden AR, Perera R, Sheikh A, Symmonds-Abrahams M (2007) Neuraminidase inhibitors for preventing and treating influenza in children. Cochrane Database Syst Rev. CD002744 10. Beigel J, Bray M (2008) Current and future antiviral therapy of severe seasonal and avian influenza. Antiviral Res 78: 91–102. 11. Conly J, Johnston B (2006) Ode to oseltamivir and amantadine? Can J Infect Dis Med Microbiol 17: 11–14. 12. Lackenby A, Thompson CI, Democratis J (2008) The potential impact of neuraminidase inhibitor resistant influenza. Curr Opin Infect Dis 21: 626–638. 13. Krishnan MN, Ng A, Sukumaran B, Gilfoy FD, Uchil PD, et al. (2008) RNA interference screen for human genes associated with West Nile virus infection. Nature 455: 242–245. 14. Hirsch AJ (2010) The use of RNAi-based screens to identify host proteins involved in viral replication. Future Microbiol 5: 303–311. 15. Leung RK, Whittaker PA (2005) RNA interference: from gene silencing to gene- specific therapeutics. Pharmacol Ther 107: 222–239. Human Genes Required for Influenza Replication blot. B: Protein levels of the protease genes were determined by densitometry. Band density is shown as a percentage of the untreated A549 cells. (TIF) Figure S3 An Ingenuity pathway analysis linked global cellular pathways and the host protease genes of interest. Hit protease genes (ADAMTS7, CPE, DPP3, MST1, and PRSS12) are shown in red and relevant influenza proteins are shown in green. Nodes indicating global cellular pathways linked with the hit genes are shown in yellow (CREB, NF-kB, caspases). (TIF) Figure S4 miRNAs interact with host protease genes. An Ingenuity pathway analysis implicated several miRNAs connected with host protease genes of interest. Hit protease genes are shown in red and miRNAs are shown in green. (TIF) Table S1 Expression ratios for pro- and anti-apoptotic genes in A549 cells treated with siRNA for protease gene targetsa,b. aGene expression in A549 cells treated with 50 nM of the appropriate siRNA was compared to cells treated with 50 nM of siNEG. The experiment was performed as described in Figure 4. bGene expression was normalized to GAPDH. *p,0.05. (DOCX) Acknowledgments The authors wish to thank Drs. Peter Ghazal, Juergen Haas, and Amy Buck for helpful discussions and host gene pathway analysis. Author Contributions Conceived and designed the experiments: VAM LEA AB SMT RAT. Performed the experiments: VAM LEA PB XY JKC. Analyzed the data: VAM LEA RAT. Contributed reagents/materials/analysis tools: SMT RAT. Wrote the paper: VAM RAT. 16. Tripp RA, Tompkins SM (2009) Therapeutic applications of RNAi for silencing virus replication. Methods Mol Biol 555: 43–61. 17. Haasnoot J, Westerhout EM, Berkhout B (2007) RNA interference against viruses: strike and counterstrike. Nat Biotechnol 25: 1435–1443. 18. Kim DH, Rossi JJ (2007) Strategies for silencing human disease using RNA interference. Nat Rev Genet 8: 173–184. 19. Wiznerowicz M, Szulc J, Trono D (2006) Tuning silence: conditional systems for RNA interference. Nat Methods 3: 682–688. 20. Alvarez R, Elbashir S, Borland T, Toudjarska I, Hadwiger P, et al. (2009) RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy. Antimicrob Agents Chemother 53: 3952–3962. 21. Barik S (2004) Control of nonsegmented negative-strand RNA virus replication by siRNA. Virus Res 102: 27–35. 22. Bitko V, Musiyenko A, Shulyayeva O, Barik S (2005) Inhibition of respiratory viruses by nasally administered siRNA. Nat Med 11: 50–55. 23. DeVincenzo J, Cehelsky JE, Alvarez R, Elbashir S, Harborth J, et al. (2008) Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antiviral Res 77: 225–231. 24. Zhang W, Tripp RA (2008) RNA interference inhibits respiratory syncytial virus replication and disease pathogenesis without inhibiting priming of the memory immune response. J Virol 82: 12221–12231. 25. Zhang W, Yang H, Kong X, Mohapatra S, San Juan-Vergara H, et al. (2005) Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nat Med 11: 56–62. 26. Barakat MR, Kaiser PK (2009) VEGF inhibitors for the treatment of neovascular age-related macular degeneration. Expert Opin Investig Drugs 18: 637–646. 27. Konig R, Stertz S, Zhou Y, Inoue A, Hoffmann HH, et al. (2009) Human host factors required for influenza virus replication. Nature 463: 813–817. 28. Pan HY, Yamada H, Chida J, Wang S, Yano M, et al. (2011) Up-regulation of ectopic trypsins in the myocardium by influenza A virus infection triggers acute myocarditis. Cardiovascular research 89: 595–603. PLoS ONE | www.plosone.org 10 May 2012 | Volume 7 | Issue 5 | e37169