Human Genes Required for Influenza Replication Figure 1. RNAi of 5 host protease genes down-regulated influenza virus replication. A: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cytotoxicity was determined by adenylate kinase (AK) release. Cells treated with the siTOX control were considered 100% cytotoxic and all values were normalized to siTOX. RLU = relative luciferase units. * p,0.05 vs siNEG, ** p,0.01 vs siNEG, ***p,0.005 vs siNEG; siTOX vs all samples: p,0.001 (not shown). Line shows 20% of siTOX control. B: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. 48 hours post-infection, cells were fixed in 4% formaldehyde and stained with an anti-NP (green) monoclonal antibody followed by counterstain with DAPI (blue.) Positive (+) control: siMEK, negative (2) control: siNEG. Magnification is 206 (bar is 100 microns). C: Cells were transfected with 100 nM of a novel siRNA targeting a different seed site from the SMARTpool used in the primary screen and infected as in B. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID50. Data is expressed as TCID50/ml. Data is representative of two independent experiments. (*p,0.05 vs siNEG). doi:10.1371/journal.pone.0037169.g001 to variations in methodologies, viruses, and cell lines used among the studies [27,29,30,35]. Of the host genes known to affect influenza virus, the proteases are important for infection and replication. Proteases may affect virus infection and replication in several ways including viral entry and hemagglutinin (HA) processing [36,37,38,39], degradation of viral components for MHC presentation [40], cap-snatching [41], induction of apoptosis [42], and by increasing vascular perme- ability aiding in the development of systemic infection in cases of severe infection [43]. However, it remains unclear which protease genes are essential in the biology of influenza virus replication. In addition to host gene involvement during viral infection, the tempo of host gene expression may be altered by a variety of factors, such as by microRNAs (miRNA). Host miRNAs are similar to siRNAs in their silencing mechanism, but miRNAs are generated in the nucleus from short hairpin precursors and must be processed and exported before entering the RISC pathway [44]. miRNAs can be generated by the virus [45,46] or the host and can silence genes by similar mechanisms to siRNAs; however, unlike siRNA silencing, miRNA sequences do not need to be homologous to the target mRNA [44]. Little is known about the role of miRNAs during virus infection, however host-derived miRNAs have been shown to negatively affect influenza replica- tion [47] and miRNAs have even been used as therapeutics [48,49]. We considered that host miRNAs might also have a role in host gene modulation during influenza infection. To comprehensively determine the host proteases required by influenza virus for infection and replication, a siRNA screen of all known 481 protease genes was performed in human lung epithelial (A549) cells using the influenza virus, A/WSN/33. Twenty-four primary gene hits were found in the siRNA screen of which five (ADAMTS7, CPE, DPP3, MST1, and PRSS12) were validated as PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e37169