siRNA screen: For the siRNA screen we employed an arrayed library targeting 17,877 genes (Dharmacon siARRAY siRNA Library (Human Genome, G-005000-05, Thermo Fisher Scientific, Lafayette, CO). siRNAs were transiently reverse transfected into the U2OS cells in triplicate at a 50 nM final concentration, using a final concentration of 0.32% Oligofectamine (Invitrogen) in a 384-well format. After 72 h, the medium was removed and the cells were infected with the Influenza A/Puerto Rico/8/34 (PR8, ATCC VR-1469), MOI of ~ 0.2-0.3 in 40 uL complete media. After 12 h, media was removed, cells were fixed with 4% formalin and stained with anti-HA antibodies (Hybridoma HA36-4-5.2, Wistar Institute), followed by an Alexa Fluor 488 goat anti-mouse secondary at 1:1,000 (A11001, Invitrogen). Cells were imaged on an automated Image Express Micro (IXM) microscope (Molecular Devices) and analyzed using the Metamorph Cell Scoring software program (Molecular Devices Inc.). The validation round for single siRNAs was done as described previously (Brass et al., 2008
) with 20 nM siRNA. All Dharmacon siRNAs and plasmids used for generating stable cell lines are shown in Dataset S1 and the Supplemental Experimental Procedures
Cell lines and culture conditions: U2OS, A549, MDCK, 293T, Huh7.5.1, primary Chicken fibroblast cells (ChEFs, Charles River Labs), Vero E6 and HeLa cells were grown in DMEM (Invitrogen Cat#11965) with 10% FBS (Invitrogen). WI-38 cells were cultured in DMEM (Invitrogen Cat#10569), containing 1X MEM non-essential amino acids (Invitrogen Cat#11140, 10 mM stock/100×) and 15% FBS. Adult IfitmDel+/-
mice (Lange et al., 2008
) were intercrossed and fibroblasts (MEFs) derived from embryos at day 13.5 of gestation, as described previously (Nagy et al., 2003) and in the Supplemental Experimental Procedures
Viral propagation and titration: influenza A (H1N1) A/PR/8/34 (ATCC VR-1469), influenza A (H1N1) A/WS/33 (ATCC VR-1520), influenza A (H1N1) A/WSN/33 and influenza (H3N2) A/Udorn/72 were propagated and viral infectivity was titrated as previously described (Huang et al., 2008
). Hybrid Moloney/Amphotropic murine leukemia virus (MLV, ATCC VR-1450) was propagated in NIH3T3 cells. WNV (strain 2741) and DNV serotype 2 (New Guinea C strain) viruses were grown on Vero cells.
West Nile (WNV) and dengue (DNV) virus infections: West Nile (strain 2741) and dengue serotype 2 (New Guinea C strain) viruses were used to infect the IFITM3-silenced HeLa cells at an MOI of 0.1 for 24 or 30 h respectively, as reported previously (Krishnan et al., 2008
). Infected cells were fixed in 4% PFA and immuno-stained with antibodies detecting viral E-proteins (Chemicon), and imaged by fluorescence microscopy (Zeiss). IFITM3 over-expressing or vector control-A549 or -U20S cells were infected with WNV at an MOI of one.
Influenza A virus and MLV infection: Influenza A virus A/PR/8/34 (H1N1) (MOI =5), A/Udorn/72 (H3N2) (MOI=1), and MLV were used to infect A549 cells expressing different IFITM proteins. 24 h later, infected cells were labeled with murine anti-influenza viral H1 IgG2a (C179), anti-influenza viral H3 IgG1 (F49) (Takara Bio. Cat#M145 and M146), or goat anti-MLV env polyclonal antibodies (ATCC), and stained with PE-conjugated anti -mouse or anti-goat secondary antibodies. Cells were then fixed with 1% formaldehyde and analyzed by flow cytometry.
VLP and Pseudotyped Virus: MLV-GFP pseudoviruses were made as described (Huang et al., 2006
; Huang et al., 2008
). Flavivirus VLP were as described (Hanna et al., 2005), except plasmids encoding structure proteins of WNV (strain NY99), yellow fever virus (strain D17), or Omsk hemorrhagic fever virus (Ref. Seq.: NP_878909.1) were used. VLP and pseudovirus entry in A549 or Vero E6 cells expressing IFITM proteins was assessed 2 d later by measuring GFP expression by flow cytometry. The infection level of siRNA-transfected U2OS cells after 2 d infection was determined by calculating the percent GPF positive cells by IF after fixation with 4% PFA and staining of nuclei with Hoechst 33342.
Intracellular HA-staining was performed as above with the exception that after PFA fixation, cells were incubated in 0.1-0.2% Tween 20 (Sigma), then blocked in 1% BSA with 0.3M glycine in D-PBS, prior to staining with the primary antibody. This identical protocol was used with staining for NP (Abcam, AA5H, ab20343, 1:1000), M2 (Abcam, 14C2, ab5416, 1:1000), Anti-HA7 from Sigma-Aldrich (Product code H 3663, which recognizes the HA nonapeptide tag on IFITM3R6
, but not PR8's HA), and monoclonal antibody against Human Influenza A virus (H1N1, H2N2) (Takara, C179, Cat#M145, 1:1000), which recognizes the HA of WS/33, but not of PR8. Sialic acid staining has been described (Huang et al., 2006
; Huang et al., 2008
Statistical analysis of gene enrichment was performed using a hypergeometric distribution as decribed in the GOhyperGAll module of Bioconductor for gene ontology terms (Gentleman et al., 2004
). A map of the viral lifecycle was created by connected keywords. Genes were mapped to these keywords using a database that integrates annotation information from UniProt (Bairoch et al., 2005
), KEGG (Kanehisa et al., 2004
), Reactome (Vastrik et al., 2007
), Gene Ontology (Ashburner et al., 2000
), NCBI GeneRIF (Mitchell et al., 2003
) and OMIM Human orthologs were mapped to other species using NCBI HomoloGene (Wheeler et al., 2005
)(see Supplemental Experimental Procedures
for more details).