A) U2OS cells were transfected with the indicated siRNAs, then infected with influenza A virus (PR8) and immuno-stained 12 hours later for hemaggutinin (HA, green: HA). NP, siRNA targeting flu nucleoprotein, C, Nontargeting siRNA negative control. Magnification, 4×.
B) Quantification of samples in A. Relative fold infection is normalized to non-targeting (C) control. Values represent the mean ± SD, N=4.
C) The results of the screen are shown with the siRNA SMARTpools ranked in order of average Z-score, from lowest (decreased infection) to highest (increased infection). The position of known influenza A virus-host factors and several newly identified genes from the screen are indicated.
D, E and F) U2OS cells were transfected with the indicated siRNAs for 72 h, and then infected with PR8. Twelve hours after infection the cells were analyzed by IF for the following viral proteins, HA (surface or entire cell), NP and M2. Relative fold infection is normalized to non-targeting (C) control. Values represent the mean ± SD, N =4.
G, H and I) Western blots for cells in D, E and F. C, Non-targeting siRNA negative control. Ran levels are provided to demonstrate relative protein loading when cross-reacting bands were not present.

Using the influenza A virus lifecycle as a guide (Lamb and Krug, 2001), the candidate proteins from the human and fly screens were placed at the position most likely to be relevant to the virus using a database of annotations from Gene Ontology, KEGG, Reactome and OMIM (see methods). Computational mapping and supporting evidences were reviewed and refined manually (Dataset S1C-F). The known molecular functions of the host factors were determined with the use of bioinformatics and multiple data sets (gray ovals). Host factors identified in the human siRNA screen (blue); the human orthologues of proteins identified in the fly-based screen (pink), factors which were found in both human and fly screens (green), and bridging proteins that were not detected, but none-the-less generate potentially insightful interactions (gray). Double borders signify the candidate is present in the Reactome influenza A virus infection pathway (Vastrik et al., 2007). Solid lines between genes indicate a protein interaction in human or other speicies. Dotted lines indicate inferred interaction from literature or annotation. Viral RNA (vRNA), viral complementary RNA (cRNA).

A) U2OS cells were transfected with the indicated siRNAs, then infected with PR8. Infection was assessed by IF for HA (surface or entire cell), NP or M2, 12 h after viral addition. Relative fold infection is normalized to non-targeting (C) control. Values represent the mean ± SD, N=3 throughout.
B) U2OS cells transfected with the indicated siRNAs were assessed for IFITM3 levels by Western blotting.
C) WI-38 human primary fibroblast cells stained for basal levels of IFITM3 expression (left panel), or after 24 h treatment with IFN-γ, or IFN-α (red: IFITM3, blue: nuclei), 63×.
D) U2OS cells stably expressing either IFITM3 with a C-terminal HA-epitope tag (IFTIM3-HA^6R) lacking the target site for siRNA IFITM3-6, or the vector alone, were transfected with the indicated siRNAs (x-axis). After 72 h the cells were incubated without (no virus) or with influenza A (PR8) for 12 h, then stained for HA expression. The anti-HA antibody used to detect infection does not recognize the HA epitope tag on IFITM3-HA^6R (no virus, uninfected control). C, non-targeting siRNA negative control.
E) U2OS cells stably expressing either IFTIM3-HA^6R or vector alone were transfected with the indicated siRNAs and assessed 72 h after transfection by Western blot with the indicated antibodies.
F) U2OS cells were untreated (-), or incubated with either IFN-γ, or IFN-α. After 24 h the levels of IFITM1, 2 or 3 were checked by Western blot.
G) U2OS cells were transfected with the indicated siRNAs, then left untreated or incubated with IFN-γ 48 h later. After 24 h of IFN incubation, the cells were infected with increasing amounts of PR8. Twelve h after infection the cells were stained for HA expression.
H) IFITM3 is required for the anti-viral effect of IFN-γ. U2OS cells stably expressing either IFTIM3^6R or vector, were transfected with the indicated siRNAs (x-axis) and treated with IFN-γ 48 h later. After 24 h, cells were incubated without or with influenza A (PR8) and 12 h after infection cells were checked for HA surface expression.
I) IFITM3 loss enhances infection by the H1N1 strain, WS/33. U20S cells were transfected with the indicated siRNAs for 72 h, infected with WS/33 for 12 h, and stained for HA expression (green, blue: nuclei). Numbers indicate percent infection normalized to control /± SD, N=3.

A) A549 cells were transduced with retroviruses containing C9-tagged cDNAs for the indicated IFITM proteins, or empty viral vector alone. After 2 d cells were infected with one of the following viruses: PR8 [H1(PR)], influenza A virus A/Udorn/72 (H3N2) [H3(Udorn)], or Moloney murine leukemia virus (MLV). 24 h after infection, cells were checked for HA surface expression by flow cytometry. Values represent the mean ± SD, N=3.
B) The expression of IFITM proteins in (A) was checked by Western using anti-C9 antibody. β-actin levels show protein loading.
C) A549 or U2OS cells stably over-expressing IFITM3 protein or vector alone, were infected with influenza A H1N1 WSN/33. 12 h. later, cells were fixed and stained for surface HA expression. Values represent the mean /± SD, N=3. (green: HA, blue: nuclei. 4×).
D) A549 and U2OS cells stably over-expressing IFITM3 were tested for expression by Western.
E) Primary Chicken fibroblast cells (ChEFs) stably over-expressing IFITM3 protein or vector alone, were infected with influenza A H1N1 WSN/33. 12 h. later, cells were fixed and stained for surface HA expression. Values represent the mean /± SD, N=3. (red: HA, blue: nuclei. 4×).
F) ChEF cells stably over-expressing IFITM3 were tested for expression by Western.
G) MDCK cells stably over-expressing IFITM3 protein or vector alone, were infected with influenza A H1N1 WSN/33 at a multiplicity of infection (moi) of 0.005. 72 h. later, cells were washed with fresh media, and then imaged live to assess cytopathic effect. Bright field images shown are representative of 4 independent experiments (10×).
H) MDCK cells stably over-expressing IFITM3 were tested for expression by Western.
I) A549 cells were transduced with retroviruses containing the indicated IFITM proteins, or empty vector. 48 h later, the cells were incubated with MLV-EGFP virus pseudotyped with the indicated envelope proteins. HA proteins from various influenza A virus strain including H1 (PR): A/PR/8/34 (H1N1), H3 (Udorn): A/Udorn/72 (H3N2), H5(Thai): A/Thailand2(SP-33)/2004 (H5N1), H7(FPV): A/FPV/Rostock/34 (H7N1), VSV-G: VSV G protein, MLV: MLV env protein, or MACH: Machupo virus glycoprotein. Viral entry is expressed as mean EGFP fluorescence relative to vector control cells, as measured by flow cytometry. Values represent the mean ± SD, N=3.
J) U2OS cells transfected with the indicated siRNAs for 72 h, were then incubated with MLV-GFP virus pseudotyped with the VSV-G or the HA protein of PR8, H1(PR). Entry, represented as percent green fluorescing cells relative to mock-transfected cells, was determined by IF microscopy two days post-infection. Values represent the mean ± SD, N=4. C, Non-targeting siRNA negative control.
K) A549 cells were transduced with the indicated retroviruses. 48 h later, the cells were tested for surface expression of sialic acid (SA). Values represent the mean ± SD, N=3.

A) MEFs derived from the indicated IfitmDel mice, were left untreated (buffer), or treated with interferon-α or γ. After 24 h, the cells were incubated with influenza A virus H1N1 (PR8). Twelve h after infection, the cells were checked for HA surface expression. Values represent the mean ± SD, N=3.
B) MEFs from A) were assessed by Western blot for the presence of Ifitm3 protein. GAPDH levels are provided to show protein loading.
C) MEFs were left untreated (buffer), or incubated with either IFN-γ, IFN-α, or PR8 virus. After 24 h the levels of Ifitm3 were checked by Western blot.
D) IfitmDel +/+ MEFs, or IfitmDel -/- MEFs were incubated in the absence (buffer) or presence of IFN-α for 24 h, prior to staining with α-Ifitm3 (red: Ifitm3, nuclei: blue). 63×.
E) IfitmDel +/+ MEFs, or IfitmDel -/- MEFs stably expressing Ifitm2, 3 or the empty vector, were challenged with PR8 virus. 12 h later, the cells were fixed and imaged for HA expression. Values represent the mean ± SD, N=3.
F) The indicated MEFS were assessed for Ifitm2 and 3 expression by Western blot. GAPDH demonstrates protein loading.

A) Vero E6 cells were transduced with retroviruses expressing the indicated IFITM proteins, or the empty viral vector. Two days later, the cells were incubated with flaviviral viral like particles (VLPs), expressing EGFP, and coated in envelope proteins from WNV, yellow fever virus (YFV) or Omsk virus (OMSK), or with EGFP-expressing MLV viruses pseudotyped with the indicated viral envelope proteins. Viral infection is expressed as mean EGFP fluorescence relative to vector control cells, as measured by flow cytometry 48 h post-infection. Values represent the mean ± SD, N=3.
B) A549 or U2OS cells stably expressing either IFITM3 protein or the vector alone (also shown in 4C, D), were infected with infectious WNV (strain 2741). 24 h. later, the cells were fixed and stained for viral E protein expression by IF. Values represent the mean ± SD, N=3
C) Images of A549 cells in B (red: WNV E protein, blue: nuclei), 4× magnification.
D) HeLa cells were transfected with the indicated siRNAs for 72 h, then infected with WNV. 24 h later, the cells were fixed and stained for viral E protein. Values represent the mean ± SD, N=3. C, Non-targeting siRNA negative control.
E) Images of HeLa cells in D (red: WNV E protein, blue: nuclei), 4× magnification.
F) HeLa cells were transfected with the indicated siRNAs for 72 h, then infected with dengue virus (New Guinea C strain). 30 h. post-infection, the cells were fixed and stained for viral E protein expression by IF. Values represent the mean ± SD, N=3. C, Non-targeting siRNA negative control.
G) Images of HeLa cells in F, 4×.

A schematic model of the influenza A virus life cycle, the induction of IFITM proteins and their role in blocking Influenza A virus infection. IFITM1, 2 and 3 are represented by the three multi-transmembrane proteins. Red legends indicate possible mechanisms of restriction, IFITM proteins may; A) sequester the incoming viruses at or near the surface, B) block viral receptors from interacting with host receptors (shown in orange), C) prevent endocytosis or viral membrane fusion D) act as receptors, and after binding the virus, signal to effectors.