The IFITM Proteins Mediate Cellular
Resistance to Influenza A H1N1 Virus,
West Nile Virus, and Dengue Virus
Abraham L. Brass,1,2,4,9,* I-Chueh Huang,5,9Yair Benita,3,10Sinu P. John,1,10Manoj N. Krishnan,6Eric M. Feeley,1
Bethany J. Ryan,1Jessica L. Weyer,5Louise van der Weyden,8Erol Fikrig,6,7David J. Adams,8Ramnik J. Xavier,2,3
Michael Farzan,5,* and Stephen J. Elledge4,*
1Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard Medical School,
Charlestown, MA 02129, USA
3Center for Computational and Integrative Biology
Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
4Department of Genetics, Harvard Medical School, Division of Genetics, Brigham and Women’s Hospital,
Howard Hughes Medical Institute, Boston, MA 02115, USA
5Department of Microbiology and Molecular Genetics, Harvard Medical School, New England Primate Research Center,
Southborough, MA 01772, USA
6Section of Infectious Diseases, Department of Internal Medicine
7Howard Hughes Medical Institute
Yale University School of Medicine, New Haven, CT 06520, USA
8Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton Cambridge CB10 1SA, UK
9These authors contributed equally to this work
10These authors contributed equally to this work
*Correspondence: firstname.lastname@example.org (A.L.B.), email@example.com (M.F.), firstname.lastname@example.org (S.J.E.)
Influenza viruses exploit host cell machinery to repli-
cate, resulting in epidemics of respiratory illness. In
turn, the host expresses antiviral restriction factors
to defend against infection. To find host cell modi-
fiers of influenza A H1N1 viral infection, we used
a functional genomic screen and identified over 120
influenza A virus-dependency factors with roles in
endosomal acidification, vesicular trafficking, mito-
chondrial metabolism, and RNA splicing. We discov-
ered that the interferon-inducible transmembrane
enza A viral replication. The IFITM proteins confer
basal resistance to influenza A virus but are also
inducible by interferons type I and II and are critical
for interferon’s virustatic actions. Further character-
ization revealed that the IFITM proteins inhibit the
early replication of flaviviruses, including dengue
virus and West Nile virus. Collectively this work
identifies a family of antiviral restriction factors that
mediate cellular innate immunity to at least three
major human pathogens.
Influenza epidemics exact a formidable toll on world health.
Moreover, viral super-infections can produce antigenic shifting,
resulting in more virulent pathogens (Monto, 2009). At present,
the emergence of a novel influenza A H1N1 viral strain has
created a pandemic, producing illness in over 200 countries
and territories (World Health Organization Pandemic [H1N1]
strain, H5N1, represents a potentially catastrophic global health
risk (Maines et al., 2008).
The influenza A viral genome encodes for 11 proteins and
consists of 8 segments of negative single-stranded RNA
(Lamb and Krug, 2001). Each subgenomic segment is coated
by viral nucleoprotein (NP) and bound to a single viral RNA-
dependent RNA-polymerase holoenzyme (RdRp), composed
of PA, PB1, and PB2 subunits. Infection begins with the binding
of the viral hemagglutinin (HA) protein to sialyated host cell-
surface glycoproteins (Skehel and Wiley, 1995). Following endo-
cytosis, viral particles are trafficked through both early and late
endosomes, with the acidification of the latter compartment
altering the conformation of HA, leading to host-viral membrane
fusion, entry of the viral ribonucleoproteins (vRNPs) into the
cytosol (Sieczkarski and Whittaker, 2003), and nuclear import.
Once in the nucleus, the RdRp commandeers 50caps from
host mRNAs to prime transcription of viral mRNA (vmRNA; Bou-
loy et al., 1978) and creates positive sense complementary RNA
(cRNA) from which it makes new viral genomes (vRNAs). The
vRNAs are coated by NP and exported though the nuclear pore
complex (NPC) by the viral factors M1 and NEP/NS2 (nuclear
export protein) working in concert with the host nuclear export
machinery. The viral envelope proteins HA, M2, and neuramini-
dase (NA) are translated on the rough endoplasmic reticulum
(ER) and trafficked to the cell surface where they, along with
Cell 139, 1243–1254, December 24, 2009 ª2009 Elsevier Inc. 1243
the soluble factors M1, RdRp, and eight distinct vRNPs, are
packaged into budding virions.
To defend against infection, the host mobilizes factors to
confront the virus. Interferons (IFN) orchestrate a large compo-
nent of this antiviral response (Takaoka and Yanai, 2006). Over
2000 gene products are induced after IFN stimulation, including
the antiviral effectors MxA, PKR, RIG-I, and 2050-OAS (Haller
et al., 2009; Nakhaei et al., 2009; Takaoka and Yanai, 2006).
for influenza A virus are primarily enacted by the viral protein
NS1 (Hale et al., 2008). To identify host factors that modify viral
replication we undertook an siRNA screen. In this screen we
identified over 120 host factors required for influenza A replica-
for the viral life cycle as well as potential antiviral drug targets.
Our studies also revealed a family of antiviral factors, the IFITM
proteins, that mediate interferon’s innate immune role in
combating not only all pathogenic influenza A virus strains
examined but also West Nile virus and dengue virus.
An siRNA Screen for Influenza A Virus
Infection-Modifying Host Factors
We used a single round infection screen of osteosarcoma cells
(U2OS) to find host proteins that modify the life cycle of influenza
A virus A/Puerto Rico/8/34 H1N1 (PR8). After 12 hr the cells were
stained for surface expression of HA as an indirect surrogate
marker for viral infection (Figure 1A). Although measuring viral
protein levels is an indirect measurement of viral replication, in
three previous viral screens where we measured HIV, West
Nile virus, and HCV replication by immunofluorescence and by
titering, the viral protein reduction strongly correlated with
reduction in viral titers. Thus we feel that this indirect assay
can provide an accurate measurement of viral impairment. This
approach detects viral-host receptor binding, endocytosis and
fusion of the virion, vRNP trafficking and nuclear import, the
transcription, nuclear export, and translation of the viral HA
mRNA, and the trafficking of HA to the surface. The screen
Figure 1. The siRNA Screen for Influenza A Virus Infection-Modifying Host Factors
(A) U2OS cells were transfected with the indicated siRNAs, then infected with influenza A virus (PR8) and immunostained 12 hr later for hemaggutinin (HA, green).
NP, siRNA targeting flu nucleoprotein; C, nontargeting siRNA negative control. Magnification, 43.
(B) Quantification of samples in (A). Relative fold infection is normalized to nontargeting (C) control. Values represent the mean ± standard deviation (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–F) U2OS cells were transfected with the indicated siRNAs for 72 hr, 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 nontargeting (C) control. Values represent the mean ± SD,
n = 4.
(G–I) Western blots for cells in (D)–(F). C, nontargeting siRNA negative control. Ran levels are provided to demonstrate relative protein loading when cross-
reacting bands were not present.
1244 Cell 139, 1243–1254, December 24, 2009 ª2009 Elsevier Inc.
was optimized using siRNAs against NP and the host factor
NXF1, an mRNA exporter required for virus replication (Ge
et al., 2003; Hao et al., 2008). siRNAs against either NP or
NXF1 resulted in inhibition of infection (NXF1 10-fold, NP 4- to
6-fold, Figures 1A and 1B and Figure S1A available online).
We screened the Dharmacon siRNA library in triplicate. siRNA
pools were selected for further evaluation if the percentage of
HA-positive cells was less than 55% of the plate mean, and
cell numbers were not less than 40% of the plate mean. These
criteria were fulfilled by 312 pools (1.7% of the total genes
screened, Figure 1C). Pools that increased HA expression
>200% of the plate mean were also selected for validation
(22 pools, 0.1%). We next rescreened the four unique siRNAs
from each pool separately. In this screen, 260 out of 334 total
pools confirmed with at least one siRNA scoring and 133
confirmed with two or more siRNAs (40%), reducing the proba-
bility of off-target effects (Datasets S1A and S1B). We employed
this gene set (Dataset S1C). Ninety-two gene ontology (GO) bio-
logical process terms, assigned to 109 genes, were significantly
enriched (Dataset S2). Of these, 17 are nonredundant and as-
signed to less than 500 genes, suggesting that they are informa-
tive and specific. The most significant terms include RNA
splicing (22 genes, p = 2e-12), proton transport (7 genes,
p = 2e-5), and mRNA transport (4 genes, p = 9e-3, Figure S1B).
Analysis of GO molecular functions identified enrichment for
60 statistically significant terms assigned to 152 genes. Twelve
terms were nonredundant and assigned to less than 500 human
genes. The most significant terms include RNA binding
(15 genes, p = 0.014), ATPase activity (6 genes, p = 0.008),
and NADH dehydrogenase activity (4 genes, p = 0.016).
Multiple biological pathways and complexes were also de-
tected, concordant with known elements of the viral life cycle
(Figure 2). Influenza A viral infection depends on sialic acid (SA)
residues on the host cell surface, and we found that depletion
Figure 2. Integrated Model of Influenza A Virus Host Factors
Using the influenza A virus life cycle 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 Experimental Procedures). Computa-
tional mapping and supporting evidences were reviewed and refined manually (Datasets S1C–S1F). The known molecular functions of the host factors were
determined with the use of bioinformatics and multiple datasets (gray ovals). Host factors identified in the human siRNA screen (blue), the human orthologs
of proteins identified in the fly-based screen (pink), factors that 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) are shown. Double borders signify that 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 species. Dotted lines indicate inferred
interaction from literature or annotation. Viral RNA (vRNA), viral complementary RNA (cRNA).
Cell 139, 1243–1254, December 24, 2009 ª2009 Elsevier Inc. 1245
confirmed the functional role of two small GTPases, RAB5A
(surface internalization to early endosome trafficking) and
RAB7L1 (late endosome trafficking), for viral infection (Sieczkar-
ski and Whittaker, 2003). We also found that lowering RAB10
levels inhibited infection (Hao et al., 2008). RAB10 regulates
the movement of endosomes generated from endocytosis
downstream of RAB5 (Glodowski et al., 2007). Loss of each of
four subunits of the multimeric vacuolar-ATPase proton pump
(e.g., ATP6AP1, ATP6V0B, ATP6V1G1, ATP6V0E2) impeded
infection, consistent with the low pH needed for fusion. Once
released from the endosome, the vRNPs are transported into
the nucleus though the NPC (Boulo et al., 2007), and multiple
NPC factors were found in the screen.
Several splicing complexes were needed for viral HA pro-
tein surface expression, including three components of the U2
smallnuclear RNP(snRNP), SF3B1,2,and 3,andtheU2 snRNP-
interacting proteins, PRPF8, PTBP1, and FUS (Figure 2; Data-
sets S1A and S1C). The U4/U6.U5 tri-snRNP, including SART1,
was also required (Stevens et al., 2001). Four out of four siRNAs
targeting SART1 resulted in lower levels of HA (surface-ex-
pressed and total protein), NP, and M2 proteins (Figures 1D
and 1G). The decreased levels of all thee viral proteins, products
of bothspliced (M2)and unspliced(HA,NP)messages, suggests
a general block in viral protein production with loss of SART1,
perhaps secondary to effects on host protein expression.
The vesicular transport complex, coatomer 1 (COP1), scored
with multiple components (p value = 1e-7). COPI directs both
retrograde intra-Golgi and Golgi-to-ER transport (Cai et al.,
2007). Depletion of six of seven components of COPI (ARCN1,
COPA, COPB1, COPB2, COPG, and COPZ1) inhibited HA
surface expression, perhaps by interfering with secretion of the
host cell receptor(s) and/or trafficking of HA protein to the cell
surface. Although COPB1 siRNAs decreased NP and M2 protein
levels, they had a greater effect on surface-expressed versus
was partly responsible for the phenotype (Figures 1E and 1H).
CALCOCO2 (NDP52) was also required for infection (Figures
1F and 1I). CALCOCO2 localizes to the Golgi and interacts
with the host proteins, TR6BP and Myosin VI, and may function
in regulating secretion (Morriswood et al., 2007).
Identification of IFITM3 as an Influenza A Virus
In the validation round, the depletion of four genes, interferon-
inducible transmembrane protein 3 (IFITM3), PUSL1, TPST1,
and WDR33, resulted in increased viral infection with two or
more siRNAs (Dataset S1B). We focused on IFITM3 because of
its link to interferon (Friedman et al., 1984). Eight out of eleven
distinct siRNAs targeting IFITM3 increased infection, with the
levels of knockdown correlating with the phenotype (Figures
tion was also observed in primary lung fibroblasts after IFITM3
depletion (Figure S3B) and in HeLa cells (Figures S3C and
S3E), with newly budded virus from HeLa cells increased >5
fold in titering assays (Figure S3D). Lowering IFITM3 levels simi-
larly increased infection by the influenza A H1N1 viral strain WS/
33 (Figure 3I) but had no effect on HIV infection (Figure S3F).
Stable expression of a C-terminal HA-tagged protein, IFITM3-
HA6R, lacking the 30-untranslated region targeted by siRNA
IFITM3-6 rescued resistance to the virus (Figures 3D and 3E).
Thus, IFITM3 is required for basal levels of cellular resistance
to influenza A virus infection.
The mRNAs for IFITM3, and the closely related and linked
genes IFITM1 and 2 (70% and 91% amino acid identity, respec-
tively, Figure S6), are inducible by both IFN types I (a) and II (g)
(Friedman et al., 1984; Lewin et al., 1991), which we confirmed
by immonofluorescence (IF) and western blot (Figures 3C and
3F). In unstimulated cells, the majority of IFITM3 resides in the
ER (based on colocalization with SA and N-acetylglucosamine-
conjugated proteins stained by wheat germ agglutinin, WGA;
Figure S3G). IFN exposure, in contrast, triggers the distribution
of IFITM3 in a vesicular pattern throughout the cell (Figures 3C,
S3H, S3I, and S5D).
IFITM3 Is Required for Interferon’s Antiviral Activity
against Influenza A Virus
In view of these dynamic changes, we examined IFITM30s func-
tional role in the IFN response. Either IFN-a or -g strongly
decreased basal levels of influenza A virus infection in both
restored with the stable expression of IFITM3-HA6R(Figure 3H).
Thus we conclude that IFITM3 is required both for basal levels of
resistance as well as for the heightened defenses elicited by
IFN-g and -a.
IFITM1, IFITM2, and IFITM3 Inhibit the Early
Replication of Influenza A Virus
We then tested if overexpression of IFITM3, or its paralogs,
IFITM1 and 2, could alter viral infection. A549 lung epithelial cells
were transduced with retroviruses expressing IFITM1, 2, or 3.
Two days later, the transduced cells showed increased resis-
tance to infection with influenza A viruses PR8 (H1[PR]) or
H3N2 A/Udorn/72 (H3 [Udorn], Figures 4A and 4B). Profound
restriction was also seen when IFITM3 was stably overex-
pressed in either A549, U2OS, or primary chicken fibroblast cells
of IFITM3 in a canine cell line used for propagating influenza A
viruses (Madin-Darby canine kidney [MDCK] cells) strongly in-
hibited the cytophathic effect of sequential rounds of viral infec-
tion (Figures 4G and 4H) and also blocked infection by two
current seasonal vaccine strains, A/Brisbane/59/07 H1N1 and
A/Uruguay/716/07 H3N2, and by A/Aichi/2/68 H3N2, a viral
isolate from the Hong Kong flu pandemic of 1968 (Figure S4D).
This restriction was not universal because IFITM proteins did
not inhibit Moloney Leukemia virus (MLV, amphotropic enve-
lope, Figure 4A).
To address where in the life cycle the block was occurring, we
used viralpseudoparticles. The pseudoparticles each contain an
MLV genome encoding the enhanced green fluorescence
protein (EGFP); however, each strain is uniquely coated with
theenvelope proteins fromone of thefollowing viruses: influenza
A virus (strains H1, H3, H5, H7), Machupo virus (MACH), or MLV
(Figure4I).Overexpression ofeachofthe IFITMproteins blocked
infection by all four influenza A enveloped pseudoviruses, with
1246 Cell 139, 1243–1254, December 24, 2009 ª2009 Elsevier Inc.
less restriction seen against VSV-G protein, and none against
MLV (g-retrovirus) or MACH (arenavirus) envelope proteins
(Figures 4I, S4E, and S4F).
To complement these gain-of-function results, we depleted
IFITM3 in U2OS cells, then infected with pseudoparticles ex-
pressing either influenza A virus envelope (H1[PR]) or VSV-G
(Figure 4J). Decreased IFITM3 levels increased infection of the
influenza A H1 pseudoviruses, with VSV-G entry elevated to
a lesser extent only with the most potent siRNA, IFITM3-6 (Fig-
ure 4J). Because the life cycles of the pseudoviruses differ only
in the means of entry mediated by their respective viral enve-
lopes, these data are consistent with the IFITM proteins blocking
influenza A virus infection early in the viral life cycle, somewhere
between and including viral-host receptor binding and entry of
the vRNP into the cytosol.
Influenza A virus infection begins with the viral envelope
proteins interacting with sialylated glycoproteins on the host
cell’s surface (Lamb and Krug, 2001). We found no reduction,
and even a slight increase, in the levels of SA with IFITM proteins
overexpression, pointing away from a reduction in SA underlying
the actions of the IFITM proteins (Figure 4K). When the trans-
duced cells were examined by flow cytometry, the N-terminal
Figure 3. IFITM3 Silencing Increases Influenza A Virus Infection and Is Required for the Antiviral Actions of Interferon
(A) U2OS cells were transfected with the indicated siRNAs, then infected with PR8. Infection was assessed 12 hr after viral addition by IF for HA (surface or entire
cell), NP, or M2. Relative fold infection is normalized to nontargeting control, C. 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 hr treatment with IFN-g or IFN-a (red: IFITM3, blue:
(D)U2OScells stably expressing eitherIFITM3 with aC-terminal HA-epitope tag (IFTIM3-HA6R)lacking thetarget site for siRNAIFITM3-6or the vector alone were
transfected withthe indicated siRNAs (xaxis). After 72 hrthe cells were incubated without(no virus)or with influenza A (PR8) for 12hr,thenstained for HA expres-
sion. The anti-HA antibody used to detect infection does not recognize the HA-epitope tag on IFITM3-HA6R(no virus, uninfected control). C, nontargeting siRNA
negative control. Values represent the mean ± SD, n = 3.
(E) U2OS cells stably expressing either IFTIM3-HA6Ror vector alone were transfected with the indicated siRNAs and assessed 72 hr after transfection by western
blot with the indicated antibodies.
(F) U2OS cells were untreated (?) or incubated with either IFN-g or IFN-a. After 24 hr, 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-g 48 hr later. After 24 hr of IFN incubation, the cells were
infected with increasing amounts of PR8. Twelve hours after infection the cells were stained for HA expression. Values represent the mean ± SD, n = 3.
(H) IFITM3 is required for the antiviral effect of IFN-g. U2OS cells stably expressing either IFITM3-HA6Ror vector were transfected with the indicated siRNAs (x
axis) and treated with IFN-g 48 hr later. After 24 hr, cells were incubated without or with influenza A (PR8), and 12 hr after infection cells were checked for HA
surface expression. Values represent the mean ± SD, n = 3.
(I) IFITM3 loss enhances infection by the H1N1 strain WS/33. U20S cells were transfected with the indicated siRNAs for 72 hr, infected with WS/33 for 12 hr, and
stained for HA expression (green, blue: nuclei). Relative fold infection is normalized to nontargeting control. Values represent the mean ± SD, n = 3.
Cell 139, 1243–1254, December 24, 2009 ª2009 Elsevier Inc. 1247
epitope tag was bound by antibody without membrane permea-
bilization, revealing that the N terminus is extracellular (Fig-
ure S5A). In addition, the C terminus could also be detected in
IF studies using nonpermeabilized cells, demonstrating that it
is also extracellular (Figure S5B, lower right panel).
Deletion of the Murine Ifitm Locus Leads to Increased
Influenza A Virus Infection In Vitro
Human and murine IFITM proteins display a high degree of inter-
species homology (Figure S6). Thus, to examine the evolutionary
conservation of IFITM protein function, we derived murine
Figure 4. The IFITM Protein Family Restricts Influenza A Virus Infection
(A) A549cells weretransduced withretrovirusescontainingC9-tagged cDNAsfor theindicatedIFITM proteinsor emptyviralvector alone.After2days, cellswere
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). Twenty-four
hours 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. b-actin levels show protein loading.
(C) A549 or U2OS cells stably overexpressing IFITM3 protein or vector alone were infected with influenza A H1N1 WSN/33. Twelve hours later, cells were fixed
and stained for surface HA expression. Values represent the mean ± SD, n = 3 (green: HA, blue: nuclei; 43).
(D) A549 and U2OS cells stably overexpressing IFITM3 were tested for expression by western.
cells were fixed and stained for surface HA expression. Values represent the mean ± SD, n = 3 (red: HA, blue: nuclei; 43).
(F) ChEF cells stably overexpressing IFITM3 were tested for expression by western.
(G) MDCK cells stably overexpressing IFITM3 protein or vector alone were infected with influenza A H1N1 WSN/33 at a multiplicity of infection (moi) of 0.005.
Seventy-two hours later, cells were washed with fresh media, then imaged live to assess cytopathic effect. Bright-field images shown are representative of
four independent experiments (103).
(H) MDCK cells stably overexpressing IFITM3 were tested for expression by western.
(I) A549 cells were transduced with retroviruses containing the indicated IFITM proteins or empty vector. Forty-eight hours later, the cells were incubated with
MLV-EGFP virus pseudotyped with the indicated envelope proteins. HA proteins from various influenza A virus strains 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), 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 hr 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 2 days post-infection. Values
represent the mean ± SD, n = 4. C, nontargeting siRNA negative control.
(K) A549 cells were transduced with the indicated retroviruses. Forty-eight hours later, the cells were tested for surface expression of sialic acid (SA). Values
represent the mean ± SD, n = 3.
1248 Cell 139, 1243–1254, December 24, 2009 ª2009 Elsevier Inc.
embryonic fibroblasts (MEFs) from a mouse strain, IfitmDel,
deleted for all of the Ifitm genes (Ifitm1, 2, 3, 5, and 6; Lange
et al., 2008). In spite of the loss of these genes, the IfitmDel
mice develop normally (Lange et al., 2008). Comparison of
IfitmDel+/+, +/?, and ?/? MEFs revealed a marked increase in
PR8 infection in the ?/? cells (Figure 5A). The differences in
infection were more pronounced when the MEFs were
cultured with IFN-a or -g prior to viral infection (Figures 5A–
5C). As in human cells, we observed a vesicular staining
pattern for Ifitm3 (Figures 5D and 3C). Forced expression of
Ifitm2 or 3 in the IfitmDel?/?cells restored resistance to influ-
enza A H1N1 infection (Figures 5E and 5F). We conclude that
the Ifitm protein family accounts for a significant proportion of
the anti-influenza actions of types I and II IFNs in mice, and the
majority of this function can be restored by the stable expres-
sion of Ifitm2 or 3.
IFITM3 Inhibits the Early Replication of West Nile Virus
and Dengue Virus
testing a panel of viral-like particles (VLPs) and pseudotyped
viruses, each expressing a unique viral envelope protein. The
Figure 5. Ifitm Knockout Cells Are More Suscep-
tible to Influenza A H1N1 Virus Infection and Are
Protected by the Reinstatement of Ifitm2 or 3
(A) MEFs derived from the indicated IfitmDel mice were left
untreated (buffer) or treated with interferon-a or -g. After
24 hr, the cells were incubated with influenza A virus
H1N1 (PR8). Twelve hours 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-g, IFN-a, or PR8 virus. After 24 hr the levels of
Ifitm3 were checked by western blot.
the absence (buffer) or presence of IFN-a for 24 hr, prior to
staining with a-Ifitm3 (red: Ifitm3, nuclei: blue); 633.
(E) IfitmDel+/+MEFs or IfitmDel?/?MEFs stably expressing
Ifitm2, 3, or the empty vector were challenged with PR8
virus. Twelve hours 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
VLPs expressed the envelope proteins of one
of three flaviviruses, West Nile virus (WNV),
yellow fever virus (YFV), or the Omsk hemor-
rhagic fever virus (OMSK). These VLPs can
undergo a single round of infection and are
produced by transiently expressing the respec-
tive envelope proteins together with the WNV
structural genes in cells stably expressing
subgenomic WNV replicons containing EGFP
(Yoshii and Holbrook, 2009). As observed with
influenza A pseudoparticles, all three VLPs
were blocked by each of the IFITM proteins, demonstrating
that these restriction factors impede first round infection
(Figure 6A). Again, the IFITM proteins were found to inhibit
VSV-G-mediated infection to a lesser extent (Figure 6A). In
contrast, pseudoparticles expressing the envelope proteins of
three arenaviruses, lymphocytic choriomeningitis virus (LCMV),
Lassa virus (LASV), and MACH, or the MLV retrovirus, were not
affected by IFITM expression. We next tested the effects of
IFITM protein levels on two pathogenic flaviviruses, WNV and
DNV. The replication of the 2741 strain of WNV was dramatically
decreased in either A549 or U2OS cells stably overexpressing
IFITM3 (Figures 6B and 6C). Furthermore, siRNA depletion of
IFITM3 protein also led to an increase in replication of both
WNV (Figures 6D and 6E) and DNV serotype 2 (New Guinea C
strain, Figures 6F and 6G). However, although IFITM3 did not
inhibit hepatitis C virus (HCV), a more distantly related member
of the Flaviviridae family, it did block influenza A virus infection
in the same HCV-permissive liver cell line (Huh 7.5.1, Figures
S5D and S5E). We conclude that IFITM proteins restrict the repli-
cation of two additional human pathogens, DNV and WNV, and
are likely to also limit YFV and OMSK infection based on the
Cell 139, 1243–1254, December 24, 2009 ª2009 Elsevier Inc. 1249
Integrated Model of Influenza A Virus Host Factors
Although considerable knowledge exists regarding the function
of viral proteins, the role of host factors in modifying infection
is less understood. Therefore, we executed a genetic screen
and identified over 120 human proteins needed by influenza A
virus. The screen enriched for multiple host cell pathways
including endosomal acidification, vesicular trafficking, mito-
chondrial metabolism, nucleocytoplasmic
export nuclear transport, and RNA processing.
Our findings both support and extend those of a previous
screen for influenza A virus-dependency factors that used
Drosophila cells (Hao et al., 2008). Among the factors identified
in our primary screen are the human orthologs of 11 insect-cell
host factors previously reported to be required for flu infection
control, NXF1, as well as NUP98, EIF4A2, ARCN1, COPG, PGD,
RAB5A, and RAB10. In addition to these exact candidate
matches, there was strong overlap between the screens within
several biologic pathways and macromolecular complexes and
those of the Reactome’s influenza A virus infection database
(Figure 2). This synthesis demonstrates the collective functional
insights that unbiased large-scale mammalian and fly RNAi
studies can provide in combination and outlines many central
functions and interactions required for the flu life cycle as well
as new antiviral drug targets.
We have also constructed an enrichment analysis for host
comparison of enriched GO categories for three whole-genome
screens for host-factor modifiers of viral infection (HIV, [Brass
et al., 2008], influenza A virus [this study], and WNV [Krishnan
et al., 2008]), wherein each screen’s respective candidate gene
list was analyzed separately for enriched GO categories. GO
terms that were enriched for in one more of the screens are
provided, with overlapping viral dependencies apparent for
components involved in spliceosome activity, Golgi function,
vesicular trafficking, and proton transport (Dataset S2). These
represent a core set of functions we now know to be shared
among a very diverse set of viruses.
The IFITM Protein Family
Our screen identified the IFITM proteins as viral restriction
factors. IFITM proteins were originally described 25 years ago
based on their expression after IFN treatment (Friedman et al.,
Figure 6. The IFITM Protein Family Restricts West Nile Virus and Dengue Virus Infections
(A)Vero E6cells weretransduced withretrovirusesexpressingtheindicatedIFITMproteinsortheemptyviralvector.Twodayslater,thecellswereincubatedwith
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-
expressingMLV virusespseudotypedwiththeindicated viralenvelope proteins. Viralinfectionisexpressedas mean EGFPfluorescencerelative tovector control
cells, as measured by flow cytometry 48 hr 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 Figures 4C and 4D) were infected with infectious WNV (strain
2741). Twenty-four hours 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), 43 magnification.
(D) HeLa cells were transfected with the indicated siRNAs for 72 hr, then infected with WNV. Twenty-four hours later, the cells were fixed and stained for viral
E protein. Values represent the mean ± SD, n = 3. C, nontargeting siRNA negative control.
(E) Images of HeLa cells in (D) (red: WNV E protein, blue: nuclei), 43 magnification.
(F) HeLa cells were transfected with the indicated siRNAs for 72 hr, then infected with dengue virus (New Guinea C strain). Thirty hours 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); 43.
1250 Cell 139, 1243–1254, December 24, 2009 ª2009 Elsevier Inc.
1984). The human IFITM1, 2, 3, and 5 genes lie adjacently on
chromosome 11. IFITM1, 2, and 3 are nearly ubiquitously ex-
pressed whereas IFITM5 is expressed in osteoblasts. The IFITM
proteins have been ascribed roles in immune cell signaling, cell
adhesion, oncogenesis, germ cell homing and maturation, and
bone mineralization (Evans et al., 1993; Imai and Yoshie, 1993;
Lange et al., 2003; Smith et al., 2006). However, with the excep-
tion of IFITM50s role in bone mineralization, we know of no other
functional studies clearly demonstrating an additional function
for an IFITM family member (Moffatt et al., 2008). Indeed, in
our hands, transformed and primary cells either overexpressing
or depleted for IFITM3 display no growth perturbations, and as
noted, the IfitmDel mice develop and age normally (Lange
et al., 2008).
The IFITM proteins belong to a protein domain superfamily
consisting of over 30 proteins, each possessing two transmem-
brane domains and an intervening highly conserved intracellular
loop (pfam04505, CD225, Interferon-induced transmembrane
protein). Expression of members of the CD225 protein family
have been reported in zebrafish, Xenopus, invertebrates, and
bacteria, and homologs are found in frog, fish, fowl, and
mammals (mouse, rat, dog, swine, cow, primate, and human;
Figures S6 and S7). Now that an antiviral role has been demon-
strated, it will be interesting to determine if any of the related
factors participate in host-pathogen interactions and, if so,
how early in evolution this protein domain became associated
with innate immunity.
The IFITM Proteins Are IFN-Inducible Restriction
Factors that Inhibit Influenza A and Flaviviral
Infection at Entry
A vital component of the innate immune response to viral infec-
tion is mediated by restriction factors. The antiviral action of the
IFITM proteins was observed in primary human, chicken, and
D: Signals to
Figure 7. IFITM Proteins Act as Antiviral
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 multitrans-
possible mechanisms of restriction: IFITM proteins
may (A) sequester the incoming viruses at or near
the surface, (B) block viral receptors from interact-
ing with host receptors (shown in orange), (C)
prevent endocytosis or viral membrane fusion, (D)
act as receptors and, after binding the virus, signal
mouse cells, in addition to multiple trans-
formed cell lines, including dog cells
permissive for influenza A virus replica-
tion. We also found that the IFITM
proteins broadly inhibit the replication of
all influenza virus strains tested, including
two current vaccine strains. Furthermore,
the IFITM proteins inhibit two highly path-
ogenic flaviviruses, WNV and DNV, and likely control YFV and
OMSK, indicating a very general antiviral role. Previous work
suggested that overexpression of human IFITM1 in murine fibro-
blasts partially blocked infection by VSV but not by influenza A
virus, although no loss-of-function experiments were reported
(Alber and Staeheli, 1996). We also detected modest inhibition
of VSV-G pseudoviruses but very strong inhibition of influenza
A virus with all three IFITM proteins. However, the more distantly
related hepacivirus, HCV, was not inhibited by IFITM3 levels
(Figures S5D and S5E), nor were HIV (Figure S3F) or pseudopar-
ticles bearing the envelope proteins of multiple arenaviruses.
Basal levels of IFITM2 and 3 were required to resist initial viral
infection and so may act as first-line defenders that prevent or
slow infections until the IFN system can reinforce them, by
both upregulating their levels and inducing the expression of
additional factors, such as IFITM1 and MxA (Takaoka and Yanai,
2006). Consistent with this notion, we observed that depletion of
IFITM3 resulted in loss of 40% to 70% of IFN’s protective effect,
with a similar diminution also detected in the IfitmDel mouse
cells. Thus, IFITM proteins are critical for the innate immunity
to influenza A virus afforded by IFNs. These results contribute
to our understanding of IFN action in that they demonstrate
that these antiviral cytokines block viral entry by inducing the
expression of the IFITM proteins.
Possible Mechanisms of IFITM Restriction of Influenza
and Flavivirus Infection
As schematized in Figure 7, the IFITM proteins could act at any
stage of viral entry, for example by directly binding the virus
and inhibiting interactions with host cell receptors (A); blocking
receptor access (B); inhibiting endocytosis, preventing viral
end (C); or acting as pattern recognition receptors on the cell
surface or on endocytosed vesicles where they could signal to
downstream antiviral effectors (D). Logic would further predict
Cell 139, 1243–1254, December 24, 2009 ª2009 Elsevier Inc. 1251
that IFITM proteins could target a common step in the viral life
cycle. While influenza A virus and flaviviruses bind distinct
receptors, both are endocytosed through a clathrin-dependent
pathway, although influenza A also has a clathrin-independent
pathway (Chen and Zhuang, 2008; Sieczkarski and Whittaker,
2002). After endocytosis, early endosomes containing influenza
A virus fuse with late endosomes where a pH < 5.5 triggers the
HA-directed fusion of the viral and endosomal membranes,
permitting vRNP entry (Figure 7; Lamb and Krug, 2001). In
contrast, flaviviruses undergo fusion in early endosomal
compartments and at a considerably higher pH of 6.5 (Krishnan
et al., 2007; Sanchez-San Martin et al., 2009). Thus, a very
general overlap involving clathrin-mediated endocytosis and
trafficking into early endosomes exists between influenza A virus
However, these common entry steps are shared to varying
degrees by several of the viruses not impacted by IFITM
proteins. For instance, sparing of the arenaviruses (MACH,
LCMV, LASV) argues against a block to general endocytosis or
clathrin-mediated endocytosis. Moreover, like flaviviruses, the
arenaviruses also traffic in early endosomes, where they
undergo pH-dependent fusion, thereby leaving no unique entry
step shared by the two viral families impacted by the actions
of the IFITM proteins. However, we cannot rule out that these
resistant viruses may have multiple entry or endosome escape
pathways or may have evolved to circumvent terminal rerouting
by IFITMs. We would also note, however, that, coculture of
IFITM3 overexpressing cells with parent control cells did not
confer protection, suggesting a cell-autonomous mode of action
(data not shown). Therefore, higher-resolution studies will now
berequired to determine theprecise mode(s) of viralinterference
employed by the IFITM proteins.
We identified three additional candidate influenza A virus
restriction factors: PUSL1, TPST1, and WDR33. It is possible
that these might work together with IFITM proteins to execute
viral resistance, and it remains to be determined whether they
block at the same point in the life cycle. WDR33 is an orphan
WD40 repeat protein about which little is known. TPST1 is
a Golgi-localized transmembrane tyrosylprotein sulfotransferase
secretion (Hoffhines et al., 2006). PUSL1 shares homology with
pseudouridylate synthase genes that modify RNA by converting
uridine into pseudouridine, a glycosylated form of uracil, in
several cytosolic and mitochondrial transfer RNAs (tRNAs)
(Massenet et al., 1999). While further validation is warranted,
it is tempting to speculate that PUSL1 could potentially block
influenza by directly modifying viral RNA.
The IFITM proteins and other innate cell-intrinsic defenders
present opportunities not just for a greater understanding of
fundamental questions but also as tools to actively combat
current and emerging pathogens. Variations in the basal and
inducible levels of these factors as well as the dependency
factors within a population might predict the severity of flu or
flaviviral infections among individuals or across species. The
discovery of the roles of IFITM proteins in innate immunity has
relevance to ongoing and future influenza pandemics. Not only
could elucidation of the IFITM restriction mechanism prove
important in designing new antiviral therapies, but the proteins
themselves could be used in multiple ways to fight influenza A,
WNV, and dengue virus. If IFITM proteins work on the plasma
membrane, they could possibly be delivered to tissues suscep-
tible to initial infection by liposomal transfer. Transgenic animals
such as fowl or swine could be developed that overexpress
IFITM proteins to provide resistance to influenza A virus and
otherpathogens,thereby preventingthespreadof theseviruses,
as well as limiting their ability to recombine with human influenza
A strains to produce strains dangerous to human populations.
Indeed, by creating animals with multiple transgenic restriction
factors, we can confront the virus with a more intractable barrier.
Finally, if IFITM proteins are also rate limiting for influenza A virus
infection in other organisms such as chickens, whose embryos
areemployed topassage attenuatedviruses forvaccine produc-
tion, the inhibition of IFITM protein expression could reduce the
amount of time it takes to produce vaccine and thereby boost
yields. This has been a critical issue confounding vaccine
production in the current influenza pandemic. The discovery of
the role of IFITM proteins as antiviral agents for multiple devas-
tating pathogenic viruses has given us new insights into innate
immunity and has provided us new tools with which to counter
viral propagation in the future.
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 hr, the medium was removed and the cells were infected with the Influenza
A/Puerto Rico/8/34 (PR8, ATCC VR-1469), multiplicity of infection (moi) of
?0.2–0.3 in 40 ul complete media. After 12 hr, media were removed, and 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 auto-
mated 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). All Dharmacon siRNAs and plasmids used for
generating stable cell lines are shown in Dataset S1 and the Supplemental
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 wereculturedin DMEM (In-
vitrogen Cat#10569), containing 13 MEM nonessential amino acids (Invitro-
gen Cat#11140, 10 mM stock/1003) 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 NIH 3T3 cells. WNV (strain
2741) and DNV serotype 2 (New Guinea C strain) viruses were grown on
1252 Cell 139, 1243–1254, December 24, 2009 ª2009 Elsevier Inc.
West Nile and Dengue 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
30hr,respectively, asreportedpreviously (Krishnan etal., 2008).Infected cells
were fixed in 4% PFA and immunostained with antibodies detecting viral
E-proteins (Chemicon) and imaged by fluorescence microscopy (Zeiss).
IFITM3 overexpressing or vector control-A549 or -U20S cells were infected
with WNV at an moi of 1.
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.
Twenty-four hours 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.
Viral-like Particles and Pseudotyped Virus
MLV-GFP pseudoviruses were made as described (Huang et al., 2006, 2008).
Flavivirus VLP were as described (Hanna et al., 2005), except plasmids encod-
ing 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 as-
sessed 2 days later by measuring GFP expression by flow cytometry. The
infection level of siRNA-transfected U2OS cells after 2 days of infection was
determined by calculating the percent of 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-
HA7from Sigma-Aldrich(Productcode H3663),whichrecognizestheHAnon-
apeptide tag on IFITM3R6but 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, 2008).
Statistical analysisof gene enrichment was performed using ahypergeometric
distribution as described in the GOhyperGAll module of Bioconductor for gene
ontology terms (Gentleman et al., 2004). A map of the viral life cycle 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), and NCBI GeneRIF (Mitchell et al.,
2003); in addition, OMIM Human orthologs were mapped to other species
using NCBI HomoloGene (Wheeler et al., 2005) (see Supplemental Experi-
mental Procedures for more details).
Additional Experimental Procedures are included in the Supplemental Data.
Supplemental Data include Supplemental Discussion, Supplemental Experi-
mental Procedures, seven figures, and two datasets and can be found with
K. Rudnicki, D. Wrobel, M. Ocana, and Z. Cooper; Ragon Institute; L. White-
man, K. Hartman, A. Piechocka-Trocha, J. Proudfoot, and T. Diefenbach.
We thank J. Philips, A. Mehle, M. Franti, F. Diaz-Griffero, J. Mabry (CDC),
his gratitude to the Phillip T. and Susan M. Ragon Foundation for their
generous support. A.L.B. (MGH GI Unit, Harvard Center for AIDS Research);
M.N.K. and E.F. (NIH grants AI 50031 and AI070343); Y.B. and R.J.X. (CSIBD,
CCIB, Genetics and Genomics Core, and NIH grants AI062773, DK060049,
and DK043351); I.C.H. and M.F. (NERCE U54 AI057159). D.J.A. is supported
by Cancer Research UK and the Wellcome Trust. L.v.d.W. is supported by
a fellowship from the Kay Kendall Leukaemia Foundation. This study was sup-
ported by the New England Regional Center of Excellence for Biodefense and
Emerging Infectious Diseases (NIH grant U54 AI057159 to D. Kasper). S.J.E.
and E.F. are Investigators with the Howard Hughes Medical Institute.
Received: November 5, 2009
Revised: December 1, 2009
Accepted: December 9, 2009
Published online: December 17, 2009
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