IFITM3 inhibits influenza A virus infection by preventing cytosolic entry.

Abstract

To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats.

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IFITM3 Inhibits Influenza A Virus Infection by Preventing
Cytosolic Entry
Eric M. Feeley1, Jennifer S. Sims1., Sinu P. John1., Christopher R. Chin1, Thomas Pertel1, Li-Mei Chen2,
Gaurav D. Gaiha1, Bethany J. Ryan1, Ruben O. Donis2, Stephen J. Elledge3,4, Abraham L. Brass1,5*
1Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Charlestown, Massachusetts, United States of America,
2Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 3Department of Genetics, Harvard Medical School,
Department of Genetics, Brigham and Women’s Hospital, Boston, Massachusetts, United States of America, 4Howard Hughes Medical Institute, Chevy Chase, Maryland,
United States of America, 5Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, United States of America
Abstract
To replicate, viruses must gain access to the host cell’s resources. Interferon (IFN) regulates the actions of a large
complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane
protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile
virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and
that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring
their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further
demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this
phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading
viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are
vulnerable to IFITM3’s actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition.
Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN.
Therefore these findings are likely to have significance for the intrinsic immune system’s neutralization of a diverse array of
threats.
Citation: Feeley EM, Sims JS, John SP, Chin CR, Pertel T, et al. (2011) IFITM3 Inhibits Influenza A Virus Infection by Preventing Cytosolic Entry. PLoS Pathog 7(10):
e1002337. doi:10.1371/journal.ppat.1002337
Editor: Michael S. Diamond, Washington University School of Medicine, United States of America
Received May 17, 2011; Accepted September 13, 2011; Published October 27, 2011
Copyright: ? 2011 Feeley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The source of funding for this work was from the Charles H. Hood Foundation and the Phillip T. and Susan M. Ragon Institute Foundation. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: abrass@partners.org
. These authors contributed equally to this work.
Introduction
The 2009 H1N1 pandemic provided a strong reminder of the
threat that influenza A virus poses to world health (http://www.
cdc.gov/h1n1flu/cdcresponse.htm). The most effective means of
protection against influenza is the seasonal vaccine. However, if
the vaccine does not match the viral strains, its effectiveness can be
reduced to 50% or less [1,2]. Among small molecules, only two
approved influenza drugs remain effective, zanamivir (Relenza)
and oseltamivir (Tamiflu). Although resistance to zanamivir is
rare, there has been an increase in oseltamivir-resistant flu strains
[3]. Of concern, both drugs target viral neuraminidase (NA),
precluding combinatorial therapy to minimize resistance [4,5].
Thus, research to identify new anti-influenza strategies would be
useful.
The influenza A virus is 50–100 nm in size, encodes for up to 11
proteins, and contains eight segments of negative single-stranded
genomic RNA (3). Influenza A virus infection initiates with the
cleavage and activation of the viral hemaglutinnin (HA) envelope
receptor by host proteases [6,7,8,9]. HA then binds to sialylated
proteins on the cell surface, eliciting endocytosis of the viral
particle. Endocytosed viruses are transported through the early
and late endosomes, with late endosomal acidification triggering a
conformational change in HA which results in viral-host
membrane fusion [6,10]. Fusion transitions from a hemifusion
intermediate into a fusion pore through which the virus’ eight viral
ribonucleoproteins (vRNPs) enter the cytosol. The vRNPs are
subsequently guided by the host cell’s karyopherins into the
nucleus [11,12,13], wherein the viral RNA-dependent RNA
polymerase synthesizes viral genomes (vRNA) and mRNAs, both
of which are exported to the cytosol, culminating in the production
of viral progeny.
Genetic screens have identified multiple host factors and
pathways which modulate influenza A virus infection in vitro
[14,15,16,17]. Using such a genetic screen, we identified the
IFITM protein family members IFITM1, 2 and 3 as antiviral
factors capable of blocking influenza A viruses [14]. We further
tested the antiviral activity of IFITM3 protein using the seasonal
influenza A strains, A/Uruguay/716/07 (H3N2) and A/Bris-
bane/59/07 (H1N1), and found similar levels of IFITM3-
mediated viral inhibition [14]. IFITM3 accounts for a significant
portion (50–80%) of IFN’s (type I or II) ability to decrease
influenza A virus infection in vitro, and IFITM3 resides in vesicular
compartments that are IFN-inducible [14]. In addition, the
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IFITM family inhibits infection by the flaviviruses, dengue virus
and West Nile virus [14,18], as well as the filoviruses, Ebola and
Marburg, and the SARS coronavirus [19]. The IFITM proteins
also block vesicular stomatitis virus-G protein (VSV-G)-mediated
entry, but do not substantially alter the replication of Moloney
leukemia virus (MLV), several arena viruses, or hepatitis C virus
(HCV, [14,20]).
The human IFITM proteins were identified 26 years ago based
on their expression after IFN stimulation [21,22,23]. The IFITM1,
2, 3 and 5 genes are clustered on chromosome 11, and all encode
for proteins containing two transmembrane domains (TM1 and 2),
separated by a conserved intracellular loop (CIL, [22]), with both
termini extra-cellular or intra-vesicular [24,25]. TM1 and the CIL
are well conserved between the IFITM proteins and a large group
of proteins representing the CD225 protein family. CD225 family
members exist from bacteria (125 members) to man (13 members,
with 156 members in chordata), with no in depth functional data
available for any member other than the IFITM proteins.
IFITM1, 2 and 3 are present across a wide range of species
including amphibians, fish, fowl and mammals. The IFITM
proteins have been described to have roles in immune cell
signaling and adhesion, cancer, germ cell physiology, and bone
mineralization [25,26,27,28,29,30]. IFITM3 expression can in-
hibit the growth of some IFN-responsive cancer cells [31]. Genetic
evidence also points to IFITM5/Bril being required for early bone
mineralization [30,32]. IfitmDel mice, which are null for all five of
the murine Ifitm genes, display a 30% perinatal mortality among
null pups, but thereafter grow and develop normally in a
controlled setting [26]. However, cells derived from these IfitmDel
mice are more susceptible to influenza A virus infection in vitro
[14]. IFITM3 inhibited infection by all influenza A virus strains
tested including a 1968 pandemic isolate and two contemporary
seasonal vaccine viruses [14]. We have found IFITM3 to be the
most potent of the IFITM protein family members in decreasing
influenza A virus replication [14].
Viral pseudoparticles are differentially inhibited by the IFITM
proteins based on the specific viral receptors expressed on their
surfaces [14,19]. Therefore, we have hypothesized that IFITM
proteins inhibit susceptible virus families (Orthomyxoviridae,
Flaviviridae, Rhabdoviridae, Filoviridae, and Coronaviridae)
during the envelope-dependent early phase of the infection cycle,
which extends from viral binding to cell surface receptors through
the creation of the fusion pore between viral and host membranes
[14,19,20]. In support of this notion, recent work demonstrated
that IFITM protein overexpression did not prevent influenza A
virions from accessing acidified compartments [19]. Consistent
with its acting on endocytosed viruses, a portion of IFITM3 resides
in structures that contain host cell endosomal and lysosomal
proteins [19]. Furthermore, inhibition of influenza A virus
infection depends on the palmitoylation of IFITM3, a post-
translational modification that targets proteins to membranous
compartments [33].
Here we directly test the idea that IFITM3 restricts influenza A
viral infection during the envelope-dependent early phase of the
viral lifecycle. Consistent with previous studies, we find that
IFITM3 inhibits influenza A viral infection after viral-host binding
and endocytosis, but prior to primary viral transcription [19,20].
Moreover, using a combination of assays, we find that either IFN
or high levels of IFITM3 impede influenza A viruses from
transferring their contents into the host cell cytosol, and that
IFITM3 is necessary for this IFN-mediated action. Therefore, we
conclude that IFN is acting predominantly through IFITM3 to
block viral fusion. We also find that IFN expands the late
endosomal and lysosomal compartments, and that IFITM3
overexpression is sufficient for this phenotype. This study also
presents data showing that IFITM3 overexpression leads to the
expansion of enlarged acidified compartments consisting of
lysosomes and autolysosomes. Interestingly, we observe that
viruses trapped in the endocytic pathway of IFITM3-overexpress-
ing cells are trafficked to these expanded acidified compartments.
Based on these results and those of others [19,20], we present a
model whereby IFN acts via IFITM3 to prevent viral fusion,
thereby directing endocytosed viruses to lysosomes and autolyso-
somes, for subsequent destruction. Collectively this study expands
our understanding of how IFITM3 restricts a growing number of
viruses by exploiting a shared viral vulnerability arising from their
use of the host’s endocytic pathway.
Results
IFITM3 inhibits influenza A viral infection after viral-host
binding but prior to viral transcription
The inhibition of HA-expressing pseudoparticles by the IFITM
proteins pointed towards restriction occurring during the enve-
lope-dependent phase of the viral lifecycle [14]. Therefore we
tested IFITM3’s impact on the most proximal phase of infection,
viral binding, by incubating influenza A virus A/WSN/33 H1N1
(WSN/33, multiplicity of infection (moi) 50) with A549 lung
carcinoma cells either stably overexpressing IFITM3 (A549-
IFITM3) or an empty vector control cell line (A549-Vector,
Fig. 1A). Samples were incubated on ice to permit viral binding
but prevent endocytosis. After incubation, cells were washed with
cold media, fixed and stained for HA. When analyzed by flow
cytometry, we observed no appreciable difference in surface bound
HA between the vector and IFITM3 cells. There was also no
difference in surface-bound virus over a series of ten-fold dilutions
of viral supernatant (data not shown). We also determined that the
stable expression of IFITM3 did not alter the surface levels of (a2,
3) or (a2,6) sialylated cell-surface proteins (Fig. S1).
To investigate IFITM3’s impact on initial viral mRNA
production, we infected canine kidney cells, either expressing
IFITM3 (MDCK-IFITM3) or the empty vector (MDCK-Vector),
with influenza A virus (A/Puerto Rico/8/34 H1N1 (PR8), moi
500). We used PR8 because of the purified high titer stocks
available. Next, the viral supernatant was removed and warm
media was added (0 min). At the indicated times, cells were
processed and stained for the positive stranded NP mRNA of PR8
using a specific RNA probe set (red, Fig. 1B), then imaged on a
Author Summary
Influenza epidemics exact a great toll on world health.
Thus research to identify new anti-influenza virus strate-
gies would be useful. Each of our cells contains antiviral
factors that work to inhibit infection. A large component of
this antiviral program is regulated by the interferon family
of signaling molecules. Here, we seek to better understand
how one of these antiviral factors, IFITM3, contributes to
both baseline, as well as interferon-induced, antagonism of
influenza A viral infection. We found that interferon
prevents influenza A virus from entering our cells by
blocking the virus’ fusion with the cellular membrane.
Furthermore, we learned that IFITM3 is required for this
antiviral action of interferon, and that high levels of IFITM3
alone can produce a similar viral inhibition. Together, these
results improve our understanding of how IFITM3 serves to
defend us against viral invasion at a very early stage of
infection.
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confocal microscope. Based on NP mRNA staining, primary viral
transcription begins by 60 min. p.i. in the vector control, with the
NP mRNA signal increasing through to 180 min., when the
export of viral mRNAs to the cytosol can be observed. A decrease
in primary viral transcription can be seen when comparing the
IFITM3 cells to the vector control line. Therefore, IFITM3
inhibits influenza A viral infection after viral-host binding but
before primary viral mRNA transcription.
IFN interferes with vRNP nuclear entry and IFITM3 is
necessary and sufficient for this antiviral defense
We next used confocal imaging to track the nuclear transloca-
tion of vRNPs (Fig. 2 [34,35]). At the start of infection, the NP
within infected cells is complexed with viral genomic RNA
forming vRNPs. Therefore, immunostaining for NP permitted us
to follow vRNP distribution intracellularly [16,34,36]. Normal
diploid human lung fibroblasts (WI-38 cells) were stably
transduced with empty vector (Vector), IFITM3 cDNA (IFITM3),
orshort hairpinRNAs (shRNA)
(shIFITM3) or a scrambled non-targeting control (shScramble,
Fig. 2, S2). WI-38s were chosen because of their normal karyotype
and relatively larger and flatter morphology. Cells were first
incubated on ice with PR8 (moi 500). Next, the viral supernatant
was removed and warm media was added (0 min). At the indicated
times after warming, cells were fixed, permeabilized, stained for
NP and DNA, and imaged on a confocal microscope. Image
analysis software was used to create an outline of each cell’s
periphery (white lines) and nucleus (blue lines). Based on NP
staining, vRNPs arrive in the nuclei by 90 min in the vector
control, shIFITM3, and in the shScramble cells, with the NP
signal increasing through to 240 min (Fig. 2A, S2A–D).
In contrast, we observed decreased nuclear and increased
cytosolic NP staining in the IFITM3 cells (Fig. 2, S2C). Moreover,
either againstIFITM3
in the IFITM3 cells greater than 60% of the cytosolic NP
colocalized with Lysotracker Red (LTRed), a dye which marks
acidiccellularcompartments
pH#5.5), and which was added to the warm media at time zero
(Fig. S2A, D). The increased NP in the cytosol of the IFITM3 cells
likely arises in part from an increase in the local concentration of
viruses because a-NP Western blots (after trypsinizing the cells to
remove adherent NP) did not show substantial differences in
internalized NP levels between cell lines for up to 90 min post
infection (p.i., data not shown). Because IFITM3 is required for
the anti-viral actions of IFN in vitro [14], we performed a
companion experiment with the WI-38 cells treated with IFN-a
(Fig. 2B). IFN-a treatment also decreased NP nuclear staining in
the WI-38-Vector cells, however this block was not as complete
nor was it associated with similar levels of cytosolic NP staining as
those seen with high levels of IFITM3. Consistent with the gain-of-
function data, the depletion of IFITM3 decreased IFN’s ability to
block vRNP trafficking to the nucleus (Fig. 2A and B, compare top
and bottom rows).
Similar results were obtained either using A549 cells (Fig. S3) or
using MDCK cells, with the latter experiments employing
additional influenza A viral strains (X:31, A/Aichi/68 (Aichi
H3N2), Fig. S4A–C, WSN/33 and A/Victoria/3/75 H3N2, data
not shown). It is important to note that the levels of IFITM3
protein in the A549-IFITM3 cells are higher than those seen after
treatment with IFN-a or -c (Fig. S3C). However, we have not
observed that other overexpressed proteins have either protected
against viral infection or expanded the lysosome/autolysosome
compartment (data not shown), arguing that this is a specific effect.
To better assess the expanded LTRed compartments observed
with IFITM3 overexpression, we created MDCK cells stably
expressing the lysosomal protein, LAMP1, fused to a red
fluorescence protein (LAMP1-RFP) and IFITM3. As compared
(late endosomes,lysosomes,
Figure 1. IFITM3 inhibits infection after viral binding but before viral transcription. A) A549 cell lines were incubated on ice with H1N1
WSN/33 to permit viral-host binding. Cells were washed, fixed and immunostained for surface-bound HA protein, then analyzed by flow cytometry.
Values given are percentage of cells staining for surface HA. Values are representative of three independent experiments. B) MDCK cells transduced
with the empty vector control (Vector) or IFITM3 were incubated with A/Puerto Rico/8/34 H1N1 (PR8) on ice. Warm media was added at time zero.
Cells were then fixed at the indicated time points and hybridized with RNA probes against the viral NP mRNA (red) and stained for DNA (blue), then
imaged by confocal microscopy. Images are representative of three independent experiments. (Scale bar: 20 mm).
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Figure 2. IFN prevents vRNP nuclear entry, and IFITM3 is necessary and sufficient for this action. A) Normal diploid human fibroblasts
(WI-38 cells) were stably transduced with retroviruses containing either IFITM3 (IFITM3), a shRNA against IFITM3 (shIFITM3), an empty viral vector
alone (Vector), or a non-targeting control shRNA (shScramble, Fig. S2). Cells were incubated with PR8 on ice, and then warm media was added at time
zero. Cells were fixed at the indicated times p.i. and stained for NP (green) and DNA and analyzed by confocal microscopy. Image analysis software
was used to define each cell’s cytosolic (white lines) and nuclear peripheries (blue lines, based on DIC images and DNA staining, respectively). Red
arrows: cytosolic compartments containing NP. Images are representative of four independent experiments. (Scale bar: 15 mm). B) As in (A) except
that cells were treated with IFN-a prior to infection.
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to control cells, the IFITM3 cells demonstrated extensive
colocalization (.60%) between the NP and LAMP1-RFP signals,
revealing that the entering viruses are trafficked to lysosomal
compartments (Fig. S5).
We extended this analysis by directly tracking the location of
the vRNA contained in the incoming vRNPs. MDCK cells
stably expressing an empty vector or IFITM3, were used in
time-course experiments as above (Fig. 3A–D). At the indicated
times, cells were processed and stained for the negative stranded
NP vRNA of PR8 using a specific RNA probe set (green). As
seen with the WI-38 cells, we observed the nuclear translocation
of vRNA by 80 min p.i. in the MDCK-vector cells (Fig. 3A).
The nuclear vRNA signal was strongly decreased with IFITM3
overexpression based on the average number of vRNA particles
present per nucleus (Fig. 3C). Consistent with the WI-38 results,
the vRNAs accumulated in the cytosol of the IFITM3 cells, with
.50% co-localizing with LTRed-staining acidic structures
(Fig. 3D). Similar levels of retained cytosolic vRNPs were
observed in experiments without LTRed (data not shown).
Interestingly, we observed the loss of the vRNA signal in the
acidic inclusions of the MDCK-IFITM3 cells between 80 and
240 min. p.i. (Fig. 3B). By comparison, the vRNAs in the
control cells increased in number in both the nucleus and
cytosol, as would be expected with the nuclear export of newly
synthesized viral genomes [36].
We next evaluated vRNP translocation in murine embryonic
fibroblasts (MEFs) derived from animals that have had all five Ifitm
genes deleted (IfitmDel2/2, [14,26]). Compared to wild-type
(WT) matched litter mate controls, the IfitmDel2/2 MEFs
displayed 5–10 fold more nuclear NP staining, with or without
IFN-c treatment (Fig. 4, S6C). IFN-mediated viral restriction was
restored when we transduced the null MEFs with a retrovirus
expressing Ifitm3 (IfitmDel2/2 Ifitm3, Fig. S6). Similar to what
was observed with the IFITM3 overexpressing cell lines, the
majority of the vRNP signal in the IFN-c-treated WT and Ifitm3-
rescued cells localized to acidic compartments (red, Fig. S6B). An
increase in acidic compartments occurred after IFN-c treatment
with either the WT or the IfitmDel2/2Ifitm3 MEFs, but not in the
IfitmDel2/2 cells, suggesting that Ifitm3 is required for this event
(Fig. 4, S6). Similar results were obtained with IFN-a (data not
shown). We conclude from these experiments using orthologous
reagents (cell lines and influenza A viruses) and methods, that IFN
impedes vRNP nuclear entry, and IFITM3 is necessary and
sufficient for this activity.
Figure 3. IFITM3 overexpression leads to both a retention of viral genomes in the cytosol, and a decrease in viral genomes entering
the nucleus. MDCK cells transduced with the empty vector control (A) or IFITM3 (B) were incubated with PR8 on ice. Warm media containing
lysotracker red dye (LTRed, red) was added at time zero. Cells were then fixed at the indicated time points and hybridized with RNA probes against
the viral NP genome (NP vRNA, green) and stained for DNA, then imaged by confocal microscopy. Image analysis software was used to define the
nuclear boundaries (blue lines) based on DNA staining. Images are representative of four independent experiments. (Scale bar: 20 mM). C)
Quantitation of nuclear vRNA particles. The number of viral RNA particles per nucleus of the MDCK-Vector and IFITM3 cells at the indicated time
points are shown. Values represent the mean +/2 the SD of three independent experiments. D) Percent colocalization of vRNA and LTRed-containing
compartments for MDCK-Vector and IFITM3 cells lines treated as in A and B, at the indicated time points.
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Viral pseudoparticle fusion mediated by either HA or
VSV-G envelope proteins is decreased by IFN, and IFITM3
is necessary and sufficient for this activity
To further characterize the mechanism of IFITM3-mediated
restriction, we used an established viral fusion assay [37,38].
Lentiviral pseudoparticles containing the b-lactamase protein
fused to the HIV-1 accessory protein Vpr (BLAM-Vpr) and
expressing either HA and NA (H1N1, WSN/33), or VSV-G
envelope proteins, were incubated for 2 h with cells, which were
then loaded with the b-lactamase flourogenic substrate, CCF2.
Upon viral pseudoparticle fusion, BLAM-Vpr enters the cytosol
and cleaves CCF2, producing a wave length shift in emitted light
(from green to blue) when analyzed by flow cytometry (Fig. 5A,
[37]). In MDCK-IFITM3 cells we observed a decrease in both
HA- and VSV-G-directed fusion, which was comparable to the
block produced by poisoning of the host vacuolar ATPase
(vATPases) with a low dose of bafilomycin A1 (Baf, Fig. 5B).
The inhibition of vATPases prevents the low-pH activation
required by these two viral envelope proteins to produce
membrane fusion. A block to fusion of pseudoparticles expressing
H1 (PR8), H3 (A/Udorn/72), H5 (A/Thai/74) or H7 (A/FPV/
Rostock/34) subtypes of HA was also detected with MDCK cells
Figure 4. Ifitm knockout cells are more vulnerable to vRNP nuclear entry and are rescued by the restoration of Ifitm3 expression.
MEFs, either A) wild type (WT) or B) IfitmDel2/2, which are missing all five of the mouse Ifitm proteins, were either left untreated (left panels, Buffer),
or treated (right panels) with IFN-c. The following day cells were incubated with PR8 on ice. Cells were next incubated in warm media containing
LTRed. Cells were then fixed at the indicated times and immunostained with anti-NP antibodies (green), stained for DNA (blue), and imaged by
confocal microscopy. Image analysis software was used to define the nuclear boundaries (blue lines). Images are representative of three independent
experiments. (Scale bar: 12 mm).
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Figure 5. HA or VSV-G-mediated fusion is inhibited by IFN or IFITM3. A) Schematic model of the established viral fusion assay [37,38]
comprised of lentiviral pseudoparticles (pps) containing the b-lactamase protein fused to the HIV-1 accessory protein Vpr (BLAM-Vpr, shown in
orange/red) and expressing HA and NA (WSN/33) on their surfaces. The H1N1pps were incubated for 2 h with cells, which were subsequently loaded
with the b-lactamase flourogenic substrate, CCF2. Upon viral fusion, BLAM-Vpr enters the cytosol and can cleave CCF2, producing a wavelength shift
from green to blue in emitted light when analyzed by flow cytometry ([37]). B) MDCK cells stably overexpressing IFITM3 (MDCK-IFITM3) or empty
vector control cells (MDCK-Vector) were exposed for 2 h to viral pseudoparticles containing a BLAM-Vpr and expressing either the HA and NA
envelope proteins of Influenza A virus (WSN/33, H1N1pp) or the VSV-G envelope protein (VSV-Gpp), then loaded with CCF2. After incubation with the
indicated pseudoparticles, the cells were fixed and assayed for cleavage of CCF2 by determining the conversion of the fluorescence emission from
520 nm (uncleaved CCF2) to 447 nm (cleaved CCF2) using flow cytometry. Fusion of the pseudoparticles was inhibited by bafilomycin A1 (Baf). These
results are representative of six independent experiments. C) IFITM3 inhibits fusion of H1N1pps in normal diploid fibroblasts. WI-38 fibroblasts stably
transduced with IFITM3 (WI-38 M3) or the empty vector (WI-38 V) were exposed for 2 h to serial dilutions of H1N1pps containing BLAM-Vpr, with or
without Baf. These results are representative of four independent experiments. D) Fusion of H1N1pps increases after IFITM3 knockdown. WI-38
fibroblasts stably transduced with a shRNA against IFITM3 (WI-38 shM3), a shRNA control with a scrambled sequence (WI-38 shScr), or the IFITM3
cDNA (WI-38 M3) were exposed to either no virus, H1N1pps or VSV-Gpps containing BLAM-Vpr. These results are representative of two independent
experiments. E) Fusion of H1N1pps is inhibited by IFN-c. WI-38 fibroblasts were treated with IFN-c for 24 h or buffer alone prior to incubation with
H1N1pps containing BLAM-Vpr. These results are representative of three independent experiments.
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or with chicken embryonic fibroblasts (ChEFs), in which IFITM3
strongly inhibited viral replication (Fig. S7A, B, C). In the case of
the MDCK cells, the block to fusion closely paralleled the level of
inhibition seen when the pseudoparticles were tested for
productive infection using HIV-1 p24 expression as a readout
(Fig. S7E). Consistent with earlier findings, pseudoparticles
expressing an amphotropic MLV envelope protein were insensi-
tive to IFITM3, showing the specificity of these results (Fig. S7D).
Similarly to its effect on H5-expressing pseudoparticles, IFITM3
inhibited replication of infectious avian H5N1 influenza A virus,
A/Vietnam/1203/04 (VN/04), isolated from a fatal human
infection (Fig. S7F–H).
To enhance our analysis, we tested two additional cell lines, WI-
38 and HeLa cells. A strong block to fusion in WI-38-IFITM3
cells, similar to that of the Baf and uninfected control samples, was
seen at a range of serial dilutions of pseudoparticles, as well as an
increase in fusion with IFITM3 depletion (shIFITM3, Fig. 5C, D).
IFN treatment inhibited fusion of the H1N1 pseudoparticles, albeit
to a lesser extent than IFITM3 overexpression (Fig. 5E), and this
effect was largely absent when IFITM3 was stably depleted in
HeLa cells (Fig. S8). Similar results were obtained with IFN-a
(data not shown). Based on these experiments using multiple cell
lines and HA, VSV-G, and MLV envelope-expressing pseudo-
particles, we conclude that IFITM3 is required and sufficient for
an IFN-mediated block of viral pseudoparticle fusion. Importantly,
the increase in pseudoparticle fusion seen when endogenous
IFITM3 was depleted in either the HeLa or WI-38 shIFITM3 cell
lines argues that fusion inhibition underlies the first line defense
provided by endogenous, as well as overexpressed, IFITM3.
MxA is an IFN-inducible large GTPase which interferes with
secondary transcription during influenza A viral replication [39].
A549 cells express MxA and have been used extensively in
influenza A viral replication studies [40]. Therefore to clarify the
antiviral roles of IFITM3 and MxA, we tested the levels of viral
replication in A549 cells stably expressing one of three shRNAs
targeting IFITM3 (shIFITM3-1, -2, or -3). All three shIFITM3 cell
lines showed increased infection (WSN/33 strain) and strong
IFITM3 knockdown, when compared to the negative control cell
line expressing a shRNA against firefly luciferase (shLuc), with or
without IFN treatment (Fig. S9A, B). The majority of the
protective effect of either IFN-a or c was lost in the shIFITM3
cell lines. We next confirmed both the baseline levels, as well as the
IFN-inducibility of MxA in the A549 cells (Fig. S9C). We also
determined that MxA was both present and IFN-inducible in WI-
38 normal fibroblasts, another cell line used in loss-of-function
experiments in this work (Fig. S9D). Furthermore, IF studies of
WI-38 cells showed that MxA is expressed in an IFN-inducible
vesicular pattern and that these structures did not appreciably co-
localize with vesicles containing IFITM3 (Fig. S9E, [39]). We
conclude that MxA is expressed in the A549 and WI-38 cell lines,
but cannot fully compensate for loss of the antiviral actions of
IFITM3.
IFITM3 is present in endosomes and lysosomes and these
compartments are expanded with IFITM3 overexpression
or IFN treatment
Our data demonstrate that IFN or IFITM3 inhibit viral fusion.
Influenza A virus fuses with the host membrane in late
endosomes when the pH decreases to 5 [6,7,41]. Rab7 is a late
endosomal/lysosomal small GTPase that is required for the
fusion of many pH-dependent viruses, including influenza A virus
[6,41]. Previous reports have shown that IFITM3 colocalizes with
LAMP1 and CD63, components of lysosomes and multivesicular
bodies, respectively [19]. However, the relationship of IFITM3
and Rab7 within the host cell infrastructure remains unknown.
Therefore we investigated the location of IFITM3, by undertak-
ing immunoflourescence (IF) studies using antibodies that
recognize IFITM3, Rab7, or LAMP1 [42]. Although the baseline
level of IFITM3 in the A549-Vector cells was low, there was
partial colocalization observed with either Rab7 or LAMP1
(Fig. 6A–D, 7A,). IFITM3 also partially colocalized with LAMP1
and LTRed-containing structures seen with IFITM3 overexpres-
sion (Fig. 6A, B, 7A). Interestingly, either IFITM3 overexpression
or IFN increased the staining intensity of Rab7 and LAMP1
(Fig. 7A, B, S10A). Partial colocalization of IFITM3 was also seen
with either endogenous LAMP1, or an exogenously expressed
Rab7-yellow fluorescence fusion protein (Rab7-YFP) in MDCK
cells (Fig. 6E–I). However, in all cases, co-localization was not
complete because cells contained areas that uniquely labeled for
each of the proteins. Western blots indicated that IFITM3 over-
expression led to modest increases in both LAMP1 and Rab7
proteins in the A549-IFITM3 cells (Fig. 7C). However, these blots
also showed that while IFN treatment of the A549-Vector cells
increased IFITM3 protein levels as expected, the amount of
Rab7 and LAMP1 remained unchanged. We conclude that
IFITM3 partially resides in the late endosomal and lysosomal
compartments along with Rab7 and LAMP1, and that IFITM3
overexpression or IFN treatment expands these compartments
through a mechanism that cannot be fully explained by increased
protein expression alone.
IFITM3 overexpression leads to the expansion of the host
cell’s acidified compartments
Our assays showed that incoming influenza A viruses were
retained in the expanded acidic compartments of both the
IFITM3 overexpressing cell lines as well as the IFN-c-treated
MEFs, and that IFITM3 partially localized to these structures
(Fig. 2–4, S2–4, S6). Therefore, we extended our investigation
of these compartments. An increase in acidic structures was
seen in MDCK and A549 cells overexpressing IFITM3 as
compared to control cell lines, using either the vital acidophilic
stain, acridine orange (AO), LTRed, or a cathepsin-L substrate
that fluoresces only after it is proteolyzed, when compared to
the corresponding vector control cells (Fig. 8A, B, a, b).
Cathepsins are a family of lysosomal zymogens active in acidic
environments (pH#5.5) which are required for both the
degradation of endocytic substrates and for the entry of several
IFITM3-susceptible viruses [19]. Flow cytometry revealed an
increase in the total LTRed fluorescent signal in both the
MDCK and A549 IFITM3 cell lines when compared to
controls (Fig. 8C). This expanded compartment represents a
heterogeneous population of lysosomes and autolysosomes,
based on confocal imaging showing the colocalization of the
autophagosome marker, microtubule-associated protein 1 light
chain 3 (LC3), with either LTRed or with CD63, with the latter
being a resident of multivesicular bodies, amphisomes and
autolysosomes (Fig. 8D, E). Furthermore, MDCK-IFITM3 cells
stably transduced with an LC3 protein fused to both a red
fluorescent protein (mCherry) and an enhanced green fluores-
cence protein (EGFP) showed a predominantly red signal,
which occurs when the mCherry-EGFP-LC3 protein resides
inside the acidified interior of an autolysosome (Fig. 8F, [43]).
In keeping with previous reports that IFN-c induces autophagy
[44,45], we detected enhanced LTRed staining in either IFN-c
treated MEFs or A549 cells (Fig. 4A, S10A). We conclude that
increases in IFITM3 levels expand the lysosomal/autolysoso-
mal compartment.
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Figure 6. IFITM3 partially colocalizes with Rab7 and LAMP1, and compartments containing these proteins are amplified with
IFITM3 overexpression. A) A549 cells stably transduced with either IFITM3 or with the empty vector alone, were incubated with LTRed (red) at
37uC, then fixed and immunostained for confocal imaging of IFITM3 (endogenous and overexpressed, gold), and LAMP1 (endogenous, green).
DNA=blue. (Scale bars: 20 mM throughout). B) Percent colocalization of IFITM3, LTRed and LAMP in A549-Vector (blue) or IFITM3 (red) cells in (A). C)
A549 cells stably transduced with either IFITM3 or with the empty vector alone were immunostained for confocal visualization of IFITM3 (endogenous
and overexpressed, red) and Rab7 (endogenous, green). DNA=blue. D) Percent colocalization of IFITM3 and Rab7 in either the A549-Vector or A549-
IFITM3 cells in (C). E) MDCK-Vector or MDCK-IFITM3 cells stained for exogenous IFITM3 (overexpressed, red) and LAMP1 (endogenous, green).
DNA=blue. F) Percent colocalization of IFITM3 and LAMP1 in MDCK-Vector or MDCK-IFITM3 cells in (E). G) MDCK cells stably overexpressing Rab7-YFP
and either IFITM3 (MDCK-IFITM3) or the empty vector control (MDCK-Vector) were immunostained and confocally imaged for IFITM3 (overexpressed,
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Discussion
Here we report several novel findings regarding the antiviral
actions of IFN and the transmembrane IEG, IFITM3. First, this
study demonstrates that IFN inhibits the nuclear translocation of
vRNPs, and that IFITM3 is required for this IFN-mediated block,
with both endogenous and overexpressed IFITM3 inhibiting
vRNP nuclear entry. Second, either endogenous or overexpressed
IFITM3, as well as IFN treatment, block the fusion of viral
pseudoparticles expressing various influenza A virus envelope
proteins (H1, H3, H5 and H7 subtypes of HA), or the VSV-G
envelope protein; this block is specific because the fusion of
pseudoparticles expressing MLV envelope is not inhibited by
IFITM3. Third, our work reveals that IFITM3 partially resides
with Rab7 in late endosomes, thus placing it in position to block
influenza A virus’ cytosolic access. Fourth, IFITM3 overexpression
or IFN induce the expansion of late endosomal and lysosomal
compartments containing Rab7 and LAMP1. Fifth, we show that
similar to IFN-c treatment, IFITM3 overexpression expands the
number and size of autolysosomes, and it is into these
compartments that trapped viruses are trafficked and subsequently
degraded. Consistent with previous reports, our data show that
high levels of IFITM3 do not prevent viral access to acidified
compartments and that IFITM3 colocalizes with CD63 and
LAMP1 [19]. This is in contrast to a report noting the exclusion of
overexpressed IFITM3 from LAMP1-containing structures [33].
Therefore, this work adds substantially to our interpretation of
previous reports by demonstrating that key downstream events in
the viral lifecycle, fusion and vRNP nuclear translocation, are
prevented by either IFN or IFITM3. IFITM3 thus represents a
previously unappreciated class of anti-viral effector that permits
viral entry into the endosomal compartment, but prevents egress
into the cytosol. These studies also raise new questions including i)
how do IFN and IFITM3 prevent viral fusion? ii) how do IFN and
IFITM3 alter the endosomal and autolysosomal compartments?
and iii) is the latter action required for viral restriction, or
alternatively does it arise as an outcome of IFITM3’s potential
cellular role?
Based on the substantial loss in IFN’s potency observed when
IFITM3 is depleted (50–80% loss of viral inhibition, Fig. S9A, B,
[14]) we conclude that inhibition of viral emergence from the
endosomal pathway is a prominent component of IFN’s
antagonism of influenza A virus replication in vitro. Our data also
show that MxA cannot fully compensate for the loss of IFITM3 in
IFN-treated cells challenged with influenza A virus. Recent work
by Dittmann et al. [46] and Zimmermann et al. [47] reveal that
human influenza A viral strains have evolved a means to evade
MxA, suggesting a possible explanation for the cellular reliance on
IFITM3 for protection in vitro. Similarly the IEG, IFIT1, prevents
viral replication by targeting viral 59 triphosphate-RNAs (PPP-
RNA) for destruction [48,49]. Given that IFITM3 is necessary for
the majority of IFN-mediated restriction of influenza A virus in
vitro, it may be that the virus has also evolved a means to at least
partially nullify IFIT1, perhaps via the massive production of short
‘‘decoy’’ PPP-RNAs, as previously postulated [49,50].
IFITM3 primarily resides in the endosomal compartment and
partly colocalizes with Rab7 and LAMP1. IFITM3 overexpression
or IFN stimulation caused the endocytosed viruses to accumulate
in acidic compartments that contained both IFITM3 and LAMP1.
Together with the BLAM-Vpr fusion assay data, these results
reveal that IFITM3 prevents viral-host membrane fusion within
late endosomes, and likely within lysosomes as well, in light of
studies showing IFITM-mediated restriction of filoviruses and
coronaviruses, which depend on cathepsin-mediated activation
prior to fusion [19]. In doing so, IFITM3 traps the virus on a path
which terminates in a degradative environment [51]. In support of
this, our experiments show the eventual loss of a detectable vRNA
signal in the LTRed-positive compartments of the IFITM3-
transduced cells, thus revealing the fate of viral fitness under those
conditions.
These studies also reveal that elevated levels of IFITM3
correlate with the expansion of host cell structures containing
Rab7 and LAMP1, and that IFITM3 was also present in these
structures. In the MEF and A549 experiments, IFN produced
increased Rab7 and LAMP1 immunostaining, in addition to an
increase in acidic structures. At present, we cannot explain the
increased Rab7 and LAMP1 signals seen after IFN stimulation or
IFITM3 overexpression solely on the slight elevations in the
abundance of these proteins detected by immunoblotting. Two
possible explanations for the increased immunostaining observed,
are that IFN stimulation induced these proteins to cluster together
or alternatively unmasked sequestered epitopes; we find the latter
possibility less likely since LAMP1 and Rab7 flourescent fusion
proteins also showed larger and more intense signals under similar
conditions. We envision that IFITM3-mediated clustering of
organelles and their protein cargoes might contribute to the host
cell’s antiviral state. Earlier work reported no correlation between
the size of the IFITM3-induced acidified compartments and the
level of viral restriction [19], however, we observe that increasing
levels of IFITM3 result in both an expansion of lysosomes/
autolysosomes and increased viral inhibition. These observations
might be explained by a common mechanism underlying the
increase in these structures and viral inhibition, in addition to
raising the possibility that they play a role in IFITM-mediated
viral restriction.
Is there a common characteristic shared by IFITM3-susceptible
viruses? The late endosomal- and lysosomal-associated small
GTPase, Rab7, is required for influenza A virus infection [7,41].
The IFITM3-resistant viruses previously tested (MLV, the arena
viruses and the hepacivirus, HCV) are all Rab7-independent,
while the entry of the IFITM3-susceptible viruses (influenza A,
dengue,Ebola,Marburg, and
[14,19,41,52,53,54]. Standing against this hypothesis, is the lack
of effect on VSV-G-mediated entry with expression of a dominant
negative Rab7 [41,55,56]). However, additional studies have
shown that VSV-G-directed entry is dependent on transport to the
late endosomes [57,58]; these latter results, together with those of
Huang et al. and Weidner et al. [19,20], are consistent with the
prediction that viruses that fuse in late endosomes or lysosomes are
vulnerable to IFITM3’s actions, while viruses whose genomes
enter at the cell surface or in the early endosomes may avoid
IFITM3’s full effect. Of note, we have been unable to demonstrate
that IFITM3 blocks HIV-1 replication using TZM-bl HeLa cells
and are working to address these differences with a published
study ([59], data not shown).
This study, together with previous work, demonstrates that
IFITM3 permits endocytosis of viruses, but prevents viral fusion
and the subsequent entry of viral contents into the cytosol [19,20].
SARS)relies on Rab7
red) and Rab7-YFP (fluorescent signal from exogenous protein, green). Nuclear peripheries are represented by blue lines. H) Percent colocalization of
Rab7-YFP and IFITM3 in either the MDCK-Vector or IFITM3 cells in (G). I) Enlarged view of images outlined by white boxes shown in (G), with MDCK-
IFITM3 cells stably overexpressing both IFITM3 (red) and Rab7-YFP (green).
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While the BLAM-Vpr fusion assay demonstrates inhibition of
fusion by IFN or by IFITM3, we note that this assay uses an
indirect readout to assess entry of viral contents. Therefore several
possibilities could explain the containment and neutralization of
viruses within the endosomal pathway, including alterations in
endosomal trafficking, acidification, or the host membrane’s fusion
characteristics (bending modulus, elasticity). While additional work
is required to further define the mechanism, the lack of toxicity
seen with cells stably overexpressing high levels of IFITM3
suggests that gross alterations in endogenous trafficking or pH
control are unlikely (data not shown). Therefore overexpressing or
activating IFITM3 to produce an enhanced antiviral state may be
an effective prevention strategy during high risk periods in
vulnerable populations.
We propose that IFN causes the degradation of endocytosed
viruses by preventing their contents from entering the host cytosol,
Figure 7. IFN treatment or IFITM3 overexpression expands late endosomes and lysosomes. A549 cells stably expressing IFITM3 (IFITM3)
or empty vector (Vector) were (A) left untreated (Buffer) or (B) treated with IFN-a, then fixed, permeabilized and immunostained for IFITM3
(endogenous and overexpressed, red), Rab7 (endogenous, gold), LAMP1 (endogenous, green), and for DNA (blue, merged image). Images were
obtained using a confocal microscope. Similar results were observed with IFN-c (data not shown). (Scale bars: 20 mm throughout). C) Whole-cell
lysates from A549-IFITM3 or A549-Vector cells in (A) and (B) treated or untreated with IFN-a or c were subjected to immunoblotting against the
proteins indicated. GAPDH levels are provided to demonstrate comparable protein loading. Molecular weights in kDa are provided to the left. These
images are representative of three independent experiments. D) A549 cells stably expressing Rab7-YFP (fluorescent signal from exogenous protein,
green) were left untreated (Buffer) or treated with IFN-a, then fixed, permeabilized and immunostained for IFITM3 (endogenous, red) and imaged
confocally. DNA=Blue. Similar results were obtained for IFN-c (data not shown). E) Percent colocalization of IFITM3 and Rab7-YFP in the A549 cells in
(D), with or without IFN-a treatment.
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and that IFITM3 is necessary and sufficient for this defense
(Figure 8G). IFITM3’s mode of defense could be envisioned as an
effective means to neutralize pathogens during an organism-wide
threat. Such actions might confer an advantage to the host because
if IFITM3 simply decreased viral attachment and/or entry, the
repulsed viruses would be free to attack neighboring cells. Of
course while there are considerable differences between this simple
scenario and the directed phagocytosis of pathogens by specialized
immune cells, i.e. macrophages, the similarities none-the-less
suggest an early prototype for a more evolved defense mechanism.
Materials and Methods
Cell lines and culture conditions
U2OS, A549, MDCK, HeLa cells (all from ATCC), and
chicken embryonic fibroblasts (ChEFs, from Charles River Labs)
were grown in complete media (DMEM, Invitrogen Cat#11965)
with 10% FBS (Invitrogen). WI-38 cells (ATCC) were cultured in
DMEM (Invitrogen Cat#10569), containing non-essential amino
acids (Invitrogen Cat#11140) and 15% FBS. Wild type and
matched IfitmDel2/2 MEFs were from adult IfitmDel+/2 mice
[26] that were intercrossed and MEFs derived from embryos at
day 13.5 of gestation, as described previously [14]. The MEFs
were genotyped by PCR and Western blot, and the generation of
the IfitmDel2/2 Ifitm3 cells have been previously described [14].
Plasmids
The IFITM3 retroviral vector, pQCXIP-IFITM3 and empty
vector control (Clontech) have been previously described [14].
The shRNA lentiviral vectors, pLK0.1-Scramble and pLK0.1-
shIFITM3-3 (clone ID HsSH00196729) are available from the
Dana Farber DNA core, Harvard Medical School, Boston, MA.
pCAGGS-HA WSN/33 and pCAGGS-NA WSN/33 were kind
gifts of Dr. Donna M. Tscherne and Dr. Adolpho Garcia-Sastre,
Microbiology Dept., Mt. Sinai School of Medicine, NY, NY [38].
pBABE-mCherry-EGFP-LC3B was fromAddgene(Plasmid
Figure 8. IFITM3 overexpression results in the expansion of acidified organelles. A) MDCK or (B) A549 cell lines, stably overexpressing
IFITM3 or the empty vector alone, were incubated with either the acidophilic dye acridine orange (AO), LTRed, or a flourogenic cathepsin-L substrate
(Cath-L). All cells were also stained for DNA (blue). After incubation cells were imaged on a confocal microscope. Middle panels show enlarged images
of the IFITM3 cells. (Scale bars: 20 mm throughout). C) Vector (blue) or IFITM3 (red) transduced cell lines, either MDCK (left) or A549 (right), were
incubated with LTRed then analyzed by flow cytometry. D) A549 cells stably transduced with IFITM3 or with the vector alone were incubated with
LTRed (red), then immunostained for confocal imaging of LC3 (endogenous, green). DNA=blue. E) MDCK cells stably transduced with IFITM3 or with
the vector alone were immunostained for confocal imaging of LC3 (endogenous, red) and CD63 (endogenous, green). DNA=blue. F) Confocal images
of MDCK cells overexpressing IFITM3 or the empty vector alone showing the distribution and fluorescence intensities of a stably expressed mCherry-
EGFP-LC3B fusion protein using fluorescence channels that detect light emitted from the mCherry protein, EGFP or both (merge). DNA=blue. G)
Model of IFITM3-mediated restriction of virus replication. Endocytosed viruses enter late endosomes where IFITM3 is present. IFITM3 prevents viral
fusion within the endosomes and likely lysosomes via an unknown mechanism, perhaps by altering pH, membrane characteristics, lipid composition,
transport speed or destination. Trapped viruses are trafficked to lysosomes and/or autolysosomes where they undergo degradation.
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#22418) and was kindly deposited by Jayanta Debnath. pLZS-
Rab7-YFP and pLVX-RFP-LAMP1 were generously provided by
Walther Mothes, Section of Microbial Pathogenesis, Yale
University School of Medicine. The following shRNA sequences
(sense strand sequence provided) were cloned into the pAPM
shRNA-expression lentiviral vector [60], to create the viruses used
to generate the A549 IFITM3 knockdown cell lines in Fig. S9:
IFITM3-1: 59-TCCTCATGACCATTCTGCTCAT-39
IFITM3-2: 59-CCCACGTACTCCAACTTCCATT-39
IFITM3-3: 59-TTTCTACAATGGCATTCAATAA-39
Viral propagation and titration
Influenza A virus A/Puerto Rico/8/1934 (H1N1) (PR8,
Charles River Labs) and A/WSN/33 (H1N1) (kind gift of Dr.
Peter Palese, Microbiology Dept., Mt. Sinai School of Medicine,
NY, NY) were propagated and assessed for viral infectivity as
previously described [14]. Influenza A virus A/Vietnam/1203/
2004 (H5N1) was propagated and characterized as previously
described [61].
Cytokines
Human interferon (IFN)-c (Invitrogen) was used at 100–
300 ng/ml, human IFN-aA2 (PBL Interferon Source) was used
at 500–2500 U/ml. Cells were incubated with cytokines for 16–
24 h prior to IF or viral infection experiments unless otherwise
noted. Murine IFN-c (PBL Interferon Source) was used at 100–
300 ng/ml.
Western analysis
Whole-cell extracts were prepared by cell lysis, equivalent
protein content boiled in SDS sample buffer, resolved by SDS/
PAGE, transferred to Immobilon–P membrane (Millipore), and
probed with the indicated antibodies.
Time course infection experiments and confocal
microscopy
Cells were seeded on glass coverslips for Influenza A virus
infection experiments. Cells were incubated on ice with PR8 for
40 min. At time zero, the viral supernatant was removed and 37uC
media was added with or without Lysotracker Red DND-99
(Invitrogen). At the indicated time points post-warming, cells were
washed twice with D-PBS (Sigma) and incubated for 30 seconds
with room temperature 0.25% trypsin (Invitrogen). The cells were
then washed with complete media twice and fixed with 4%
formalin (PFA, Sigma) in D-PBS. Image analysis for quantitation
of vRNP nuclear translocation was done using Imaris 7.1 (bitplane
scientific software). We generated a mask of the nucleus and
applied this mask to the channel containing the viral signal
(puncta) to determine vRNA puncta contained in each nucleus.
Live cell imaging experiments
Cells were incubated at 37uC and 5% CO2for 60 min. with
either Lysotracker Red DND-99 or acridine orange (Immuno-
Chemistry Technologies). Hoechst 33342 (DNA stain, Invitrogen)
was incubated (1:10,000) with the cells for the final 15 min. The
Cathepsin L flourogenic substrate assay was performed as per the
manufacturer’s instructions (Cathepsin L -Magic Red, Immuno-
Chemistry Technologies). Cells were visualized live by confocal
microscopy.
Immunoflourescence protein
Cells were fixed in 4% PFA in D-PBS, and then incubated
sequentially in 0.25% Tween 20 (Sigma), then 1% BSA with
0.3 M glycine (Sigma), both in D-PBS. Primary and secondary
antibodies are listed below. Slides were mounted in Vectashield
with DAPI counterstain (Vector Labs). Slides were imaged using a
Zeiss LSM 510, laser scanning inverted confocal microscope
equipped with the following objectives: 406Zeiss C-APOCHRO-
MAT UV-Vis-IR water, 1.2NA, 636 Zeiss Plan-APOCHRO-
MAT DIC oil, 1.4NA, and 1006 Zeiss Plan-APOCHROMAT
DIC oil, 1.46NA. Image analysis was performed using ZEN
software (Zeiss). Laser intensity and detector sensitivity settings
remained constant for all image acquisitions within a respective
experiment. Nuclear outlines were generated using Metamorph
software suite (Molecular Devices) using the Kirsch/Prewitt filter
to define boundaries and then subtracting out the original binary
images.
Antibodies
The following antibodies were used in this study for either
Western blotting (WB) or immunoflourescence (IF), or both as
indicated, along with their respective source and catalogue
number: Primary antibodies: Actin (Sigma A5316, WB), CD63
(Developmental Studies Hybridoma Bank (DSHB) clone H5C6,
IF), Fragilis (mouse Ifitm3) (Abcam ab15592, WB, IF), GAPDH
(BD Biosciences 610340, WB), HA (Wistar collection, Coriell
Institute, clone H18-S210, WC00029, IF), IFITM3 (Abgent
AP1153a, WB, IF), IFITM3 (Abgent AP1153c, IF), LAMP1
((DSHB) clone H4A3, WB, IF), LC3 (Nanotools Mab LC3-5F10,
WB, IF), MX1 (Proteintech 13750-1-AP, WB, IF), NP (Millipore
clone H16-L10-4R5 MAB8800, IF), RAB7 (Abcam 50533, WB,
IF). Secondary antibodies for IF (all from Invitrogen): Alexa Fluor
488 and 647 (goat anti-rabbit and goat anti-mouse). The LAMP1
[H4A3] and CD63 [H5C6] antibodies were developed by J.T.
August and J.E.K. Hildreth and were obtained from the DSHB
and developed under the auspices of the NICHD and maintained
by The University of Iowa, Department of Biology, Iowa City, IA.
Immunoflourescence RNA
These experiments employ the QuantiGene ViewRNA slide-
based assay kit from Affymetrix (Cat #QV0096) with all
components from that source unless noted. RNA was visualized
following a modified manufacturer protocol; changes made
include the omission of the ethanol dehydration step, and use of
Vectashield mounting media. Post-fixation with 4% PFA, cells
adherent on coverslips were incubated with 16detergent solution
or incubated in 0.25% PBS-Tween20. Cells were then incubated
with Proteinase K. Next cells were incubated at 40uC in
hybridization solution A containing a viewRNA probe set
designed against either the negative stranded RNA NP genome
(vRNA) of PR8 (Affymetrix VX1-99999-01 QG ViewRNA TYPE
1 Probe Set against NP Influenza A virus (A/PuertoRico/8/
34(H1N1)) at 1:100) or a probe set against the positive stranded
NP mRNA. Cells were then incubated in hybridization pream-
plifiers (1:100 in hybridization buffer B) at 40uC. Finally cells were
incubated with labeled probes (1:100 in hybridization buffer C),
washed and imaged as above. All steps were followed by two D-
PBS washes.
BLAM-Vpr pseudoparticle fusion assays
Pseudotyped lentiviral particles expressing the HA envelope
were produced by plasmid transfection of HEK 293T cells with an
HIV-1 genome plasmid derived from pBR43IeG-nef+ (NIH AIDS
Research and Reference Reagent Program (Division of AIDS,
NIAID, NIH, Cat#11349, from Dr. Frank Kirchhoff) modified
with a deletion which abolishes expression of Env without
disrupting the Rev-responsive element, pCAGGS-HA WSN/33,
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pCAGGS-NA WSN/33 and pMM310, which encodes a hybrid
protein consisting of b-lactamase fused to the HIV accessory
protein, Vpr (NIH AIDS Research and Reference Reagent
Program, Division of AIDS, NIAID, NIH (Cat#11444) from
Dr. Michael Miller). pCG-VSV-G together with pBR43IeG-nef+
and pMM310 were transfected to produce VSV-G pseudotyped
lentiviral particles. For the H5N1, H3N1, and H7N1 pseudopar-
ticles, pCAGGS-HA5 (A/Thailand2(SP-33)/2004) pCAGGS-
HA3 (A/Udorn/72), and pCAGGS-HA7 (A/FPV/Rostock/34)
expression plasmids were co-transfected with the pCAGGS-NA
WSN/33, pMM310, and the pBR43IeG-nef+ lentiviral backbone.
Cultures for pseudoparticle fusion assays, including stably
transduced MDCK cells and WI-38 fibroblasts, were plated in
24-well dishes with 90,000 cells per well at the beginning of each
assay. At the time of assay, 0.5 mL of virus stock was added to cells
and incubated for 2–3 h (depending on cell type) at 37uC. In
experiments using bafilomycin A1 (Sigma), the inhibitor was
added at 0.1 nM final concentration (low dose) at 37uC for 1 h
prior to incubation with virus. After infection, viral media was then
aspirated and replaced with complete DMEM containing CCF2-
AM (Invitrogen) along with 1.7 mg/mL probenecid (Sigma). Cells
were incubated in the dark for 1 h, followed by dissociation from
the dish using Enzyme Free PBS-based Dissociation Buffer, and
fixation in 2% PFA. Flow cytometry was conducted on a Becton
Dickinson LSRII using 405 nm excitation from the violet laser,
and measuring 450 nm emission in the Pacific Blue channel and
520 nm emission in the Pacific Orange channel. Data was
analyzed using FACSDiva and FlowJo8.8.7.
Sialic acid linkage expression studies
A549 cells stably transduced to overexpress IFITM3 or with
empty expression vector (pQCXIP, Clontech) were grown to
,50% confluency, dissociated with trypsin-free EDTA-based
dissociation buffer (Invitrogen) for 10 min. at 37uC. Cells were
incubated at 4uC with FITC-conjugated Sambucus nigra lectin
(SNA, Vector Labs #FL-1301) to detect (a-2,6) sialic acid linkages,
and biotinylated Maackia amurensis lectin II (MAL, Vector Labs
#B-1265) to detect (a-2,3) sialic acid linkages, followed by
streptavidin-PE-Cy7 (Invitrogen). Cells were incubated with lectins
individually and in combination, and the results of staining were
indistinguishable. All cells were stained with violet cell-imperme-
able dye (Invitrogen #L34955), and cells were included in the
analysis if viable by FSC/SSC and viability dye.
Binding assay
A549 cells transduced with IFITM3 or the empty vector
pQXCIP were detached using Enzyme Free PBS-based Dissoci-
ation Buffer, and then washed in cold PBS extensively. Cells and
virus (WSN/33) were pre-chilled on ice for 30 min. and mixed at a
moi of 50 and incubated at 4uC for 1 h with rotation. Cells were
washed extensively with ice cold PBS and then fixed using 4%
PFA. The cells were then probed with anti-HA mouse monoclonal
antibody (Wistar collection, Coriell Institute, clone H18-S210,
WC00029, IF) for 1 h at room temperature, followed by anti-
mouse AlexaFlour-488 conjugated antibody (Invitrogen) for 1 h
with PBS washes in between, then analyzed by flow cytometry.
Supporting Information
Figure S1
surface levels of (a-2,3) or (a-2,6) sialylated proteins.
A549 cells stably transduced with IFITM3 or the empty vector
were incubated with biotinylated Maackia Amurensis lectin II (MAL)
to detect (a-2,3) sialic acid linkages, followed by streptavidin-PE-
IFITM3 overexpression does not alter the
Cy7, as well as FITC-conjugated Sambucus Nigra lectin (SNA) to
detect (a-2,6) sialic acid linkages. A) The percentage of IFITM3 or
vector cells staining positive for both sialic acid linkages (upper
right hand quadrant), compared to unstained controls. B) IFITM3
overexpressing and vector cells are compared with regard to each
sialic acid linkage in the double-stained populations.
(PDF)
Figure S2
cytosolic inclusions preventing vRNP nuclear transloca-
tion. A) Normal diploid human fibroblasts (WI-38 cells) were
stably transduced with retroviruses containing IFITM3 (WI-38
IFITM3) or (B) a non-targeting control shRNA (WI-38 shScram-
ble). Cells were incubated with PR8 on ice, and then warm media
containing LTRed (red) was added at time zero. Cells were fixed
at 150 min. p.i. and stained for NP (green) and DNA, then
analyzed by confocal microscopy. Image analysis software was
used to define each cell’s cytosolic (white lines) and nuclear
peripheries (blue lines, based on DIC images and DNA staining,
respectively). Images are representative of four independent
experiments. (Scale bar: 12 mM). C) Quantitation of nuclear
vRNP particles. The number of vRNP particles per nucleus of the
WI-38 cell lines (with or without IFN treatment) at the indicated
time points are shown. Values represent the mean +/2 the SD of
three independent experiments. D) Percent colocalization of
vRNPs and LTRed compartments in WI-38 shScramble,
shIFITM3 or IFITM3 expressing cells at the indicated times p.i.
Values represent the mean +/2 the SD of three independent
experiments. E) Western blot of lysates from WI-38 cells probed
with the indicated antibodies. shIFITM3-3 is referred to as
shIFITM3 in the preceding figures and was selected for use based
on its superior knockdown of the target protein.
(PDF)
IFITM3 arrests influenza A virus in acidic
Figure S3
vRNP nuclear entry. A549 cells overexpressing the empty
vector control (A) or IFITM3 (B) were incubated with PR8 on ice
(moi 500). At time zero warm media was added along with LTRed
(red). At the indicated times, cells were processed and stained for
NP (green) and DNA (blue lines represent the nuclear periphery
based on staining), then imaged using a confocal microscope.
(Scale bar: 20 mM). These images are representative of three
independent experiments. C) Whole cell lysates of A549 cells used
in (A) and (B) treated with either buffer, IFN-a or IFN-c, were
subjected to immunoblotting using the indicated antibodies.
(PDF)
A549 cells overexpressing IFITM3 inhibit
Figure S4
A virus in acidic cytosolic inclusions prior to vRNP
nuclear translocation. MDCK cells stably expressing (A) the
empty vector control or (B) IFITM3 were incubated with Aichi
H3N2 virus on ice, and then warm media was added at time zero
along with LTRed (red). Cells were then fixed at the indicated
times p.i. and stained for NP (green), and DNA (blue lines denote
nuclear periphery), then imaged by confocal microscopy. Images
are representative of three independent experiments. (Scale bar:
20 mm). B) Quantitation of nuclear vRNP particles. The number
of vRNP particles per nucleus of the MDCK cell lines at the
indicated time points are shown. Values represent the mean +/2
the SD of three independent experiments. C) Percent colocaliza-
tion of vRNP and LTRed compartments in MDCK-Vector and
MDCK-IFITM3 cell lines at the indicated times p.i.
(PDF)
IFITM3 overexpression halts H3N2 influenza
Figure S5
organelles in cells overexpressing IFITM3. A) MDCK-
vRNPs are retained in LAMP1-containing
IFITM3 Inhibits Influenza A Virus’ Cytosolic Entry
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Page 15

Vector or IFITM3 cells stably expressing a LAMP1-red
fluorescence protein (LAMP1-RFP) were challenged with PR8 as
in Fig. S4. Cells were immunostained for NP (green), stained for
DNA, and then imaged confocally along with the collection of
LAMP1-RFP fluorescence (orange). Images are representative of
three independent experiments. Blue lines represent the nuclear
margins based on DNA staining. (Scale bar: 20 mM). B)
Quantitation of nuclear vRNP particles. The number of vRNP
particles present per nucleus of the MDCK cell lines at the
indicated time points are shown. Values represent the mean +/2
the SD of three independent experiments. C) Percent colocaliza-
tion of vRNP particles and LAMP1-RFP-containing compart-
ments in MDCK-Vector and MDCK-IFITM3 cell lines at the
indicated times p.i. Values represent the mean +/2 the SD of
three independent experiments.
(PDF)
Figure S6
inhibition of vRNP nuclear translocation in IfitmDel2/
2 MEFs. A) IfitmDel2/2 MEFs stably overexpressing Ifitm3
(IfitmDel2/2Ifitm3), were left untreated (left panels, Buffer), or
treated (right panels) with IFN-c. The following day cells were
incubated on ice with PR8 (moi 500). Cells were next incubated in
warm media containing LTRed (0 min.). Cells were then fixed at
the indicated times p.i., immunostained with anti-NP antibodies
(green) and imaged by confocal microscopy. Image analysis
software was used to define the nuclear boundaries (blue lines).
Images are representative of three independent experiments.
(Scale bar 12 cM). B) Percent colocalization of vRNP and LTRed
compartments in the indicated MEF cell lines, with or without
IFN-c treatment, are shown for the indicated times p.i. C)
Quantitation of nuclear vRNP particles. The number of vRNP
particles per nucleus of the MEF cell lines, with or without IFN-c
treatment, at the indicated time points are shown. Values
represent the mean +/2 the SD of three independent experi-
ments. D) Western blot of whole cell lysates from the indicated
MEFs probed with anti-mouse Ifitm3 and using GAPDH as a
loading control.
(PDF)
Ifitm3 expression rescues IFN-c-mediated
Figure S7
HA envelope subtypes, but not a MLV envelope, is
decreased by IFITM3. IFITM3 inhibits the replication of
infectious H5N1 virus. A) MDCK cells stably transduced with
IFITM3 or empty vector were incubated with pseudoparticles
expressing N1 and HA subtypes (H1N1pp, H3N1pp, or H5N1pp).
Cells were then fixed and assayed for cleavage of CCF2 using flow
cytometry. These results are representative of three independent
experiments. B) Chicken embryonic fibroblasts (ChEF) cells stably
expressing the empty vector control or IFITM3 were incubated with
pseudoparticles expressing N1 and either of the two avian influenza
A viral HA subtypes, H5 or H7, as in (A). These data are
representative of three independent experiments. C) ChEF cells
stably transduced with the empty vector control or overexpressing
IFITM3, were infected with WSN/33 for 12 h then stained for HA
protein (red) and DNA (blue). Average percent infection is given for
three independent experiments +/2 SD. 46 magnification. D)
MDCK-Vector or MDCK-IFITM3 cells were incubated with
pseudoparticles expressing the amphotropic MLV envelope protein
(MLVpp) and then assayed for cleavage of CCF2 using flow
cytometry. These results are representative of two independent
experiments. E) Infectivity of HA-expressing pseudoparticles is
decreasedby IFITM3. MDCK-Vectoror MDCK-IFITM3celllines
were infected with the indicated pseudoparticles for 48 h. Cells were
then immunostained for expression of HIV-1 p24 protein expressed
Fusion of viral pseudoparticles expressing
from the integrated lentiviral genomes. Percent infection is provided.
These results are representative of three independent experiments.
46 magnification. F) A549 cells were stably transduced with
retroviruses containing IFITM3 or empty viral vector alone, then
infected with A/Vietnam/1203/04 (H5N1) influenza A virus (VN/
04). After 12 h, the cells were fixed and stained for viral NP
expression (green) and for DNA (blue). Values given are percentage
infectedcellsandarerepresentativeoftwoindependentexperiments.
46magnification. G) Western blot of lysates from A549-IFITM3 or
A549-Vector cell lines probed with the indicated antibodies. H)
A549 cell lines were infected with increasing amounts of H5N1 VN/
04. Twelve hours after infection the cells were immunostained for
NP expression and scored for infection status. Values are
representative of two independent experiments.
(PDF)
Figure S8
HA-mediated fusion. A) HeLa cells were stably transduced
with retroviruses containing either IFITM3, a shRNA against
IFITM3 (shIFITM3), or a non-targeting control shRNA (shScr).
Cells were left untreated (left panels), or treated with IFN-c (right
panels), then exposed for 2 h to H1N1pps (WSN/33) containing
BLAM-Vpr. After incubation with the pseudoparticles, the cells
were fixed and assayed for cleavage of CCF2 by flow cytometry.
These results are representative of three independent experiments.
B) The indicated HeLa cell lines were treated with IFN-c for 24 h
then infected with increasing amounts of WSN/33. After 12 h of
infection the cells were stained for HA expression. These results
are representative of three independent experiments. C) Western
blot of the indicated HeLa cell line lysates probed with the
indicated antibodies.
(PDF)
IFITM3 is required for IFN’s inhibition of
Figure S9
susceptibility to influenza A virus infection. MxA is
expressed and is IFN-inducible in A549 and WI-38 cells.
A) A549 cells, stably transduced with retroviruses expressing
IFITM3, a negative control shRNA against firefly luciferase
(shLuc), or one of three shRNAs against IFITM3 (1, 2 or 3), were
treated with buffer, IFN-a or IFN-c for 24 h, then challenged with
WSN/33. After 12 h of infection, the cells were fixed and
immunostained for HA and stained for DNA. IF images were
captured and the percentage of infected cells determined based on
HA staining. Values represent the average of three independent
experiments +/2SD. B) Western lysates of A549 cells from (A)
probed with the indicated antibodies. Western lysates of (C) A549
cells or (D) WI-38 cells, treated with buffer, IFN-a or -c, then
probed with the indicated antibodies. E) Confocal images of WI-
38 cells treated with buffer or IFN-a, then fixed, permeabilized
and immunostained for either IFITM3 (endogenous, red), or MxA
(endogenous, green), and for DNA (blue, scale bar: 20 mM).
(PDF)
A549 cells depleted of IFITM3 show increased
Figure S10
IFITM3-containing structures, and increases the size
and number of acidified organelles. A) Confocal images of
WI-38 cells treated with buffer, IFN-a, or IFN-c, and then
immunostained for either IFITM3 (endogenous, red) or Rab7
(endogenous, green), and DNA (blue). Arrows denote larger
structures staining for Rab7 and IFITM3 that were seen
predominantly with IFN-c treatment. (Scale bar: 20 mM). B)
A549 cells treated with either buffer or IFN-c, then incubated with
LTRed before fixation and DNA staining (blue) followed by
confocal imaging. Images in this figure are representative of three
independent experiments.
(PDF)
IFN treatment both expands Rab7- and
IFITM3 Inhibits Influenza A Virus’ Cytosolic Entry
PLoS Pathogens | www.plospathogens.org15October 2011 | Volume 7 | Issue 10 | e1002337