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J Virol. 2013 July; 87(14): 7837–7852.
PMCID: PMC3700195

The CD225 Domain of IFITM3 Is Required for both IFITM Protein Association and Inhibition of Influenza A Virus and Dengue Virus Replication


The interferon-induced transmembrane protein 3 (IFITM3) gene is an interferon-stimulated gene that inhibits the replication of multiple pathogenic viruses in vitro and in vivo. IFITM3 is a member of a large protein superfamily, whose members share a functionally undefined area of high amino acid conservation, the CD225 domain. We performed mutational analyses of IFITM3 and identified multiple residues within the CD225 domain, consisting of the first intramembrane domain (intramembrane domain 1 [IM1]) and a conserved intracellular loop (CIL), that are required for restriction of both influenza A virus (IAV) and dengue virus (DENV) infection in vitro. Two phenylalanines within IM1 (F75 and F78) also mediate a physical association between IFITM proteins, and the loss of this interaction decreases IFITM3-mediated restriction. By extension, similar IM1-mediated associations may contribute to the functions of additional members of the CD225 domain family. IFITM3's distal N-terminal domain is also needed for full antiviral activity, including a tyrosine (Y20), whose alteration results in mislocalization of a portion of IFITM3 to the cell periphery and surface. Comparative analyses demonstrate that similar molecular determinants are needed for IFITM3's restriction of both IAV and DENV. However, a portion of the CIL including Y99 and R87 is preferentially needed for inhibition of the orthomyxovirus. Several IFITM3 proteins engineered with rare single-nucleotide polymorphisms demonstrated reduced expression or mislocalization, and these events were associated with enhanced viral replication in vitro, suggesting that possessing such alleles may impact an individual's risk for viral infection. On the basis of this and other data, we propose a model for IFITM3-mediated restriction.


The interferon (IFN)-induced transmembrane protein 3 (IFITM3) gene is a widely expressed interferon-stimulated gene that inhibits the in vitro replication of pathogenic viruses, including influenza A and B viruses (IAV and IBV, respectively), West Nile virus, dengue virus (DENV), severe acute respiratory syndrome (SARS) coronavirus, and the filoviruses, Ebola virus and Marburg virus (14). The antiviral activity of IFITM3 was discovered using orthologous functional genomics strategies (1, 36); notably, this single protein accounts for 50 to 80% of the in vitro antiviral actions of interferon and is also vital in vivo, where its attenuation can change a mild viral infection into a life-threatening event in either mice or humans (1, 79) (see Fig. 1A and andBB).

Fig 1
Two distinct regions of IFITM3's N-terminal domain (NTD) are required for restriction of IAV. (A) A549 cells stably transduced with a shRNA targeting IFITM3 (shM3) or a negative-control shRNA (shLuc) were treated for 16 h with or without IFN-α ...

IFITM3 has four paralogs in humans (IFITM1, -2, -5, and -10) and four orthologs in mouse (IFITM1, -2, -5, and -6). Among these family members, IFITM3 is most effective against IAV, while IFITM1 protects best against filoviruses (1, 2). The IFITM proteins contain two intramembrane domains (intramembrane domain 1 [IM1] and IM2) separated by a conserved intracellular loop (CIL [see Fig. 1C] [10]). The combined homology of IM1 and the CIL defines the CD225 domain, which is shared by the greater than 300 members of the CD225/pfam04505 family ( [11]). While the IFITM family's CD225 domain is highly conserved, the family's respective N-terminal domains (NTDs) display heterogeneity in both sequence and length, suggesting that they may contribute to the family's antiviral specificities.

Additional work has revealed that IFN blocks viral fusion subsequent to endocytosis, thereby preventing the cytosolic entry and nuclear translocation of viral genomes, and that IFITM3 is both required and sufficient for this action (2, 12, 13). IFITM3 partially colocalizes with the late endosomal and lysosomal host proteins RAB7, CD63, and lysosome-associated membrane glycoprotein 1 (LAMP1), placing it in the entry pathway of IFITM3-susceptible viruses (2, 12). Specifically, viruses that are restricted by IFITM3 normally enter cells by emerging from late endosomes and/or lysosomes, with the exception of vesicular stomatitis virus (VSV) (1, 12, 14). IFITM3 thus represents a previously unappreciated class of restriction factor that traps invading pathogens within the endocytic compartment resulting in their degradation (2, 12, 13). Other additional modes of IFITM3's antiviral actions, such as signaling, remain areas of active investigation.

Recent efforts have provided insight into the structure and function of the IFITM proteins by showing that maximal inhibition of VSV depends on the first 20 N-terminal residues of IFITM3 (13). Posttranslational modifications (PTMs) of IFITM3 include both palmitoylation and ubiquitination, with the former being necessary for restriction and the latter decreasing its antiviral action (10, 15). We now present data elucidating the structure and function of IFITM3, the most potent anti-IAV IFITM. To comprehensively identify the molecular determinants of IFITM3-mediated restriction, we performed unbiased alanine scan (AS) mutagenesis along the entire protein. IFITM3 AS mutants were assessed for their restriction of an orthomyxovirus, IAV, and a flavivirus, DENV. We found that both the NTD and the CD225 domain contained multiple regions that were important for antiviral action. Further mutagenesis revealed multiple single residues involved in viral inhibition, thus providing insight into the molecular determinants of IFITM3-mediated restriction.

Of note, we learned that specific amino acids within IM1 are needed for the physical interaction of the IFITM proteins and that this activity correlates with restriction, thus providing one of the first structural and functional associations for the CD225 domain. Comparison of the mutant proteins also showed that common determinants within IFITM3 are needed for the inhibition of IAV and DENV, with the exception of R87 and Y99 in the CIL, whose loss preferentially permits IAV infection. These data suggest that IFITM3 that is properly localized to the late endocytic pathway relies on IM1-mediated interactions, in conjunction with the actions of critical residues within the NTD and CIL, to maximally inhibit viral fusion. Given the emerging data on membrane-associated proteins altering the biophysical properties of lipid bilayers, we also postulate that IFITM3 modifies the host membrane, thereby preventing viruses from successfully infecting our cells.




Cell culture conditions.

A549, HeLa, MDCK, and HEK293T cells (ATCC) were grown in Dulbecco modified Eagle medium (DMEM) (Invitrogen) with 10% fetal bovine serum (FBS) (Invitrogen). Human gamma interferon (IFN-γ) (Invitrogen) was used at 100 to 300 ng/ml. Human alpha interferon (IFN-α) (PBL) was used at 1,000 U/ml.

A549 cell lines.

Retroviral particles were produced as previously described (16). A549 cells were transduced with retroviruses and selected using puromycin (2.0 μg/ml; Clontech) or hygromycin (200 μg/ml; Invitrogen) for a week. The expression of the respective constructs was checked by immunoblotting of whole-cell lysates.


Whole-cell lysates were prepared using Laemmli buffer and resolved by SDS-PAGE. The samples were then transferred to Immobilon-P membrane (Millipore) and probed with the indicated antibodies. Anti-NTD IFITM3 antisera (catalog no. AP1153a; Abgent) was probed at a dilution of 1:250. Secondary anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (catalog no. 211-062-171; Jackson ImmunoResearch) was used at 1:5,000. Anti-CIL antibody raised against IFITM1 (catalog no. 5807; ProSci) was used at 1:500. Anti-NTD IFITM1 (catalog no. 60074-1-Ig; Proteintech) was used at 1:1,000. Antiactin was used at 1:5,000 (catalog no. A5316; Sigma) (Western blotting [WB]).

Viral propagation and infections.

IAV (H1N1) A/WSN/33 (WSN/33) was propagated as previously described (1) and was a kind gift from Peter Palese (Mt. Sinai Medical Center). DENV serotype 2 (New Guinea C [NGC] strain; ATCC) virus was grown on C6/36 cells (a kind gift from Priscilla Yang, Harvard Medical School) or Vero cells (ATCC), and infectivity was titrated in HeLa and A549 cells by immunofluorescence (IF) assays. A549 cells expressing the indicated IFITM constructs were plated at equivalent cell densities the day before infection. IAV and DENV stocks were added at multiplicities of infection (MOIs) of 0.5 to 1.0. IAV-infected cells were fixed 12 h postinfection, and DENV-infected plates were fixed 36 h postinfection, using 4% paraformaldehyde (PFA) (Sigma) in Dulbecco's PBS (D-PBS) (Invitrogen). IAV-infected cells were immunostained for HA expression using an anti-HA monoclonal antibody (Wistar Institute) (12) in D-PBS with 1% bovine serum albumin (BSA). DENV-infected cells were first permeabilized using 0.1% Triton X-100 (Sigma) and immunostained with anti-DENV E-protein monoclonal antibody (a kind gift from John Aaskov, Queensland University of Technology). Both IAV- and DENV-infected cells were then immunostained with a secondary antibody, goat anti-mouse conjugated with Alexa Fluor 488 (goat anti-mouse 488) at 1:1,000 (Invitrogen) in D-PBS with 1% BSA. Host cell DNA was stained with Hoechst 33342 (Sigma) in D-PBS (1:10,000). Cells were imaged on an automated Image Express Micro (IXM) microscope (Molecular Devices) and analyzed using the MetaMorph cell scoring software program (Molecular Devices). For Fig. 1B, the indicated A549 cell lines were infected with WSN/33 for 12 h, the supernatant was then removed, and the cells were washed once with D-PBS. Fresh complete medium was then added, and 6 h later, this supernatant was used to infect MDCK cells. After 12 h, the MDCK cells were then fixed and stained for HA expression and DNA and analyzed as described above.

Immunoprecipitation for protein association.

HEK293T cells (ATCC) were transfected with the indicated untagged wild-type IFITM3 or IFITM3 mutants, and the HA-tagged IFITM1, IFITM2, or IFITM3 plasmid DNA using TransIT-293 transfection reagent (Mirus Bio), following the manufacturer's protocol. Forty hours posttransfection, cells were washed with ice-cold phosphate-buffered saline (PBS) (Sigma) and lysed with 2 ml ice-cold lysis buffer {50 mmol/liter Tris [pH 7.4], 150 mmol NaCl, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) (Affymetrix)} supplemented with an EDTA-free protease inhibitor cocktail (Roche). Crude cell lysates were scraped off the surface of the flask, transferred to prechilled tubes, and rotated at 4°C for 20 min. The crude cell lysates were then centrifuged at 20,000 × g for 30 min at 4°C. One milliliter of the supernatant was then transferred to a prechilled microcentrifuge tube and subjected to 100,000 × g for 1 h at 4°C. Nine hundred microliters of the clarified lysate was transferred to a prechilled tube for the immunoprecipitation (IP), and the remaining clarified lysate was diluted equally in 2× Laemmli sample buffer, incubated at 100°C for 5 min, and stored at −80°C. Two micrograms of mouse anti-HA antibody (clone HA-7; Sigma) was conjugated to 50 μl of protein G Dynabeads (Invitrogen) following the manufacturer's instructions. The beads were washed 3 times with 1 ml ice-cold lysis buffer, resuspended in 100 μl lysis buffer, and added to the clarified cell lysates. After 2 h rotating at 4°C, the protein G magnetic bead immune complexes were washed 5 times with 1 ml ice-cold lysis buffer, resuspended in 100 μl of 1× Laemmli sample buffer, resolved on SDS-polyacrylamide gels, and immunoblotted with either anti-HA (clone HA-7; Sigma) or anti-IFITM3 (Abgent). For Fig. 6I, A549 cells stably expressing either an IFITM3 fused to a single HA epitope tag (M3-1HA) (cells transduced with MSCV-IFITM3-HA6R [MSCV stands for murine stem cell virus]) (1) or the empty vector alone were treated with either IFN-γ or buffer for 16 h and then processed as described above.

Fig 6
IM1 within the CD225 domain is required for IFITM protein interactions. (A) The indicated IFITM protein plasmids (HA tagged) were cotransfected into 293T cells along with untagged IFITM3. Forty-eight hours later, the cells were lysed in CHAPSO buffer, ...

Affinity purification coupled to mass spectroscopy.

A549 cells stably expressing either an IFITM3 fused to a single HA epitope tag (M3-1HA) (cells transduced with MSCV-IFITM3-HA6R) (1) or the empty vector alone were grown to 75% confluence. The cells were washed in cold PBS three times with centrifugation at 1,000 rpm and then lysed in 3 ml MCLB (50 mM Tris [pH 7.5], 150 mM NaCl, and 0.5% NP-40) containing protease and phosphatase inhibitors (MLCB-PIs) (Roche) with shaking at 4°C, followed by clarification by centrifugation. The supernatant was mixed with HA agarose beads (Sigma) prepared in MCLB-PIs and incubated overnight at 4°C. The beads were subsequently washed in cold MCLB-PIs five times followed by two cold PBS washes. The proteins were then eluted with HA peptides (Sigma) in PBS with incubation at room temperature. Supernatant was collected by centrifugation at 2,000 rpm. To remove the HA peptides, a trichloroacetic acid (TCA) (Sigma) precipitation was carried out. The pellet was washed in ice-cold 10% TCA and then washed in ice-cold acetone (Sigma). After air drying, the pellet was subjected to trypsin digestion prior to mass spectroscopy (MS) analysis.

Confocal microscopy studies.

The cells were fixed with 4% PFA in D-PBS and incubated sequentially in D-PBS containing 0.1% Triton and 0.1% Tween 20 and then D-PBS containing 1% BSA and 0.3 M glycine (all from Sigma). For γ-tubulin immunostaining, the cells were fixed in −20°C methanol for 10 min and then washed with D-PBS and processed as described above. As noted for each experiment, primary and secondary antibodies were diluted in 1% BSA in D-PBS. Antibodies were used in pairs of primary and secondary antibodies: Fragilis (1:150; catalog no. AP1153a; Abgent) and γ-tubulin (1:100; HM2569 antibody, a kind gift from Sambra Redick and Steven Doxsey, University of Massachusetts Medical School) with goat anti-rabbit 488 or 568 (1:1,000; catalog no. A-11008 and A-11011, respectively; Life Technologies), CD63 (1:50) and LAMP1 hybridoma (1:200; catalog no. H5C6 and H4A3, respectively; University of Iowa [UI] hybridoma bank) and IFITM3 (1:50; catalog no. SAB1404822; Sigma) with goat anti-mouse 488 (1:1,000; catalog no. A-11001; Life Technologies). Coverslips were mounted in Vectashield with 4′,6′-diamidino-2-phenylindole (DAPI) counterstain (catalog no. H-1200; Vector Laboratories). Coverslips were imaged using a Zeiss LSM510 laser scanning inverted confocal microscope with a 63× Zeiss Plan-Apochromat differential interference contrast (DIC) oil lens objective or Leica SP5 inverted confocal microscope. Image analysis to determine colocalizations was done using ZEN software (Zeiss) or Fiji. The same laser intensity and detector sensitivity settings were used for all image acquisitions within a given experiment.


Two distinct regions of IFITM3's NTD are required for restriction of IAV.

Previously we and others had shown that loss of IFITM3 resulted in a marked decrease in the antiviral effects of IFN up to the stage of HA protein surface expression (1, 2). To test the effect of IFITM3 depletion on the entire viral replication cycle, we first treated A549 human lung carcinoma cells expressing a short hairpin RNA (shRNA) targeting IFITM3 (shM3) or a negative-control shRNA (shLuc [Luc stands for luciferase]) with or without IFN-α and then infected them with IAV (H1N1 A/WSN/33 [WSN/33]) (Fig. 1A and andB).B). After 12 h of infection, the A549 cells were washed once and then incubated with fresh medium for an additional 6 h, at which point the resulting viral supernatants were used to infect MDCK cells. Both A549 (18 h postinfection) and MDCK cells (12 h postinfection) were fixed, immunostained for viral HA protein, stained for DNA, imaged, and analyzed to determine the percentage of infected cells under each condition. These data, together with the results of previous studies, demonstrate that depletion of IFITM3 results in a >60% loss of the in vitro protective effects of IFN-α spanning the entire viral replication cycle.

We next hypothesized that specific amino acids within IFITM3 make critical contributions to its antiviral function, and identifying these molecular determinants will provide a better understanding of IFITM3's protective actions. The CD225 domain is also largely uncharacterized. Therefore, investigating the structural basis of IFITM3's restriction will improve our understanding of a number of related proteins. With this goal in mind, we constructed AS mutants along the entire IFITM3 protein (six amino acids changed to alanine per mutant [Fig. 1C and andDD]).

We chose this unbiased approach, because no previous work has looked at the function of IFITM3 at this resolution. Furthermore, we were concerned that a deletional analysis might disrupt the protein's architecture. We stably expressed either the wild-type IFITM3 or mutant proteins (designated by AS and the number of the first amino acid of the substituted residues) in A549 cells, which express low basal levels of endogenous IFITM3 (1). The level of each AS protein was determined by immunoblotting using polyclonal anti-IFITM3 sera raised against the NTD (anti-NTD). We chose not to use an epitope tag because we have found that this results in some loss of function (data not shown).

Next, each AS protein was assessed for its impact on IAV infection. The cells were challenged with WSN/33 at a multiplicity of infection (MOI) of 0.5 to 1.0. The cells were then fixed and stained for viral HA expression and for cellular DNA. Immunofluorescent (IF) images were captured, and the percent infection and cell number were determined (Fig. 1E). These studies showed that two portions of IFITM3's NTD, amino acids 19 to 29 and amino acids 43 to 55, were both required for inhibition of IAV infection (Fig. 1E and andF).F). The 55AS protein was expressed at low levels and so was not further evaluated (data not shown).

The anti-NTD sera recognizes the IFITM3 sequence from amino acids 20 to 32 (underlined portion of NTD schematic in Fig. 1D) and was used to determine the expression levels of the wild-type (WT) or AS mutant proteins (Fig. 1H). However, because their mutations destroyed its epitope, the anti-NTD sera could not detect expression of 19AS and 25AS. Therefore, the levels of these two proteins were evaluated using an antisera generated against the CIL of IFITM1 (Fig. 1G). The two bands seen with the CIL antibody likely arise from it recognizing both the full-length protein (top band) and an IFITM3 species that lacks the first several kilodaltons (bottom band) due to proteolysis. In addition, the higher running band seen with the 25AS mutant may result from it being posttranslationally modified. For both the 19AS and 25AS mutants, we could not assess their intracellular location because the anti-CIL sera does not perform well in IF applications. The intracellular distribution of the 43AS mutant, which also exhibited decreased restriction, was visible at greater levels at the cell's periphery compared to the wild-type IFITM3 (Fig. 1F). In support of this observation, the 43AS mutant, compared to IFITM3, was found to colocalize less with the late endosomal- and multivesicular body-associated protein, CD63 (Fig. 2E). We conclude that two regions of the NTD (amino acids 19 to 29 and amino acids 43 to 54) are required for viral restriction and that amino acids 43 to 48 are also required for wild-type localization.

Fig 2
IFITM3's CD225 domain is necessary for viral inhibition. (A) Alignment of the IM1 and CIL IFITM3 AS mutant proteins, with the wild-type amino acid sequence (IFITM3) shown in the top row. (B) A549 cells stably transduced with the empty retroviral vector, ...

IFITM3's CD225 domain is necessary for viral inhibition.

Next, we assessed the role of IFITM3's IM1 and CIL in viral restriction; these regions represent IFITM3's CD225 domain (Fig. 1C and and2A).2A). Alterations across a large segment of the CD225 domain (IM1 or CIL), spanning amino acids 67 to 102, lessened IFITM3's restriction of IAV to levels approaching those of the empty vector alone (Fig. 2B). One reason for this decrease in activity was the lower levels of expression observed for the 67AS mutant (4-fold lower than IFITM3 based on densitometric analyses of immunoblot autoradiographs [Fig. 2C]) and the 73AS mutant (3-fold), as well as for the 80AS and 85AS mutants (both 2.5-fold less [Fig. 2C]). However, while lower expression probably accounts for some lost function, it would not fully explain the observed deficits based on other mutant proteins that restricted equivalently to wild-type IFITM3 when expressed at comparable levels (i.e., 121AS, H57A, and Y80A mutants [Fig. 3D and and5A]),5A]), suggesting that the levels of wild-type IFITM3 are saturating.

Fig 3
Virus-specific determinants of IFITM3-mediated restriction. (A) Alignment of the CIL, IM2, and carboxy-terminal domain (CTD) IFITM3 AS mutant proteins, with the wild-type amino acid sequence in the top row. (B) A549 cells stably transduced with the empty ...
Fig 5
Multiple residues in IFITM3's NTD and CIL are required for restriction. (A) Normalized percent infection of the indicated IFITM3 point mutant cell lines spanning the IFITM3 protein. The cells were challenged side by side with IAV WSN/33 or DENV NGC, and ...

Confocal imaging revealed that both 80AS and 85AS mutant proteins were more centrally located than WT IFITM3, suggesting that these regions influence intracellular distribution (Fig. 2D). Consistent with this observation, expression of the 80AS mutant protein resulted in a similarly patterned redistribution of CD63, as well as a diminishment in the overall size and intensity of CD63-containing structures, suggesting that this region of IFITM3 may contribute to this compartment's formation and/or stability (Fig. 2E). The 91AS mutant protein could not be expressed. The 97AS mutant protein was present at wild-type levels and localization but exhibited diminished antiviral function. Altering the last portion of the CIL in the 103AS mutant also lowered viral inhibition without perturbing location, albeit with 2-fold-lower expression than IFITM3 (Fig. 2D and and3A3A to toC).C). Alterations within IM2 (109AS and 121AS), produced moderate changes in antiviral activity (Fig. 3A to toC),C), with the exception of 115AS, which could not be stably expressed (data not shown). Last, mutation of the CTD (127AS) resulted in a 4-fold decrease in restriction.

Virus-specific determinants of IFITM3-mediated restriction.

DENV, like IAV, is inhibited by IFITM3 (1). To identify the structural determinants needed for DENV restriction, we utilized the same panel of IFITM3 AS-expressing A549 cell lines described above. The cells were challenged with DENV serotype 2 New Guinea C (NGC) strain and then immunostained for DENV E-protein expression as a measure of viral replication. These studies revealed that many of the same regions in IFITM3's NTD (amino acids 19 to 30) and CD225 domain (amino acids 67 to 90 and amino acids 103 to 108) that were necessary for IAV inhibition were also needed for DENV restriction (Fig. 3D). However, two regions of IFITM3 that were required for inhibiting IAV were dispensable for curtailing DENV: NTD amino acids 43 to 54 and CD225 domain amino acids 97 to 102. We conclude that many structural determinants are jointly required to stop either IAV or DENV; however, there are also regions of IFITM3 that are specifically required to block the orthomyxovirus.

Multiple residues in IFITM3's NTD and CIL are required for restriction.

The AS mutant proteins revealed several regions of IFITM3 that were required for viral inhibition. To more precisely define the molecular determinants within these regions that are necessary for viral inhibition, we used a panel of proteins each with a single residue changed to alanine. These mutant proteins were stably expressed in A549 cells, assessed for expression by immunoblotting, and then challenged with IAV or DENV. We predominantly mutated charged residues and/or residues that were reported to have posttranslational modifications (PTMs) on PhosphoSitePlus, an open-access resource of PTMs ( For example, when examining the residues in the NTD altered in the 19AS mutant, we found that phosphorylation of Y20 has been reported repeatedly by investigators using either human (17, 18) or mouse (19, 20) cells. These data prompted us to create an Y20A-expressing cell line, which demonstrated that removal of this tyrosine produces a loss of protection against both IAV and DENV (Fig. 4A). Furthermore, alteration of Y20 resulted in a mislocalization of the mutant protein to smaller clusters throughout the cellular periphery and surface, as well as its lack of association with CD63 (Fig. 4C) (12). The relative colocalization of Y20 with LAMP1 was also lower compared to IFITM3, with the normalized values being 1.0 ± 0.1 for IFITM3 versus 0.3 ± 0.1. for Y20A (data not shown). Our results regarding Y20A are consistent with those recently reported during the completion of this work (21). Moreover, we note that the altered expression of Y20A is similar to that of IFITM1, suggesting that this region, which IFITM1 does not share, is responsible for the differential localization of these paralogs (Fig. 4C; see Fig. 7D). The Y20A mutant protein also migrated slower than IFITM3 and the other mutant proteins in immunoblots, suggesting that this mutation may result in additional PTMs (Fig. 5B). Several other point mutations engineered within the NTD, E21A, E25A, and E26A slightly decreased restriction of IAV (Fig. 5A and andBB).

Fig 4
Multiple residues in IFITM3's NTD and CIL are required for restriction. (A) A549 cells stably transduced with the empty retroviral vector, wild-type IFITM3, or the indicated IFITM3 alanine point mutant proteins were challenged with influenza A virus WSN/33 ...

IM1 comprises the first portion of the CD225 domain, and AS mutants (67AS and 73AS) within this region were defective in intracellular localization and antiviral activity. Consistent with an earlier study, we also found that changing C72 to an alanine produced a decrease in both IAV and DENV inhibition along with a more centralized expression pattern (Fig. 4A and andBB and and5A5A and andB).B). In contrast, conversion of C71 to an A showed no effect on either localization or IAV or DENV restriction (Fig. 5A; data not shown). Further mutational analyses of IM1 will be discussed below.

Within IFITM3's CIL, Y99 has been reported to be phosphorylated on PhosphoSitePlus. However, the number of independent proteomics data sets reporting this PTM was less than Y20 (2 murine studies detected Y99-phosphate [P] versus 16 studies reporting the detection of Y20-P). A549 cells expressing the Y99A mutant protein showed that IAV replication was enhanced compared to wild-type IFITM3 (>12-fold increased replication [Fig. 4A]). Intriguingly, the cells expressing the Y99A mutant protein still restricted DENV infection, showing only a 1.8-fold increase in viral replication versus wild-type IFITM3. The Y99A mutant protein was expressed to levels similar to those of IFITM3 and has an intracellular distribution indistinguishable from wild-type (Fig. 4B and and5B).5B). Therefore, two tyrosines, Y20 in the NTD and Y99 in the CIL, each contributed to IFITM3-mediated restriction, with Y99 being preferentially required for the inhibition of IAV over DENV.

Changing a region of mostly charged residues in IFITM3's CIL (85AS [RDRKMV]) strongly decreased restriction, likely due to the mutant protein's relocation to the perinuclear area (Fig. 2D). We therefore assayed cells expressing IFITM3 proteins with single-alanine substitutions of R85, D86, R87, and K88 (Fig. 4A to toCC and and5A5A and andB).B). The D86A protein could not be expressed (data not shown). Interestingly, mutation of R85 to alanine alone was sufficient to diminish restriction of both IAV and DENV and produce the perinuclear localization seen with 85AS (Fig. 4A and andC).C). The location of the R85A mutant protein suggested it may be near the microtubule organizing center (MTOC), and this was confirmed by immunostaining for the kinetochores (Fig. 4D). R85A colocalization with CD63 and LAMP1 was somewhat less than that seen between those proteins and IFITM3, with the relative colocalization of either R85A or IFITM3 with LAMP1 being 0.6 ± 0.1 and 1.0 ± 0.2, respectively (Fig. 4C; data not shown). In contrast, R87A offered less protection from IAV than against DENV, while maintaining wild-type levels and location (Fig. 4A and andBB and and5A5A and andBB).

To assess which individual amino acids in the most C-terminal portion of the CIL (103AS) were needed for restriction, we individually substituted either K104 or C105 with alanine. Alteration of the K104 alone reproduced much of the loss-of-function phenotype seen with 103AS, along with wild-type expression and intracellular distribution (Fig. 4A and andBB and and5A5A and andB).B). Similar to C71, we found that altering C105 to alanine did not affect IFITM3's location or its inhibition of IAV or DENV infection (Fig. 5A and andB;B; data not shown). Data for additional point mutant proteins are also provided (Fig. 5A and andB).B). In conclusion, these experiments identified or confirmed specific residues within IFITM3's NTD, IM1, and CIL that were needed for antiviral activity.

IM1 within the CD225 domain is required for IFITM protein interaction.

While performing affinity purification mass spectroscopy studies with exogenously expressed HA-tagged IFITM3, we recovered peptides unique to endogenous IFITM2 in addition to the IFITM3 peptides in the spectra, revealing a physical association between the paralogs (data not shown). To explore this finding, we assayed for potential interactions using coimmunoprecipitation (co-IP) studies of lysates from cells expressing HA-tagged IFITM1, IFITM2, or IFITM3 together with untagged IFITM3 (Fig. 6A). Interactions were detected between all of the HA-tagged IFITM proteins and IFITM3 when they were cotransfected into 293T cells. Importantly, these interactions persisted in the supernatant fraction after clarification with high-speed centrifugation (100,000 × g), strongly suggesting that these associations are not due to the proteins simply residing in adjacent regions of membrane. Furthermore, the HA-tagged protein, MDA9, did not interact with IFITM3, demonstrating the specificity of this association (Fig. 6A, right blots).

To find the residues needed for these interactions, we utilized the IFITM3 AS proteins. These co-IP experiments revealed that amino acids 67 to 85 within IM1 were required for IFITM association, with the most critical residues being amino acids 73 to 78 (LGFIAF [Fig. 6B and andC]).C]). Mutating the most hydrophobic residues within the sequence from amino acids 73 to 78 showed that loss of IFITM interactions occurred only when both F75 and F78 were changed to alanines (Fig. 6D). The region from amino acids 67 to 85 includes C72, whose palmitoylation is required for restriction (10, 15). Therefore, we tested C72A, as well as two other amino acids reported to be required for restriction, C71 and C105A (10, 15), in the co-IP assay. This experiment showed that all three C-to-A mutant proteins interacted with HA-tagged IFITM3 similar to wild-type IFITM3, demonstrating that palmitoylation of any one of these cysteines is unnecessary for these interactions (Fig. 6E). In keeping with these biochemical data, when we stably expressed the single mutants (F75A or F78A), double mutant (F75A/F78A), or quadruple mutant (LFIF/AAAA) proteins in A549 cells, only the F75A/F78A- and LFIF/AAAA-expressing cell lines failed to restrict IAV replication (Fig. 6G). Confocal imaging showed that the F75A/F78A double mutant was expressed similarly to WT IFITM3, with both proteins partially colocalizing with the MVB- and lysosome-associated protein, CD63 (Fig. 6H) (12). In addition, like IFITM3, the F75A/F78A double mutant protein partially colocalized with LAMP1, with the normalized colocalization values being 0.7 ± 0.1 for the F75A/F78A double mutant and 1.0 ± 0.2 for IFITM3 (data not shown). We also detected an interaction between endogenous IFITM3 and a stably expressed IFITM3 containing a single HA tag, and this interaction was enhanced after IFN-γ treatment was used to increase endogenous IFITM3 levels (Fig. 6I, bottom bands in top blot). Attempts to test for a direct interaction have unfortunately been complicated by our inability to solubilize the recombinant full-length IFITM3 protein (data not shown).

The NTD of IFITM3 can alter the location of IFITM1 but does not increase its antiviral action.

Among the IFITM members, IFITM3 is the most potent in preventing IAV replication (1). As noted, IFITM2 and IFITM3 reside in late endosomes and lysosomes, while IFITM1 localizes more to the early endosomes and the cell surface (2, 12). IFITM3 is 59% identical to IFITM1, with the major difference residing in IFITM3's NTD, which is 22 amino acids longer than the NTD of IFITM1 (Fig. 1C). The highly similar IFITM3 and IFITM2 proteins differ in 12 amino acids, six of which reside in the NTD. Therefore, we constructed chimeric IFITM proteins to test whether the respective NTDs can influence restriction and/or protein localization. IFITM3's NTD (amino acids 1 to 50) was fused in frame with IFITM1's amino acids 29 to 245 or IFITM2's amino acids 50 to 118 to generate M3M1 and M3M2 (Fig. 7A). The NTD of IFITM1, amino acids 1 to 28, or the NTD of IFITM2, amino acids 1 to 49, was fused to IFITM3, amino acids 51 to 119, to produce M1M3 and M2M3. Stably transduced A549 cell lines were generated for each respective chimera or wild-type protein. The anti-CIL serum was employed to determine the levels of protein expressed (Fig. 7B). The cell lines were then infected with IAV (WSN/33) and immunostained for HA expression. Similar to earlier reports (1, 2), the relative strength of restriction among the wild-type proteins was observed to be IFITM3 > IFITM2 > IFITM1, with the slope of IFITM3's infectivity curve being considerably flatter above a relative MOI of 1.25 (1.9- and 2.4-fold less steep than IFITM2 and IFITM1's respective slopes) (Fig. 7C). Moreover, when we replaced IFITM1's endogenous NTD with the longer NTD of IFITM3, the resulting M3M1 chimera was found to be only slightly better at inhibiting IAV than wild-type IFITM1 (Fig. 7C). However, because M3M2 expression is 4-fold lower, the activity of the M3M2 protein is estimated to be comparable to the activity of the wild-type IFITM2. Last, since the M2M3 chimera was slightly less effective compared to IFITM3 at preventing viral replication and its levels were 3-fold higher, we infer that the NTD of IFITM2 is not functionally interchangeable with that of IFITM3.

Fig 7
The NTD of IFITM3 can alter the location of IFITM1 but does not increase its antiviral action. (A) Schematic diagrams of the IFITM chimeras. IFITM1 (M1; green), IFITM2 (M2; blue), and IFITM3 (M3; red) are shown with their IM1, CIL, and IM2 domains. The ...

Interestingly, when we looked at the intracellular distribution of the M1M3 and M3M1 chimeras, we saw that their location is partially influenced by their respective NTDs; M1M3 was seen to be more finely distributed in a pattern that extended to the cell's periphery and surface, somewhat similar to the pattern of expression of IFITM1 or that of the Y20 IFITM3 point mutant (Fig. 7D). In contrast, M3M1 was situated more centrally with a coarser staining pattern, showing its location in larger intracellular organelles that, based on earlier work, are lysosomes and autolysosomes, akin to IFITM3 (Fig. 7D) (12) (data not shown). Together, these results suggest that the intracellular location of the IFITM proteins can be influenced by their respective NTDs. However, a similar transfer of enhanced restriction was not seen with the addition of IFITM3's NTD to the remainder of the IFITM1 protein. We conclude that location alone does not explain the differing efficacies of the IFITM proteins against IAV.

IFITM3 single-nucleotide polymorphisms.

A number of putative single-nucleotide polymorphisms (SNPs) have been identified in IFITM3 including the synonymous rs12252 SNP, which alters a predicted splice acceptor site and was previously reported by us due to its association with severe influenza (7, 9). We determined the validity of nonsynonymous IFITM3 SNPs in both dbSNP and the 1000GENOMES sequencing project (Fig. 8A and Table 1; see Table S1 in the supplemental material). SNP H3Q/rs1136853 and rs12252 were present in both data resources. As previously reported, rs12252 has variable allele and genotype frequencies in different human populations with the severe influenza-associated alternative C allele being rare in Europeans (Fig. 8A and Table 1) (7). Similarly, the H3Q/rs1136853 alternative T allele is rare in all populations (minor allele frequency [MAF], 3%). Three SNPs, V31M/rs199582787, P70T/rs199749095, and G95R/rs1744108 were not present in the 1000GENOMES data most likely due to their MAFs of 0.2%, 6.4%, and 1.4%, respectively, in European and African American populations. For 7 SNPs (T4I/cosm42696, FS9S/rs56398316, H27Q/rs55888283, V31M/rs199582787, A34G/rs56188107, T42M/rs55900504, and P70T/rs199749095), the 1000GENOMES project provides evidence for miscalls of the SNPs due to misassignments occurring during human genome assembly and SNP calling; such events may have occurred because the short read 1000GENOMES data can map equally well to both IFITM3 and IFITM2. Indeed, for these 7 SNPs, IFITM2 (NCBI reference sequence NM_006435.2) encodes the alternative amino acid predicted to be a nonsynonymous SNP in its paralog, IFITM3 (Fig. 8A and Table 1 and Table S1).

Fig 8
IFITM3 single-nucleotide polymorphisms. (A) The relative positions of multiple single-nucleotide polymorphisms (SNPs) reported within the coding region of the human IFITM3 gene are shown. The predicted nucleotide and amino acid changes (if nonsynonymous) ...
Table 1

However, for 5 SNPs (H3Q/rs1136853, D56G/rs55794999, H57D/rs1553883, N69D/rs12778, and G95R/rs61744108), the amino acids at these positions are identical in IFITM3 and IFITM2, suggesting that these SNPs were accurately called as nonsynonymous in either IFITM3 or IFITM2. To appreciate any functional significance associated with these five SNPs, we individually introduced them into a retrovirus gene-encoded IFITM3, in addition to two of the polymorphisms that effectively change IFITM3 to IFITM2 (T4I/cosm42696 and P70T/rs199749095) and the Q97Stop polymorphism (rs113745243). We then used the same number of CFU of each wild-type- or SNP-encoding retrovirus to stably transduce A549 cells.

Upon challenging these stably transduced cell lines with IAV, we found that the performances of three were indistinguishable from wild-type IFITM3: H3Q, T4I, and P70T (Fig. 8B). However, the D56G, H57D, N69D, and Q97Stop cells all expressed the transduced proteins at levels much lower than those of wild-type IFITM3, H3Q, or T4I based on immunoblots and confocal images, and thus not surprisingly, they mounted less resistance to IAV (Fig. 8B to toD;D; data not shown). D56 and H57 both reside among the amino acids altered in 55AS, which was also poorly expressed. Similarly, N69 is one of two conserved asparagines in IM1, a region of IFITM3 whose alteration led to reduced expression or antiviral function (61AS unexpressible) (67AS defective in restriction [Fig. 2 and and3]).3]). In contrast, G95R was expressed and localized similarly to both wild-type IFITM3 and P70T, while conferring 3-fold-less protection than either of these proteins (Fig. 8B to toD).D). Therefore, G95R's attenuated restriction of IAV suggests that it may play a role in determining IAV resistance in individuals possessing this rare variant.


Organisms are under selection to defend themselves against the continual assault of pathogens. To survive such pressures, a diverse set of protective strategies has evolved, which in humans includes the actions of hundreds of IFN-induced genes regulated by the antimicrobial cytokine IFN. In principle, understanding how these IFN-induced genes protect cells from microbial attack may guide us in preventing and treating infectious diseases. We and others have identified IFITM1, IFITM2, and IFITM3 as a family of proteins capable of halting the replication of diverse pathogenic viruses, including IAV and DENV (1, 36). Among this family, IFITM3 plays a uniquely important role as an “early defender” prior to IFN stimulation; this role is then expanded upon IFN exposure to account for 50 to 80% of the in vitro protective effect of IFN type I or II (1, 7, 12).

IFITM3 blocks IAV replication by altering the properties of the host cell's late endosomes and lysosomes, impeding virus-host membrane fusion (12). Such a strategy leads to invading viruses being trapped in the endocytic pathway and ultimately destroyed in lysosomes and autolysosomes, both of which are expanded with IFN treatment or IFITM3 expression (12). This model accounts for both the diversity of IFITM3-sensitive viruses and the short time in which IFITM3 acts to prevent viral cytosolic entry (5 to 30 min for IAV).

Beginning with an unbiased mutagenesis strategy, we identified or confirmed multiple distinct regions and specific amino acids within IFITM3 that are required to block IAV and DENV infection (Fig. 9A). Importantly, the CD225 family is functionally uncharacterized; therefore, identifying these molecular determinants of IFITM3's action likely provides functional insights for a number of related proteins. To better interpret the functional changes observed with these mutant proteins, we assessed both the level and cellular location of each when stably expressed in A549 cells. These collective data reveal that many of the mutant proteins that decreased IFITM3-mediated restriction can be grouped into four classes depending on the property of IFITM3 they alter: location, expression, interaction, or dual restriction, with the latter class referring to IFITM3's inhibition of both IAV and DENV.

Fig 9
Structural determinants required for IFITM3-mediated restriction and a model of IFITM3's antiviral actions. (A) Schematic diagram of IFITM3 in the endosomal membrane. The structural determinants needed for the indicated IFITM3 properties are shown. The ...


To make endosomes resistant to viral entry, IFITM3 must traffic to, and remain within, the relevant cellular compartment. Palmitoylation targets proteins to the endosomes and lysosomes, in some instances by first directing them to the cellular surface and periphery (22). Abrogating the palmitoylation of three cysteines in IFITM3's IM1 and CIL (C71, C72, and C105) was reported to be needed for proper localization and IAV inhibition (15). Consistent with this, we found that changing C72 to alanine decreased IFITM3-mediated restriction of both IAV and DENV, and led to a coarser and more centralized intracellular distribution (10, 15). However, we found that alanine substitution of either C71 or C105 did not perturb antiviral action or intracellular distribution. Thus, palmitoylation of C72 appears to be a nonredundant IFITM3 PTM required for inhibition of IAV and DENV, likely because it is needed for proper trafficking of newly made IFITM3 to the appropriate compartment.

A change in IFITM3's location was also detected upon alanine scanning of the first portion of the CIL in the 80AS and 85AS mutant proteins; in each instance, mutagenesis led to a considerable decrease in viral restriction concomitant with a perinuclear location. Mutation of a single arginine to alanine (R85A) was sufficient to reproduce both the loss of restriction and the perinuclear residence seen with the AS protein; the mechanism underlying this loss of function remains to be elucidated. While alteration of R85 led to a redistribution toward the cell's interior, the opposite effect was seen with the 19AS and 43AS mutant proteins; both of these mutant proteins were situated more peripherally in the cell, up to and including the cytolemma. More precisely defining the molecular determinants underlying the mislocalization of 43AS awaits future study. However, point mutations across the span of amino acids altered in the 19AS protein revealed that mutating a single residue, Y20, altered the localization of IFITM3 to late endosomes and lysosomes. Indeed, Y20 resides in a short stretch of amino acids, PPNY, that is similar to a confirmed lysosomal sorting signal, NPXY, that is important for low-density lipoprotein receptor trafficking (23). IFITM3's Y20 has also long been reported to be phosphorylated in multiple proteomics studies (PhosphoSitePlus), and this has been confirmed and extended in a recent report (21). Thus, we infer that loss of Y20, which is not present in IFITM1, partially accounts for the differential localization of the two paralogs. Such a comparison is best made using magnified views of the stably transduced A549 cell lines (Fig. 7D). Testing of the IFITM chimeras also showed that the respective NTDs of IFITM1 and IFITM3 can partially influence location. Mutating all four lysines in IFITM3 (K24, K83, K88, and K104) was reported to decrease ubiquitination and IFITM3 turnover leading to enhanced restriction (10). In contrast, when we altered three of these lysines individually, we found that loss of K104 decreased restriction, while mutating K83 or K88 had no effect on the function or level of IFITM3.

Collectively, our results, combined with those of others, demonstrate that multiple residues within IFITM3 act in concert to position the restriction factor within the endocytic pathway. IFITM3 may therefore be classified as a lysosomal membrane protein that is palmitoylated on the trans-Golgi, resulting in its trafficking to the cell periphery and surface (15). Upon its arrival at the cell periphery, IFITM3's PPNY motif leads to the restriction factor being endocytosed and targeted to lysosomes.


Four out of 21 IFITM3 AS proteins were expressed poorly despite our creating puromycin-resistant cells at equivalent levels after transduction with comparable retroviral particles; three of these mutant proteins had alterations within the CD225 domain, 61AS (located in IM1), 91AS (in the CIL), and 114AS (in IM2), with the fourth protein, 55AS, bearing mutations immediately before the start of IM1. 61AS resides in the first portion of IM1. One possibility is that upon this segment's translation, amino acids 55 to 66 direct the initial positioning of IM1 with the membrane. Mutagenesis of this critical “signal sequence” region, and the loss of membrane association, would result in misfolding and aggregation. Among the point mutations tested, only the alteration of any of three conserved aspartates led to poor expression. D56G/rs55794999, a rare SNP-bearing protein, also was not expressed, further suggesting that the side chain properties of D56 are necessary for the proper expression and stability of IFITM3. Perhaps D56, together with H57, serves as a hydrophilic boundary element that defines the locations of the NTD and IM1 and/or interacts with other membrane-associated proteins.


The third class of IFITM3 mutants involves two critical residues in IM1, F75 and F78, whose alteration resulted in a loss of IFITM interaction and viral inhibition. The functional importance of IM1 and the data presented above suggest a model of IFITM3-mediated restriction based in part on its changing the properties of the host cell membrane using intermolecular interactions. In this model, the intramembranous interactions between adjacent IFITM3 proteins may decrease membrane fluidity, as well as alter the bending modulus of the endosomal membrane, increasing the force required by the viral fusion machinery to deform the membrane. Moreover, the four insertions of each IFITM3 protein into the trans leaflet may serve to differentially compress the endosomal membrane (Fig. 9A and andB)B) (10, 2427). By extension, IFITM3's presence in the viral envelope could decrease membrane fluidity, thereby inhibiting the rearrangement of host membrane-engaged viral envelopes, an event that has been reported as critical for HA receptor-mediated viral entry (28). Of note, we and others have observed profound changes elicited by IFITM3 in the late endosomal and lysosomal compartments, suggesting that additional IFITM-induced alterations in the physical properties of the endocytic pathway may also contribute to viral restriction (2, 12).

Dual restriction.

This class of mutations separates IFITM's characteristic dual inhibition of either IAV or DENV and included AS proteins 43AS, 49AS, and 97AS, as well as the point mutant proteins, Y99A and R87A. While 43AS was more peripherally located than wild-type IFITM3, the remaining proteins in this class demonstrated normal intracellular distributions and levels. However, they all failed in part to inhibit IAV replication, while losing little efficiency in blocking DENV. We conclude that IFITM3 restriction of DENV is less functionally demanding than for IAV. Why might this be? Perhaps differences in where IAV and DENV fuse within the endocytic pathway, and the levels of IFITM3 at these areas, play a role. In addition, these data may reveal virus-specific molecular determinants of IFITM3-mediated restriction. Alternatively, the IAV fusion machinery may be stronger than that of DENV, and thus can more readily overcome a mutant protein.

CD225 domain.

Several molecular determinants necessary for IFITM3's restriction of IAV or DENV reside in its CD225 domain: IM1 contains F75 and F78, which are important for an intermolecular interaction, as well as C72, which is needed for proper trafficking (Fig. 9A). Within the CIL, R85, R87, Y99, and K104 were all found to be needed for IFITM3's function. To compare the prevalence of these functionally important residues, we created a probability diagram for amino acids in either the CD225 (IM1 and CIL) or IM2 domain (Fig. 9C). Interestingly, the results of this analysis show that C72, F75, R85, and K104 are among the most well conserved residues. D86 and D92, two alanine mutants that could not be expressed, are also well conserved, further suggesting their importance. As mentioned, Y99 is known to be phosphorylated and therefore represents a potential means of regulation. However, Y99 is not well conserved across the CD225 domain family and may represent a specific anti-IAV determinant. On the basis of the high degree of conservation found between CD225 domain family members, we deem it likely that the functional insights reported here will hold true for a number of related proteins.

Supplementary Material

Supplemental material:


We thank the following individuals at the University of Massachusetts Medical School (UMMS): B. Hobbs, L. Benson, T. Brailey, R. Fish, and J. Barrett. We thank M. Boyarina, K. Donnelly, and P. Richtmeyer at the Ragon Institute. We are indebted to R. Iorio and A. Rittenhouse (UMMS) for helpful discussions.

This work was generously supported by a grant (1R01AI091786) from the NIAID, NIH to A.L.B. A.L.B. is grateful to the Charles H. Hood Foundation, the Phillip T. and Susan M. Ragon Foundation, the Harvard and UMMS Centers for AIDS Research (CFARs) for their generous support. S.J.E. is an investigator with the Howard Hughes Medical Institute. P.K., S.E.S., and A.R.E are supported by the Wellcome Trust.


Published ahead of print 8 May 2013

Supplemental material for this article may be found at


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