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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nat Rev Immunol. Author manuscript; available in PMC 2013 September 16.
Published in final edited form as:
Published online 2012 December 14. doi:  10.1038/nri3344
PMCID: PMC3773942
NIHMSID: NIHMS506258

The broad-spectrum antiviral functions of IFIT and IFITM proteins

PREFACE

Over the past few years, several groups have identified novel genes downstream of type I interferon (IFN) signaling that inhibit infection by individual or multiple families of viruses. Among these IFN stimulated genes (ISGs) with antiviral activity are two genetically and functionally distinct families — IFN-induced proteins with tetratricopeptide repeats (IFIT) and IFN-induced transmembrane proteins (IFITM). This review focuses on recent advances in identifying the unique mechanisms of action of IFIT and IFITM genes, which explain their broad-spectrum activity against replication, spread, and disease pathogenesis of a range of human viruses.

To control infection by viruses, host cells must recognize invasion and develop a rapid and effective antiviral response. In mammalian cells, this response is initiated after detection of non-self pathogen-associated molecular patterns (PAMPs), including single-stranded and double-stranded viral nucleic acids. These viral PAMPs are detected by specific host pattern recognition receptors (PRRs), including Toll-like receptors (TLR3, TLR7, TLR8 and TLR9), RIG-I-like receptors (MDA5 and RIG-I) and DNA sensors (DAI, IFI16, DHX9 and DHX36) in the endosome and within the cytoplasm 1, 2. Binding of viral PAMPs to these PRRs triggers signaling cascades that induce the expression of virus-responsive genes and pro-inflammatory cytokines (such as type I interferon (IFN)), which restrict virus replication and modulate adaptive immunity (Fig 1).

Figure 1
Detection of pathogen RNA and DNA in the cytoplasm and activation of IFN-β and ISGs

IFN signaling induces a broad and potent antiviral response against most viruses that infect vertebrate animals. Type I IFNs are a family of functionally and genetically related cytokines consisting of several members, with IFNα and IFNβ the most extensively studied 3. Type I IFN signaling is mediated through a common receptor, the IFNα/β receptor (IFNAR), which is composed of a heterodimer of IFNAR1 and IFNAR2 subunits 4. Signal transduction following the binding of type I IFN to IFNAR occurs via Janus kinase (JAK) and Signal transducer and activator of transcription (STAT) proteins and results in translocation of the transcription factor complex IFN-stimulated gene factor 3 (ISGF3, which is comprised of IFN regulatory factor 9 (IRF9) and phoshorylated STAT1 and STAT2) into the nucleus, which induces the transcription of hundreds (estimated 500 to 1,000 genes per cell or tissue 57) of different interferon-stimulated genes (ISGs) [G]. These ISGs encode distinct proteins with diverse biological effects that block multiple stages of the viral lifecycle including entry, translation, replication, assembly and spread. They also can have immunomodulatory functions including effects on leukocyte recruitment and priming of adaptive immunity. Beyond this, a subset of ISGs is induced in an IFN-independent manner after viral infection through the actions of transcription factors (such as IRF3) that respond directly to signals downstream of PRRs.

Although the first antiviral ISGs were discovered decades ago (reviewed in 8), until recently, most experimental effort was restricted to defining the mechanism of action of a limited number of proteins, including protein kinase R (PKR), ribonuclease (RNAse) L, myxoma resistance 1 (Mx1), and oligoadenylate synthetases (OAS). More contemporary studies have expanded the analysis to several other ISGs, including APOBEC3 9, BST2 (also known as tetherin) 10, ISG15 11 and RSAD2 (also known as viperin) 12, with progress made in understanding the mechanisms of IFN-mediated control and evasion by particular families of viruses (such as retroviruses 13). Moreover, systematic investigation of the antiviral functions of large groups of ISGs using ectopic gene screens 14, 15 has identified genes that coordinately control infection with several families of RNA and DNA viruses. There has been a resurgence of interest in defining ISGs with broad-spectrum antiviral activity, possibly as a means for identifying new classes of drugs that activate these genes directly; antiviral therapies that target host rather than viral proteins in theory could minimize the emergence of resistance and collateral effects associated with type I IFN therapy that limit its current clinical application. This Review describes recent advances in understanding the antiviral activity and mechanisms of action of two particular ISG families with broad-spectrum antiviral activity: IFIT and IFITM proteins. While genetically and functionally distinct, an analysis of IFIT and IFITM proteins clarifies more generally how specific ISGs inhibit the replication, spread and disease pathogenesis of a range of human viruses.

IFN-induced proteins with tetratricopeptide repeats (IFIT family)

Gene/protein family

IFIT genes encode a family of proteins that are induced after IFN treatment, viral infection and PAMP recognition 16 (Fig 2A). IFIT genes have a similar genomic structure with most composed of two exons, with the second exon containing virtually all of the coding sequence. IFIT gene homologues have been reported in several mammalian species as well as birds, fish and amphibians (reviewed in 17). Four family members have been characterized in humans (IFIT1 (also known as ISG56), IFIT2 (ISG54), IFIT3 (ISG60) and IFIT5 (ISG58) and are localized to chromosome 10q23, whereas three members are expressed in mice — Ifit1 (Isg56), Ifit2 (Isg54), Ifit3 (Isg49) — and located on chromosome 19qC1. Additional uncharacterized yet highly related IFIT genes (IFIT1B (human) and Ifit1b, Ifit1c and Ifit3b (mouse)) in syntenic regions of the chromosome exist, although their functional significance and expression patterns remain undefined. Moreover, a non-transcribed IFIT1-related pseudogene is present on human chromosome 13 18.

Figure 2
Genomic relationship and structure of IFIT genes

IFIT proteins localize within the cytoplasm and ostensibly lack any enzymatic domains or activity. Rather, they contain multiple tetratricopeptide repeats (TPR) (Fig 2B); this motif is present in various host proteins, and is composed of 34 amino acids that adopt a helix–turn–helix structure and mediate protein–protein interactions. Proteins containing TPR motifs regulate the cell cycle, transcription, protein transport and protein folding 19. The sequence identity between human and mouse IFIT orthologues ranges from 52% to 62%, with less relatedness (~40% to 45%) between homologues of different species 16, suggesting the duplication of a common ancestral gene. However, different IFIT family members have distinct numbers of TPR motifs (Fig 2B), which may dictate specific functions; for example, IFIT1 has six whereas IFIT2 has four.

Structure

A recent paper published the first X-ray crystallographic structure of an IFIT family member, that of human IFIT2 20 (Fig 2C). In the 2.8 Å high-resolution structure, the authors showed that IFIT2 monomers had nine TPR motifs and formed domain-swapped dimers. Moreover, IFIT2 had a positively charged C-terminal region that supported RNA binding, and mutation or deletion of charged residues in this region altered viral RNA binding and negatively affected antiviral activity against Newcastle disease virus. As this study also suggested that IFIT2 can bind RNA containing AU-rich elements, which are sometimes found in mRNA of proteins that encode cytokines or apoptotic factors, this may be a possible mechanism by which IFIT proteins regulate inflammatory responses (see below).

Expression

Most cell types do not express IFIT proteins under basal conditions, with the possible exception of some myeloid cell subsets 21. However, IFIT genes are induced rapidly to high levels in many cells after virus infection 22. This expression pattern is determined in part by the upstream promoter regions of IFIT genes, which contain IFN-stimulated response elements (ISRE) 2325. Accordingly, Ifit1 and Ifit2 are induced within two hours of exogenous IFNα treatment 24, but less so after exposure to IFNγ 5. Moreover, cell-type and tissue-specific kinetics of expression of individual IFIT genes has been reported 2629. IFIT mRNA levels after IFN stimulation also can be sustained or transient depending on the cell type. In some cells, subsets of IFIT genes are induced selectively after stimulation with type I IFN or viral infection 30. The differential expression of individual IFIT genes in a given cell or tissue is hypothesized to confer non-redundant antiviral functions against particular viral infections 28, 29.

IFIT gene expression also can be triggered independently of type I IFN, through signals generated after the ligation of PRRs (such as TLR3, TLR4, MDA5 and RIG-I) by PAMPs (such as double-stranded (ds)RNA and lipopolysaccharide (LPS)). Indeed, IFIT genes were described as viral stress-inducible genes 22 and are induced at the transcriptional level directly by IRF3 31, 32, which is activated soon after viral infection, often prior to the induction of type I IFN. Other IRF proteins (such as IRF1, IRF5, and IRF7) also can induce the expression of IFIT genes directly 33, 34, presumably after stimulation of host defense signaling cascades, although these pathways remain less well defined. Human IFIT genes also are induced by retinoic acid 35, although the kinetics are slower relative to PAMP-dependent recognition, and might be regulated in part by IFNα induction 34.

Antiviral activity and mechanisms of action of IFIT proteins

Given their rapid induction pattern after type I IFN treatment or PRR activation, IFIT proteins are poised to confer inhibitory effects after infection. Recently, progress has been made in identifying how IFIT proteins inhibit the replication of multiple families of viruses through distinct mechanisms of action.

Translation inhibition

Eukaryotic initiation factor 3 (eIF3) is a multi-subunit protein complex that functions in translation initiation at several steps, including assembly of the eIF2–GTP–Met-tRNA ternary complex, formation of the 43S pre-initiation complex, mRNA recruitment to the 43S pre-initiation complex, and scanning of the mRNA for AUG (start codon) recognition (reviewed in 36). Biochemical studies suggest that some IFIT family members reduce the efficiency of cellular cap-dependent protein translation [G] by binding subunits of the eIF3 translation initiation complex 37. Human IFIT1 and IFIT2 can block binding of eIF3 to the eIF2–GTP–Met-tRNA ternary complex by interacting with eIF3e, whereas human IFIT2, and mouse IFIT1 and IFIT2, can block the formation of the 48S pre-initiation complex by binding to eIF3c 27, 37, 38 (Fig 3).

Figure 3
IFIT proteins function as antiviral molecules by inhibiting distinct steps in the translation of viral mRNA

Hepatitis C virus (HCV), a positive-stranded RNA virus, contains an internal ribosome entry site (IRES) [G], which regulates the assembly of cap-independent translation [G] initiation complexes on viral mRNA by a sequential pathway requiring eIF3 39. Type I IFN inhibits HCV infection by blocking translation of the HCV RNA 40, 41. Examination of the cellular proteins associated with HCV-translation complexes in IFN-treated human cells showed that human IFIT1 is an eIF3-associated factor that fractionates with the initiator ribosome-HCV RNA complex 41. IFIT1 suppressed the function of the IRES of HCV, whereas a mutant IFIT1 protein lacking eIF3-binding activity failed to inhibit HCV replication. Moreover, ectopic expression of IFIT1 decreased HCV infection in hepatocytes 42. Thus, IFIT1 seems to block HCV replication through targeting eIF3-dependent steps in the viral RNA translation initiation process; these include HCV IRES-dependent recognition of the 43S pre-initiation complex and assembly of the 43S–mRNA complex (Fig 3).

2’-O methylation

The cellular mRNA of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2′-O positions of the 5′ guanosine cap by nuclear and cytoplasmic methyltransferases. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2′-O methylation [G] had remained uncertain 43, 44. Recent studies showed that a West Nile virus (WNV) mutant lacking 2’-O methyltransferase activity was attenuated in wild type cells and mice but was pathogenic in the absence of Ifit1 expression 45, 46. The mutant virus lacking 2’-O methyltransferase activity showed increased replication in peripheral tissues of Ifit1−/− mice after subcutaneous infection and a 16,000-fold decrease in lethal dose (LD50) value [G] in Ifit1−/− compared with wild type mice. 2’-O methylation of viral RNA did not affect IFN induction in WNV-infected cells but instead modulated the antiviral effects of IFIT genes. Poxvirus and coronavirus mutants that lacked 2’-O methyltransferase activity had enhanced sensitivity to the antiviral actions of IFIT proteins 45, 47. It remains unclear whether IFIT proteins inhibit viruses lacking 2’-O methylation at the stage of protein translation by directly recognizing non-2’-O methylated viral RNA, thereby preventing recognition of viral RNA by the 43S pre-initiation complex, or by serving as a scaffold for other proteins that regulate translation (Fig 3). Wild type alphaviruses of the Togaviridae family of positive-stranded cytoplasmic RNA viruses lack 2’-O methylation on their viral RNA 48 and thus, should be sensitive to IFIT-mediated restriction. Although further mechanistic studies are warranted, in support of this, ectopic expression of Ifit1 inhibited infection by the Sindbis alphavirus, and reciprocally, silencing of Ifit1 resulted in enhanced infection 49.

5’-ppp RNA recognition

A recent study indicates that human IFIT1 also can function as a sensor for viral RNA by recognizing an uncapped 5’-ppp [G] and sequestering it from the actively replicating pool 50 (Fig 4). Using a proteomics approach with 5’-ppp RNA as bait, mass spectrometry analysis identified IFIT1 as a primary binding partner. Subsequent experiments showed that only IFIT1 interacts directly with 5’-ppp on RNA, whereas IFIT2 and IFIT3 form a complex with IFIT1 that is required for function. These IFIT-dependent interactions were relevant against RNA viruses displaying a 5’-ppp, as silencing of IFIT1, IFIT2 and IFIT3 in HeLa cells to varying degrees enhanced replication of the negative strand Rift Valley fever virus (RVFV), vesicular stomatitis virus (VSV), and influenza A virus, despite the fact that the production of IFNβ mRNA was unaffected. By contrast, ectopic expression of individual IFIT proteins in cells did not confer an inhibitory effect on these viruses, suggesting that the IFIT protein complex is required for this antiviral activity. Studies with Ifit1−/− mouse fibroblasts and myeloid cells also showed enhanced replication of VSV despite wild-type production levels of type I IFN and other inflammatory cytokines. In vivo, Ifit1−/− mice were more vulnerable to infection with VSV, with higher virus-induced mortality observed. However, and in apparent conflict, experiments by a second group with the same VSV strain but an independently generated Ifit1−/− mouse revealed no difference in mortality compared with wild type mice over a wide range of VSV doses 51. Instead, VSV infection was uniformly lethal in Ifit2−/− mice, a phenotype that was associated with enhanced replication in neurons of the brain but not in cells from other organs, such as lung and liver. Finally, a third study showed that gene silencing of IFIT3 in human A549 lung adenocarcinoma cells resulted in decreased IFNα-dependent antiviral activity against VSV, whereas ectopic expression of IFIT3 inhibited infection with both VSV and encephalomyocarditis virus, the latter being a picornavirus with a virally encoded genome-linked protein (Vpg) [G] at its 5’ end that likely blocks the uncapped 5’-ppp 52. Clearly, studies with additional RNA and DNA viruses and IFIT-deficient cells and mice are warranted to establish the mechanism of control of different families of viruses by IFIT genes.

Figure 4
IFIT proteins recognize the 5’-ppp of viral RNA and inhibit infection

Binding to viral proteins

IFIT1 can inhibit infection with human papillomavirus (HPV), a large DNA virus, through a distinct mechanism by binding the viral E1 helicase, which is required for replication 53, 54. E1 is a multifunctional viral protein with ATPase and DNA helicase activities. IFIT1 sequesters HPV E1 in the cytoplasm, partitioning it from the replication complex, which is localized to the nucleus. Whereas HPV replication is sensitive to the antiviral effects of type I IFN, silencing of IFIT1 with shRNA resulted in a loss of this inhibitory activity. In contrast to the wild type E1 gene, transfection of an E1 protein mutant ([increment]399), which eliminated binding to IFIT1, supported DNA replication of HPV even in the presence of inhibitory levels of type I IFN 54.

Modulation of inflammatory responses by IFIT proteins

In addition to their antiviral effector functions, IFIT proteins may have immunomodulatory activity although the data as to the net effect of individual IFIT proteins on cellular immune responses is not consistent. Two reports suggested that IFIT proteins negatively regulate the host inflammatory and antiviral response. One showed that ectopic expression of Ifit2 in mouse macrophages inhibited LPS-induced expression of tumor necrosis factor (TNF), interleukin-6 (IL-6) and CXCL2 (also known as MIP2), and that this effect was mediated post-transcriptionally, possibly by affecting mRNA stability 55. More recently, human IFIT1 and IFIT2 were reported to bind and inhibit Stimulator of IFN gene (STING, also known as MITA), which functions as a mitochondrial adaptor protein that recruits tank-binding kinase-1 (TBK1) and IRF3 to a complex with mitochondrial antiviral signaling adaptor protein (MAVS, also known as IPS1, Cardif or VISA) resulting in the downstream induction of IFNβ in response to viral RNA or DNA56. Ectopic expression of IFIT1 in human 293T cells and macrophages inhibited the activation of IRF3, NF-κB and IFNβ mRNA transcription in response to poly (I:C) and reversed poly(I:C)-induced inhibition of VSV infection. Moreover, silencing of IFIT1 inhibited VSV infection, presumably by modulating the IRF3- and IFN-dependent responses. Biochemical analysis indicated that IFIT1 disrupted the physical interaction between STING and MAVS or TBK1.

Although provocative, these experiments conflict with results in human HeLa cells in which silencing of IFIT1 and IFIT2 resulted in increased VSV infection; also, modulation of IFIT protein levels did not alter type I IFN responses in mouse fibroblasts, macrophages or dendritic cells 50. Moreover, other groups reported recently that silencing of mouse Ifit1 suppresses downstream inflammatory gene activation by LPS-mediated TLR4 activation 57 and that ectopic expression of IFIT3 enhances IRF3-mediated gene expression 58. In the latter study, a TPR motif of IFIT3 interacted with the N-terminus of TBK1, and bridged TBK1 to MAVS on the mitochondrion, such that the host antiviral responses were boosted in the presence of IFIT3. Given these ostensibly conflicting results, more investigation is required to evaluate the network of immunomodulatory effects of individual IFIT genes in cell culture and in vivo.

Anti-proliferative effects of IFIT proteins

Type I IFN can have anti-proliferative effects in cell culture 59. Because of their ability to bind components of the eIF3 complex and inhibit host translation, IFIT proteins might contribute to the restriction of cell division imposed by IFN signaling. Independently, IFIT proteins may modulate expression of negative regulators of the cell cycle, resulting in the accumulation of cells at the G1/S phase transition 60; ectopic expression of IFIT3 in U937 human myeloid cells resulted in sequestration of c-Jun activation domain-binding protein-1 (JAB1), which limited ubiquitin and proteasome-dependent degradation of cyclin-dependent kinase inhibitor 1B (also known as p27 or KIP1). In other studies, IFIT1 was shown to bind and sequester the ribosomal protein L15 (RPL15). Ectopic expression of IFIT1 or silencing of RPL15 had an anti-proliferative effect on human gastric cancer cells with higher IFIT1 levels correlating with enhanced sensitivity to IFN-induced inhibition of proliferation 61. Finally, expression of human IFIT2, independent of IFN stimulation, was shown recently to promote cell apoptosis via a mitochondrial pathway; in this study, IFIT2 formed a complex with IFIT1 and IFIT3, with the latter protein negatively regulating apoptosis 62. Thus, IFIT proteins as a complex appear to regulate cell apoptosis after induction of type I IFN or other cell stress pathways.

Summary of IFIT functions

IFIT genes are rapidly induced in many virally infected cells through IFN-dependent and –independent pathways. Over the past decade, it has become clear that this family of related proteins inhibits viral infections through multiple mechanisms including suppression of translation initiation, binding of uncapped or incompletely capped viral RNA, and sequestering viral proteins or RNA in the cytoplasm. Moreover, recent functional studies suggest, through pathways that remain to be defined and/or corroborated, that IFIT family members additionally may regulate cell-intrinsic and cell-extrinsic immune responses. As new structural and functional insight is gained about individual IFIT family members, likely, we will begin to appreciate the basis and complexity of ligand interactions that explain their distinct functions in controlling viral pathogenesis and possibly, minimizing immune-mediated damage to the host.

IFN-induced transmembrane proteins (IFITM family)

Gene/protein family

Although IFIT and IFITM proteins have quite distinct mechanisms of antiviral activity, there are some underlying similarities in terms of family structure. For example, both families include multiple, closely related members that lack obvious enzymatic activities. Most vertebrate animals have two or more IFITM genes. The human IFITM locus is located on chromosome 11 and composed of four functional genes: IFITM1, IFITM2, IFITM3 and IFITM5. IFITM4p is a pseudogene. Murine Ifitm1, Ifitm2, Ifitm3 and Ifitm5 are located on chromosome 7 and are orthologues of their human counterparts. In addition, mice have two other IFITM genes: Ifitm6, which is syntenically located on chromosome 7, and Ifitm7, a retrogene located on chromosome 16. As in humans, murine Ifitm4 is a pseudogene 63.

IFITM proteins have a common topology with short luminal amino- and carboxy-termini, two anti-parallel transmembrane domains, and a short, conserved cytoplasmic domain (Fig 5). The first, more conserved, transmembrane domain includes two cysteine residues, at least one of which is modified by palmitylation 64. Although several groups have confirmed this topology by flow cytometric recognition of amino- and carboxy-terminal tags, an alternative topology was proposed recently. According to this model, the putative transmembrane regions associate with the inner leaflet of the membrane, and both amino- and carboxy-terminal domains are located in the cytoplasm 65. Evidence for this model (Fig 5) includes the absence of N-linked glycans in the putative ectodomains despite the presence of native or engineered N-linked glycosylation sites, and the observation that the amino-terminal domain can be ubiquitinated. N-linked glycosylation and ubiquitin modifications typically are found in the lumenal and cytosolic domains of transmembrane proteins, respectively.

Figure 5
Proposed topologies and sequence alignment of IFITM orthologues and paralogues

Expression

In contrast to the IFIT proteins, IFITM proteins are expressed basally in the absence of IFN induction in both primary tissues and cell lines 66. IFITM1, IFITM2, and IFITM3 are expressed nearly ubiquitously in humans, whereas IFITM5 is expressed primarily in osteoblasts. All four human IFITM proteins are induced robustly by both type I and type II IFNs. In mice, however, expression of Ifitm3 is the most strongly induced by IFN, whereas other IFITM genes are less responsive to IFN treatment. Human IFITM3 and murine Ifitm3 are also induced by IFNγ and by members of the gp130 family of cytokines (such as oncostatin M and IL-6), which use similar JAK–STAT signaling mechanisms. This observation suggests that more targeted, IFN-independent induction of IFITM3 expression might be possible through ligation of tissue-specific receptors by gp130-family cytokines. Studies on the induction of IFITM genes after ligation of PRRs might also identify additional IFN-independent mechanisms of expression.

Antiviral activity and mechanism of action of IFITM proteins

IFITM proteins were identified more than 25 years ago, and their responsiveness to type I and II IFNs is well described 67. IFITM proteins have been ascribed roles in diverse biological processes, such as immune cell signaling, germ cell homing and maturation, and bone mineralization 68. In B cells, human IFITM1 was shown to associate directly with the tetraspanin CD81 and indirectly with the B cell receptor components CD19 and CD21, although the significance of these interactions remain unclear 69, 70. Despite abundant evidence of their strong induction by IFNs, for years, most studies of IFITM-family proteins focused on their role in development 66. However, these investigations were called into question by the observation that mice homozygous for a deletion of the entire Ifitm locus (IfitmDel−/− mice) had no apparent developmental defects, or indeed any overt phenotype 71.

An antiviral role for IFITM3 was discovered in an RNA interference screen for factors modulating influenza A virus infection 72. Depletion of IFITM3 by siRNA or shRNA enhanced influenza A virus infection, and ectopic expression of IFITM1, IFITM2 or IFITM3 markedly inhibited influenza A virus replication. Surprisingly, retroviruses pseudotyped [G] with the influenza A virus hemagglutinin were affected similarly by IFITM depletion and ectopic expression, whereas retroviruses pseudotyped with the entry proteins of murine leukemia, Lassa, or Machupo viruses were not affected by the presence or absence of IFITM proteins. This observation localized the restriction of influenza A virus by IFITM proteins to a hemagglutinin-mediated step in the virus lifecycle. Subsequent studies established that, uniquely among antiviral proteins, IFITM proteins interfered with a step in viral replication preceding fusion [G] of the viral and cellular membranes 73, 74. There are several implications of this early restriction step. First, IFITM-mediated restriction precedes the induction of type I IFN in infected cells, which might explain the high basal level of expression of IFITM proteins in many tissues. IFN induction, however, can amplify IFITM expression and protect uninfected cells in a paracrine manner, and acute-phase cytokines such as IL-6 might induce IFITM protein expression systemically. Second, viral escape from restriction by IFITM proteins could be more challenging than for antagonizing inhibitory factors that function at later stages of the virus lifecycle. For example, viral proteins such as HIV-1 vif and vpu, which are generated after entry, evade host responses mediated by APOBEC3G or BST2 that affect replication and viral assembly by degrading these restriction factors. In comparison, because IFITM-mediated restriction precedes infection, the opportunity for de novo synthesis of viral inhibitors is not available, and the virion must carry a protein that counteracts IFITM-mediated restriction (which is less likely given the relatively small amount of viral protein that is delivered to a cell) or alter its site of fusion with host cell membranes (Fig 6).

Figure 6
Correlation between the site of virus fusion and susceptibility to IFITM-mediated restriction

In addition to influenza A virus, IFITM proteins restrict infection of several other enveloped viruses 14, 72, 7476. These include the flaviviruses (dengue and West Nile viruses), filoviruses (Marburg and Ebola viruses) and coronaviruses (such as SARS). By contrast, infection with alphaviruses, arenaviruses and murine leukemia virus (a retrovirus) seems to be unaffected by IFITM protein expression. Vesicular stomatitis virus is weakly restricted by IFITM proteins, and HIV-1 might be restricted in a cell-type specific manner 14, 77. These varying degrees of restriction also are observed for retroviruses pseudotyped with the entry proteins of different viruses. Viruses that are restricted by IFITM proteins tend to fuse with host cell membranes in a late endosome or lysosome. Indeed, when retroviruses bearing the entry protein of the SARS coronavirus were induced by trypsin to fuse at the plasma membrane, IFITM-mediated restriction was bypassed, establishing that the site of fusion is crucial for the antiviral activity of IFITM proteins 74.

There seems to be specialization among the antiviral functions of IFITM proteins 74. IFITM3 in particular, is especially effective in controlling influenza A virus, as Ifitm3−/− mice challenged with an H1N1 virus sustained higher viral loads and succumbed more rapidly to disease 78. Ifitm3−/− mice had a virological phenotype indistinguishable from IfitmDel mice (which lack Ifitm1, Ifitm2, Ifitm3, Ifitm5 and Ifitm6), which suggests that the other murine IFITM proteins do not have a significant role in controlling influenza A virus 79. Consistent with these data, patients hospitalized with severe H1N1 2009 influenza A virus infection were enriched for a single nucleotide polymorphism that decreased expression of full-length IFITM3 78. Although analogous in vivo studies of other viruses restricted by IFITM proteins remain to be carried out, cell culture experiments indicate that IFITM1 restricts filoviruses and SARS coronavirus more effectively than does IFITM3 74. More impressively, murine IFITM6 did not prevent influenza A virus infection, but efficiently limited infection mediated by filovirus entry proteins.

The mechanisms underlying the antiviral activity of IFITM proteins remain uncertain. Several possibilities, however, have been excluded 73, 74. Ectopic expression of IFITM proteins does not alter the expression of virus receptors, affect the pH of endosomal compartments, or interfere with cathepsin activity necessary for fusion of some restricted viruses. Although IFITM proteins can be detected on the plasma membrane, particularly when over-expressed or induced by IFN, they are enriched in intracellular compartments, including late endosomes, where restricted viruses fuse. Two models have been proposed to explain the antiviral activity of IFITM proteins 73, 74 (Fig 6). In the first model, IFITM proteins are hypothesized to modify endosomal or lysosomal vesicles such that they become inhospitable to viral fusion. This could occur by altering the lipid components of the vesicle membrane, by enriching vesicles with non-specific proteases that inactivate entry proteins or, as proposed recently 80, by interfering with the activity of the v-ATPase responsible for endosomal acidification. In the second model, IFITM proteins could alter the rate or pattern of vesicle trafficking such that viruses are redirected to a non-fusogenic pathway. Expression of IFITM proteins in many cell lines induces large vacuoles, suggesting some interference with vesicle trafficking, fusion, or resolution 73. However, the presence and size of these vacuoles do not correlate with the efficiency of restriction, and morphological changes were not observed when endogenous IFITM proteins were depleted, despite greater influenza A virus replication in these cells 72, 74. As with the IFIT proteins, the absence of obvious enzymatic domains in the IFITM proteins suggests that cellular cofactors are necessary for antiviral activity. Consistent with this possibility, IFITM proteins have species-specific signature sequences that are localized at the cytoplasmic base of both transmembrane domains (see Fig 5).

Summary of IFITM-protein function

IFITM proteins are a family of small transmembrane proteins that are induced strongly by IFNs, but which also are expressed basally in a number of cell types and lines. Although other functions have been suggested, the primary role of IFITM proteins appears to be antiviral. IFITM3 in particular significantly contributes to the control of influenza A virus in vivo, and tissue culture studies suggest that several of the other IFITM proteins help to restrict infection of other enveloped viruses. Expression of IFITM proteins makes cells refractory to steps in the infection cycle that precede viral fusion, but the means by which they do so remain incompletely defined. It is also remains poorly understood how IFITM proteins differentially restrict different viruses, and whether they can modulate the replication of other pathogens including non-enveloped viruses, bacteria, and parasites. As with IFIT proteins, additional work characterizing their activity and regulation may suggest more tailored approaches to controlling infection by specific pathogens.

Overall Summary

It may be unfortunate that IFIT- and IFITM-family proteins share such similar acronyms, because, although both are IFN-induced, they control virus infection through distinct mechanisms. IFIT proteins function in the cytoplasm, whereas IFITM proteins traverse the membrane and are enriched in late endosomes and lysosomes. IFIT proteins suppress translation initiation, bind and sequester uncapped viral RNA, and sequester at least one viral protein (HPV E1) in the cytoplasm. IFITM proteins, by contrast, prevent several enveloped viruses from crossing endosomal or lysosomal membranes and penetrating into the cytoplasm. Moreover, IFIT proteins are expressed poorly, if at all, in the absence of inflammatory or danger signals, whereas IFITM proteins are expressed basally in many tissues. IFITM proteins generally are induced to greater levels by IFNγ, and possibly by members of the gp130 (IL-6)-family of cytokines. However, although there are many differences, there are some parallels between IFIT and IFITM proteins. Compared with the APOBEC families of restriction factors, the IFIT and IFITM families target a wider range of viruses. Moreover, and similar to APOBEC proteins, IFIT and IFITM families have specialized paralogues, perhaps reflecting an evolutionary arms race with pathogens. A deeper understanding of the antiviral activity and mechanism of action of the members of each family may facilitate the development of broad-spectrum antiviral agents that mimic or amplify their activities.

ONLINE SUMMARY

  • -
    Induction of IFIT proteins after type I IFN or IRF3-dependent signaling contributes to an antiviral state against some viruses by binding components of the eIF3 translation initiation complex and inhibiting protein translation.
  • -
    Mutant flaviviruses, poxviruses and coronaviruses lacking 2’-O methyltransferase enzymes are attenuated in wild type primary cells and mice but pathogenic in the absence of IFIT1 expression. Thus, IFIT proteins restrict viruses lacking 2’-O methylation of the 5’ RNA cap.
  • -
    IFIT proteins form a multi-protein complex to bind viral RNA displaying 5’-ppp. By sequestering viral RNA containing 5’-ppp, IFIT proteins function as both a pathogen sensor and effector molecule.
  • -
    IFITM proteins comprise a family of small IFN-inducible proteins. Unlike IFIT proteins, IFITM proteins have two transmembrane domains and block replication of enveloped viruses including influenza A virus, dengue virus, Ebola virus and SARS coronavirus at a step before these viruses enter the cytosol.
  • -
    IFITM proteins seem to be specialized in their activity, with IFITM3 making the primary contribution to control of influenza A virus in mice and probably humans, whereas other human and murine IFITM proteins more efficiently restrict Ebola virus and SARS coronavirus infection.
  • -
    The mechanisms by which IFITM proteins prevent entry of enveloped viruses remain unclear, but likely involve altering the properties or the trafficking of intracellular compartments where these viruses traverse cellular membranes.

ACKNOWLEDGMENTS

The authors would like to thank members of their laboratory, M.Gale and G. Sen for helpful discussions. We also greatly appreciate the critical comments made by J. Hyde, J. White, M. Gack, and T. Pierson on the manuscript, and the help with figure preparation by J. Brien and S. K. Austin. NIH grants U54 AI081680 (Pacific Northwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (to M.S.D)), U54 AI057159 (New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (to M.F.)) and U19 AI083019 (M.S.D) supported this work.

GLOSSARY TERMS

IFN-stimulated genes (ISGs)
Genes induced by interferon or interferon regulatory factors, which have antiviral or immunomodulatory functions.
Cap-dependent protein translation
Initiation of translation in eukaryotic cells usually involves the interaction of certain translation initiation factors with an N7-methyl guanosine modified base or cap at the 5’-end of an mRNA molecule.
Internal Ribosome entry site (IRES)
An IRES is an RNA sequence that allows for recruitment of translation machinery independent of the 5’ end of the messenger RNA (Cap-independent translation).
2′-O methylation
A modification of cellular and/or viral RNA. In mammalian cells, this modification seems to prevent inhibition by IFIT proteins.
Lethal dose (LD50) value
The LD50 test was introduced for biological standardization of dangerous drugs or agents. It refers to the concentration or dose of a given agent that causes 50% lethality in a given population.
Uncapped 5’-ppp
Refers to the 5% end of an RNA in the absence of nucleotide cap. Uncapped 5’-ppp are generated by some RNA viruses on their negative and or/positive RNA strand intermediates and recognized specifically by host pathogen recognition receptors (e.g. RIG-I) to trigger immune responses.
Genome-linked protein (Vpg)
VPg is a protein attached to the 5′ end of RNA during RNA synthesis by several families of positive-stranded RNA viruses including Picornaviridae and Caliciviridae. These proteins play pivotal roles in the life cycle of these viruses includes effects on viral protein synthesis.
Pseudotyped virus
A virus that is produced with envelope proteins from a foreign or heterologous virus.
Viral fusion
A process required by enveloped viruses for entry and replication. Fusion often occurs in endosomal or early lysosomal compartments after pH- and/or protease-dependent changes.

Biographies

• 

Michael Diamond received his M.D. and Ph.D. from Harvard University (USA), and his post-doctoral and clinical training in infectious diseases and virology from the University of California, Berkeley and the University of California, San Francisco (USA). He is currently a Professor of Medicine, Molecular Microbiology, Pathology & Immunology at Washington University School of Medicine (USA) and the Co-Director of the Midwest Regional Center for Excellence in Biodefense and Emerging Infectious Disease Research. Michael Diamond’s research focuses on the interface between viral pathogenesis and the host immune response. More recently, his group has studied how novel innate immune response effector molecules restrict infection of multiple families of pathogenic human viruses.

• 

Michael Farzan received his A.B. and Ph.D. degrees and his post-doctoral training from Harvard University. He is currently Professor of Microbiology and Immunobiology at Harvard Medical School. Michael Farzan’s research focuses on the entry processes of enveloped viruses, how innate and adaptive immune responses control these processes, and how these immune responses can be emulated or amplified to prevent or control viral infections.

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