The broad-spectrum antiviral functions of IFIT and IFITM proteins

Abstract

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

Figure 1. Detection of pathogen RNA and DNA in the cytoplasm and activation of IFNB and ISGs.

IFN-induced protein with tetratricopeptide repeats (IFIT) genes and IFN-induced transmembrane protein (IFITM) genes are induced by host innate immune defences after pathogen infection. The figure shows a scheme of innate immune signalling triggered by viral infection. Viral RNA and DNA is detected by: cytosolic RIG-I-like receptors (RLRs), such as melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I); cytosolic DNA sensors, such as DNA-dependent activator of IRFs (DAI), IFNγ-inducible protein 16 (IFI16), DEAH box protein 9 (DHX9) and DHX36; and endosomal Toll-like receptors (TLRs), including TLR3, TLR7 and TLR9. Infection by RNA viruses produces RNA intermediates that are recognized as non-self by RIG-I and MDA5 in the cytosol and by TLR3 and TLR7 in endosomes. The RLRs interact with mitochondrial antiviral signalling protein (MAVS), leading to the recruitment of TNFR-associated factor 3 (TRAF3), TANK-binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), or of IKKγ (also known as NEMO), IKKα and IKKβ, which results in the activation and nuclear translocation of IFN-regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB), respectively. TLRs interact with the adaptor proteins TRIF and MYD88, leading to the activation of IRF3 or IRF7. IRF3, IRF7 and NF-κB bind to the interferon-β (IFNB) gene promoter and induce transcription. Secretion of IFNβ by the infected cells results in paracrine type I IFN signalling through the IFNα/β receptor, which induces hundreds of IFN-stimulated genes (ISGs). Phosphorylated IRF3 also can activate the expression of ISGs (such as IFIT and IFITM genes) independently of IFN signalling. DNA can be present in the cytoplasm and in endosomes during viral or bacterial infection and following the phagocytosis of dead cells. TLR9 recognizes CpG DNA in endosomes and activates MYD88. The binding of DNA by DAI or IFI16 results in stimulator of IFN genes (STING)-dependent activation of IRF3 and NF-κB. RNA polymerase III transcribes this DNA to produce short RNAs containing a 5′-ppp motif, which are ligands for RIG-I. DHX9 and DHX36 bind to DNA ligands (such as CpG-A and CpG-B DNA) in the cytosol and induce MYD88- and IRF7-dependent responses. ER, endoplasmic reticulum; IκB, NF-κB inhibitor.

Figure 2. Genomic relationship and structure of IFIT proteins.

a | The phylogram shows the relationships between proteins of the IFN-induced protein with tetratricopeptide repeats (IFIT) family in different species. All full-length IFIT protein sequences for eight species (human, mouse, rat, chimpanzee, dog, frog, toad and salmon) were obtained from the National Center for Biotechnology Information (NCBI) database. IFIT-like and duplicate amino acid sequences were removed manually or using ElimDupes. Amino acid alignments were generated using CLC Main Workbench. A tree was created from the alignment using the neighbour-joining method and 1,000 bootstrap replicates. The scale of branch length is shown below the tree. b | The cartoon diagram shows the structure of the human IFIT2 monomer (PDB ID: 4G1T), with α-helical structural elements shown as cylinders. The amino-terminal region (blue), domain-swapped region (green) and carboxy-terminal region (yellow) are shown. The RNA-binding region is located near the C-terminus and is labelled in red (residue K410). The figure was prepared using PyMOL and is adapted, with permission, from Ref. © (2012) Macmillan Publishers Ltd. All rights reserved.

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

IFN-induced protein with tetratricopeptide repeats (IFIT) proteins bind to subunits of the eukaryotic initiation factor 3 (eIF3) multisubunit complex that regulates translation initiation. Human IFIT1 and IFIT2 bind to eIF3E, and human IFIT2, mouse IFIT1 and mouse IFIT2 bind to eIF3C. The figure shows a schematic diagram of translation initiation and the steps putatively blocked by IFIT family members. To begin translation in mammalian cells, free 40S ribosomal subunits are stabilized by eIF3 and bind to the ternary complex (eIF2–GTP–Met-tRNA) in the presence of eIF1 (not shown). This allows the assembly of the 43S pre-initiation complex, which then binds to mRNA that is capped at the 5′ end and methylated at the N-7 and 2′-O positions. This interaction is stabilized by eIF4E and eIF4G, and results in the formation of the 43S–mRNA complex, which is competent for AUG (start codon) scanning and mRNA translation. For hepatitis C virus (HCV) genomic RNA with an internal ribosome entry site (IRES), association with eIF4E and eIF4G or other cap-binding factors is not required to stabilize the 43S–mRNA complex. IFIT proteins can inhibit translation through several mechanisms^,,,,. One, the interaction of IFIT1 and IFIT2 with eIF3E blocks the binding of eIF3E to the ternary complex (eIF2–GTP–Met-tRNA) (a). Two, the binding of human IFIT2, and mouse IFIT1 and IFIT2, to eIF3C blocks the formation of the 43S–mRNA complex (b). Three, the binding of human IFIT1 to eIF3E prevents the recognition of the HCV IRES by the 43S complex. Disruption of eIF3 binding to the HCV IRES also can prevent eIF2 recruitment and suppresses ternary complex formation (c). IFIT1 can also inhibit the translation of viral RNA lacking 2′-O methylation through two possible mechanisms. One, IFIT1 may directly recognize the type 0 cap structure (no 2′-O methylation) on viral RNA and prevent its binding to the 43S pre-initiation complex (d). Two, the binding of IFIT1 to eIF3 may preferentially prevent the formation of the 43S–mRNA complex for RNA containing type 0 cap structures (e).

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

Viral infection by negative-stranded RNA viruses (such as Rift Valley fever virus, vesicular stomatitis virus and influenza A virus) generates single- or double-stranded RNA with uncapped 5′-ppp motifs. These RNA molecules are recognized by the cytoplasmic sensors retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). RIG-I and MDA5 induce the expression of IFN-stimulated genes (ISGs) — including IFN-induced protein with tetratricopeptide repeats 1 (IFIT1), IFIT2 and IFIT3 — through both interferon-β (IFNβ)-dependent pathways and IFNβ-independent (for example, IFN-regulatory factor 3 (IRF3)-dependent) pathways. IFIT1 functions as a sensor of viral RNA containing the 5′-ppp motif, resulting in the assembly of an IFIT1–IFIT2–IFIT3 complex. This presumably inhibits viral infection by sequestering RNA from the actively replicating pool or by promoting RNA degradation. Data are conflicting regarding whether IFIT proteins also promote or inhibit the host inflammatory response, possibly by changing the relative amount of viral RNA in the cell with free 5′-ppp ends. dsRNA, double-stranded RNA; IκB, NF-κB inhibitor; IKK, IκB kinase; MAVS, mitochondrial antiviral signalling protein; NF-κB, nuclear factor-κB; ssRNA, single-stranded RNA; TBK1, TANK-binding kinase 1; TRAF3, TNFR-associated factor 3, Part of this figure is adapted, with permission, from Ref. © (2011) Macmillan Publishers Ltd. All rights reserved.

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

a | Two topologies have been proposed for proteins of the IFN-induced transmembrane protein (IFITM) family. In the first model, the amino and carboxyl termini are located in the lumen of IFITM-containing vesicles, and the hydrophobic regions fully traverse the membrane (left). Yount et al. have proposed an alternative model in which both termini are oriented towards the cytoplasm, and the hydrophobic domains are embedded in the membrane without traversing it (right). A yellow dot in both models indicates the site of a palmitoyl group that is important for protein stability and restriction activity. b | An alignment of human, mouse and chicken IFITM proteins is shown. Red indicates conservation of a residue in at least nine of the twelve IFITM proteins shown. Note that the conservation of the first transmembrane domain and the cytoplasmic domain is based on the first topology model. The site of palmitoyl addition is highlighted in orange. Green and blue highlighting indicates species-specific signature residues of humans and mice, respectively, possibly suggesting interaction with a cofactor that similarly diverged in each species.

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

Viruses fuse with host-cell membranes in different compartments within the endocytic pathway, and IFN-induced transmembrane protein (IFITM)-mediated restriction activity correlates with the site of fusion. For example, arenaviruses (such as Junin virus and Machupo virus) follow the recycling pathway of their common receptor, transferrin receptor 1 (Ref. 82). These viruses are not susceptible to IFITM-mediated restriction. By contrast, viruses such as influenza A virus fuse in late endosomes and are restricted by IFITM proteins, particularly by IFITM3 (Ref. 72). Viruses such as severe acute respiratory syndrome (SARS) coronavirus, Ebola virus and influenza A virus depend on lysosomal cathepsins and other lysosome-resident proteins for fusion, and these viruses are restricted mainly by IFITM1 (Ref. 74). Mouse IFITM6 is more specialized and restricts the entry of Ebola virus and SARS coronavirus, but not influenza A virus. Trypsin treatment of SARS coronavirus allows it to fuse at the plasma membrane and bypass IFITM-mediated restriction. Retroviruses pseudotyped with entry proteins from these viruses show identical patterns of restriction, implicating the entry process in the antiviral activity of IFITM proteins. Note that the diagram is schematic and ignores much of the diversity of cellular compartments and the complexity of cellular trafficking.