Duck Interferon-Inducible Transmembrane Protein 3 Mediates Restriction of Influenza Viruses

Abstract

Interferon-inducible transmembrane proteins (IFITMs) can restrict the entry of a wide range of viruses. IFITM3 localizes to endosomes and can potently restrict the replication of influenza A viruses (IAV) and several other viruses that also enter host cells through the endocytic pathway. Here, we investigate whether IFITMs are involved in protection in ducks, the natural host of influenza virus. We identify and sequence duck IFITM1, IFITM2, IFITM3, and IFITM5. Using quantitative PCR (qPCR), we demonstrate the upregulation of these genes in lung tissue in response to highly pathogenic IAV infection by 400-fold, 30-fold, 30-fold, and 5-fold, respectively. We express each IFITM in chicken DF-1 cells and show duck IFITM1 localizes to the cell surface, while IFITM3 localizes to LAMP1-containing compartments. DF-1 cells stably expressing duck IFITM3 (but not IFITM1 or IFITM2) show increased restriction of replication of H1N1, H6N2, and H11N9 IAV strains but not vesicular stomatitis virus. Although duck and human IFITM3 share only 38% identity, critical residues for viral restriction are conserved. We generate chimeric and mutant IFITM3 proteins and show duck IFITM3 does not require its N-terminal domain for endosomal localization or antiviral function; however, this N-terminal end confers endosomal localization and antiviral function on IFITM1. In contrast to mammalian IFITM3, the conserved YXX endocytosis signal sequence in the N-terminal domain of duck IFITM3 is not essential for correct endosomal localization. Despite significant structural and amino acid divergence, presumably due to hostvirus coevolution, duck IFITM3 is functional against IAV. IMPORTANCEImmune IFITM genes are poorly conserved across species, suggesting that selective pressure from host-specific viruses has driven this divergence. We wondered whether coevolution between viruses and their natural host would result in the evasion of IFITM restriction. Ducks are the natural host of avian influenza A viruses and display few or no disease symptoms upon infection with most strains, including highly pathogenic avian influenza. We have characterized the duck IFITM locus and identified IFITM3 as an important restrictor of several influenza A viruses, including avian strains. With only 38% amino acid identity to human IFITM3, duck IFITM3 possesses antiviral function against influenza virus. Thus, despite long coevolution of virus and host effectors in the natural host, influenza virus evasion of IFITM3 restriction in ducks is not apparent.T ype I interferons are produced in response to viral infection and rapidly upregulate hundreds of interferon-stimulated genes (ISGs) in surrounding cells to induce an antiviral state. The interferon-inducible transmembrane proteins (IFITMs) are recently recognized ISGs with antiviral activity against a broad range of viruses (1, 2). Originally identified in a cDNA screen of genes upregulated by type I interferons, their antiviral properties have since been well characterized in vitro (3, 4). IFITMs inhibit the ent...

Identification of IFITM1 and IFITM3 in Goose: Gene Structure, Expression Patterns, and Immune Reponses against Tembusu Virus Infection

“…Analysis of the tissue distribution of goose IFITM1 and IFITM3 in geese showed that both goose IFITM1 and IFITM3 were readily detected in some immune-related tissues, including the thymus, bursa of Fabricius, and Harderian gland. In addition, the expression of goose IFITM1 was restricted and primarily confined to the gastrointestinal tract tissues (cecum, small intestine, and gizzard), which was observed previously in both chickens [17] and ducks [18]. In contrast, goose IFITM3 was constitutively and ubiquitously expressed in various tissues.…”

“…We predicted that the conserved motif in goose IFITM3 is also involved in the internalization and antiviral actions. Moreover, three other YxxΦ motifs that might be required for the endosomal localization and antiviral functions of duck IFITM3 [18] are also conserved in IFITM3 of chickens and geese. However, whether these YxxΦ motifs in goose IFITM3 are critical for correct cellular localization and inhibition of virus entry has yet to be confirmed.…”

“…Previous studies demonstrated that immune-related IFITMs were significantly upregulated in highly pathogenic avian influenza-infected ducks [18,47]. However, the available information on whether IFITMs positively respond to TMUV infection is scant.…”

“…Only a small number of bird IFITM sequences have been deposited in public databases, including Gallus gallus IFITM1-like (GenBank: XM_001233949), Anas platyrhynchos IFITM1 (GenBank: KF584226), Gallus gallus IFITM3-like (GenBank: XM_420925), Anas platyrhynchos IFITM3 (GenBank: KF584228), Serinus canaria IFITM3 (GenBank: XM_009102512), and Nipponia nippon IFITM3 (GenBank: XM_009463652). In addition, it was reported that chicken IFITM3 [17] and duck IFITM3 [18] could restrict influenza viruses, but information regarding goose IFITMs was unavailable.…”

Genetic characteristics and polymorphisms in the chicken interferon-induced transmembrane protein (IFITM3) gene

“…Mutagenesis of the Yxxϕ domain augmented mislocalization of the IFITM3 protein and reduced its antiviral capacity in mammals (Jia et al 2012). However, in a recent study performed in ducks, the Yxxϕ domain was not essential for correct localization of the IFITM3 protein and did not affect antiviral capacity (Blyth et al 2015). Although mammals and birds share a very similar protein structure called CD225, which is significantly associated with antiviral ability, birds do not likely depend on the Yxxϕ domain for correct IFITM3 protein localization.…”