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 YXXθ 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 host-virus coevolution, duck IFITM3 is functional against IAV.

Importance: Immune 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.

FIG 1

Organization of duck IFITM locus with sequence alignment and maximum likelihood tree of duck, chicken, human, and mouse IFITMs. (A) The duck IFITM genes between ATHL1 and B4GALNT4 were annotated using gene synteny from the chicken IFITM locus. (B) IFITM sequences were aligned using T-COFFEE. Membrane-spanning domains are underlined in each sequence alignment. The noncanonical dibasic signal sequence in IFITM1 is indicated with a double arrow. Important residues for IFITM3 function are noted. The conserved YXXθ motif is boxed. Symbols: *, palmitoylated cysteine residues; Δ, ubiquitinated lysine residues. (C) A maximum-likelihood tree was generated using 100 bootstrap values to show similarity of orthologous IFITMs.

FIG 2

Duck IFITMs are upregulated in lung tissue in response to highly pathogenic IAV infection. Total RNA was isolated from duck lung tissue 1 and 3 days postinfection with PBS (mock), BC500 (low-pathogenicity IAV), or VN1203 (highly pathogenic IAV). IFITM1 (A), IFITM2 (B), IFITM3 (C), and IFITM5 (D) expression was measured using qPCR and compared to that of the mock-infected group.

FIG 3

Duck IFITM3 restricts replication of low-pathogenicity IAV. DF1 cells transiently overexpressing duck IFITMs or empty vector were challenged with H6N2 (A) or H11N9 (B) at an MOI of 1 or 5. Six hours after infection cells were fixed and stained for IAV nucleoprotein, and the percentage of infected cells was determined. The percentage of infected cells is expressed relative to the vector control. Statistical significance compared to the vector control cells was analyzed using an unpaired two-tailed Student's t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). The maximum percentage of infected cells for H6N2 was 40%, and for H11N9 it was 33%. (C) Expression level of each duck IFITM was determined by Western blotting.

FIG 4

Duck IFITMs localize to different cellular compartments. DF1 cells overexpressing dIFITM1, dIFITM2, dIFITM3, or dIFITM5 were fixed, stained, and imaged using confocal microscopy. Panels show staining for Hoechst 33324 (blue), LAMP1 containing endosomes (green), V5-epitope tagged dIFITM (red), and a merged image.

FIG 5

Duck IFITM3 restricts low-pathogenicity IAV but not VSV. DF1 cells stably expressing all dIFITMs were challenged with H6N2 (A) or VSV (B) at an MOI of 1. (C) DF-1 cells expressing dIFITM3 were challenged with H6N2, H11N9, PR8, or VSV at an MOI of 1. The percentage of infected cells was determined using fluorescence microscopy (A) or flow cytometry (B and C). Statistical significance compared to vector control cells was analyzed using an unpaired two-tailed Student's t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). The maximum percentage of infected cells for H6N2, H11N9, PR8, and rVSV-GFP was 57%, 15%, 12%, and 55%, respectively. (D) Supernatants of DF1 cells stably expressing empty vector or dIFITM3 were collected 12 h postinfection with H6N2, and the viral titer was determined using plaque assay. (E) Level of dIFITM protein expression of each stably expressing DF-1 cell line was determined by Western blotting. (F) Transcript abundance as determined by qPCR of each overexpressed duck IFITM is shown relative to that of its respective endogenous chicken IFITM.

FIG 6

N-terminal domain (NTD) of dIFITM3 is not necessary for antiviral activity. (A) Chimeric proteins of dIFITM1 and dIFITM3 were generated. CTD, C-terminal domain. DF1 cells stably overexpressing dIFITIM1, dIFITM3, or the chimeric proteins were challenged with H6N2 (B) or VSV (C) at an MOI of 1, and the percentage of infected cells was determined relative to empty vector-transfected cells using fluorescence microscopy (B) or flow cytometry (C). Statistical significance compared to the vector control cells was analyzed using an unpaired two-tailed Student's t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D) Expression of each dIFITM or mutant protein was determined using Western blotting. (E) Representative confocal microscopy images of DF1 cells overexpressing 1NTD-3CD225 and 3NTD-1CD225 stained for nuclei (blue), LAMP1 (green), or chimeric protein (red), with a merged image shown. (F) Colocalization of each dIFITM or chimeric protein with LAMP1 was completed using Pearson's correlation coefficient. Bars show mean values from at least 8 analyzed cells.

FIG 7

N-terminal YXXθ endocytic signal sequence of dIFITM3 is not necessary for endosomal localization or antiviral activity. (A) Amino acid sequence of duck IFITM3. Regions targeted for mutagenesis are shaded, and the CD225 domain is underlined. (B) Confocal microscopy images of DF-1 cells overexpressing IFITM3, IFITM3-Y14F, IFITM3-Y56F, IFITM3-Y82F, or IFITM3-Y94F. Cells were stained for nuclei (blue), LAMP1 (green), or dIFITM3 protein or mutant protein (red). A merged image is shown. Images are representative of multiple cells analyzed. (C) Colocalization of dIFITM3 or mutant protein with LAMP1 was completed using Pearson's correlation coefficient. Bars show mean values from at least 8 analyzed cells. Statistical significance compared to vector control cells was completed using an unpaired two-tailed Student's t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D) DF1 cells stably expressing dIFITM3 or point mutants were challenged with H6N2 at an MOI of 1, and the percentage of infected cells was determined relative to that of vector-only transfected cells by fluorescence microscopy. Statistical significance compared to vector control cells was analyzed using an unpaired two-tailed Student's t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (E) Level of dIFITM protein expression of each stably expressed DF-1 cell line was determined by Western blotting.

FIG 8

Cellular localization of duck IFITM1, IFITM3, and IFITM3-Y14F in HeLa and HEK293T cells. HeLa and HEK293T cells were grown on coverslips and transfected with N-terminally V5-tagged duck IFITM1, IFITM3, or Y14F. Cells subsequently were fixed, stained with an anti-V5 antibody (green) and Hoechst 33324 (blue), and imaged using confocal microscopy.