Chicken interferon-inducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro - PubMed (original) (raw)

Chicken interferon-inducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro

S E Smith et al. J Virol. 2013 Dec.

Abstract

Interferon-inducible transmembrane protein 3 (IFITM3) is an effector protein of the innate immune system. It confers potent, cell-intrinsic resistance to infection by diverse enveloped viruses both in vitro and in vivo, including influenza viruses, West Nile virus, and dengue virus. IFITM3 prevents cytosolic entry of these viruses by blocking complete virus envelope fusion with cell endosome membranes. Although the IFITM locus, which includes IFITM1, -2, -3, and -5, is present in mammalian species, this locus has not been unambiguously identified or functionally characterized in avian species. Here, we show that the IFITM locus exists in chickens and is syntenic with the IFITM locus in mammals. The chicken IFITM3 protein restricts cell infection by influenza A viruses and lyssaviruses to a similar level as its human orthologue. Furthermore, we show that chicken IFITM3 is functional in chicken cells and that knockdown of constitutive expression in chicken fibroblasts results in enhanced infection by influenza A virus. Chicken IFITM2 and -3 are constitutively expressed in all tissues examined, whereas IFITM1 is only expressed in the bursa of Fabricius, gastrointestinal tract, cecal tonsil, and trachea. Despite being highly divergent at the amino acid level, IFITM3 proteins of birds and mammals can restrict replication of viruses that are able to infect different host species, suggesting IFITM proteins may provide a crucial barrier for zoonotic infections.

PubMed Disclaimer

Figures

Fig 1

The chIFITM locus architecture and sequence. The IFITM gene cluster on Gallus gallus chromosome 5 is flanked by genes ATHL1 and B4GALNT4. This region is syntenic with the IFITM gene cluster on human chromosome 11 (A). Note that the orientation change of chIFITM2 and chIFITM1 makes the assignment of orthology difficult; therefore, the chicken genes are named by gene order and conservation of specific functionally defined amino acid residues. The predicted mass is shown above the gene block. The colored columns in the sequence alignment (B) show residues that are shared between all nine IFITM sequences from humans, chimpanzees, and chickens. Significant residues have been highlighted with a symbol below the sequence: △, tyrosine; ○, double cysteine; star, phenylalanine important for multimerization; ↑, conserved ubiquitinated lysine. IM1, intramembrane 1; CIL, conserved intracellular loop; IM2, intramembrane 2.

Fig 2

Cellular localization of overexpressed IFITM proteins. Confocal microscopy of DF-1 cells transfected with chIFITM proteins 1 to 3 (A, B, and C) and A549s transfected with huIFITM proteins 1 to 3 (D, E, and F) in the absence of infection. Panels show nuclei stained with DAPI (4′,6-diamidino-2-phenylindole) (blue), endosomes marked with an antibody against Lamp1 (green), IFITM protein marked by an antibody against the HA tag (red), and a merged image.

Fig 3

An increase in the expression of chIFITM3 is associated with a decrease in viral infection. A range of clonal A549 cell populations expressing increasing levels of chIFITM3 protein (bars A to G) were assessed by Western blotting of the HA tag (B). These cell lines were infected by a lentivirus pseudotyped with the Lagos bat virus (LBV) glycoprotein, and the replication was measured by GFP expression relative to that in untransduced A549s (A). Error bars show standard deviations of the means (n = 3).

Fig 4

Human and chicken IFITM proteins restrict cell infection. Stable cell lines expressing human and chicken IFITM2 and -3 were infected by pseudotyped viruses with either lyssavirus glycoprotein envelopes (RABV [CVS-11]) and LBV (LBV.NIG56-RV1) (A) or IAV hemagglutinin envelopes (H1 [human], H5 [human], H7 [bird], or H10 [bird]) (B). The relative level of infection compared to untransduced A549 cells was measured by GFP expression or luciferase activity for the lyssavirus and IAV envelope pseudotypes, respectively. Error bars represent standard deviations across two biological replicates, each performed in triplicate. The expression levels of each cell line are shown by Western blotting (C) relative to endogenous B-actin. The stable cell line expressing chIFITM3 was infected with a pseudotyped virus expressing a luciferase reporter gene and the murine leukemia virus (MLV-A) envelope as a control (D).

Fig 5

Chicken IFITM3 has an antiviral activity in DF-1 chicken cells. The expression level and log fold change of chIFITM3 were measured using quantitative RT-PCR after stimulation with IFN-α and IFN-γ or after preincubation with a nontargeting siRNA or one specific to chIFITM3 (A). The effect of knocking down endogenous chIFITM3 expression in DF-1 cells infected with influenza A virus (A/WSN/1933 [WSN/33]) was measured by flow cytometry using an antibody against nucleoprotein (NP) (B). P = 0.01, Student's t test. DF-1 cells transfected with chIFITM3-HA were infected by WSN. Expression of HA and NP was detected by flow cytometry (C and D), and viral titers were measured by PFU (E). Error bars represent standard deviations across each condition performed in triplicate.

Fig 6

Differential expression of chIFITM transcripts in chicken tissues. Expression levels of IFITM1, -2, and -3 were determined by RT-PCR across a range of chicken tissues (A) and compared to the expression level of GAPDH (B). GAPDH PCR was performed without reverse transcriptase (−RT) to control for genomic DNA contamination.

References

1. Brass AL, Huang IC, Benita Y, John SP, Krishnan MN, Feeley EM, Ryan BJ, Weyer JL, van der Weyden L, Fikrig E, Adams DJ, Xavier RJ, Farzan M, Elledge SJ. 2009. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139:1243–1254 - PMC - PubMed
1. Huang IC, Bailey CC, Weyer JL, Radoshitzky SR, Becker MM, Chiang JJ, Brass AL, Ahmed AA, Chi X, Dong L, Longobardi LE, Boltz D, Kuhn JH, Elledge SJ, Bavari S, Denison MR, Choe H, Farzan M. 2011. Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus. PLoS Pathog. 7:e1001258.10.1371/journal.ppat.1001258 - DOI - PMC - PubMed
1. Lu J, Pan Q, Rong L, Liu S-L, Liang C. 2011. The IFITM proteins inhibit HIV-1 infection. J. Virol. 85:2126–2137 - PMC - PubMed
1. Chutiwitoonchai N, Hiyoshi M, Hiyoshi-Yoshidomi Y, Hashimoto M, Tokunaga K, Suzu S. 2013. Characteristics of IFITM, the newly identified IFN-inducible anti-HIV-1 family proteins. Microbes Infect. 15:280–290 - PMC - PubMed
1. Lewin AR, Reid LE, McMahon M, Stark GR, Kerr IM. 1991. Molecular analysis of a human interferon-inducible gene family. Eur. J. Biochem. 199:417–423 - PubMed

Chicken interferon-inducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro - PubMed (original) (raw)