Interferon-Induced Transmembrane Protein 1 Restricts Replication of Viruses That Enter Cells via the Plasma Membrane

Abstract

The acute antiviral response is mediated by a family of interferon-stimulated genes (ISGs), providing cell-intrinsic immunity. Mutations in genes encoding these proteins are often associated with increased susceptibility to viral infections. One family of ISGs with antiviral function is the interferon-inducible transmembrane proteins (IFITMs), of which IFITM3 has been studied extensively. In contrast, IFITM1 has not been studied in detail. Since IFITM1 can localize to the plasma membrane, we investigated its function with a range of enveloped viruses thought to infect cells by fusion with the plasma membrane. Overexpression of IFITM1 prevented infection by a number of Paramyxoviridae and Pneumoviridae, including respiratory syncytial virus (RSV), mumps virus, and human metapneumovirus (HMPV). IFITM1 also restricted infection with an enveloped DNA virus that can enter via the plasma membrane, herpes simplex virus 1 (HSV-1). To test the importance of plasma membrane localization for IFITM1 function, we identified blocks of amino acids in the conserved intracellular loop (CIL) domain that altered the subcellular localization of the protein and reduced antiviral activity. By screening reported data sets, 12 rare nonsynonymous single nucleotide polymorphisms (SNPs) were identified in human IFITM1, some of which are in the CIL domain. Using an Ifitm1^-/- mouse, we show that RSV infection was more severe, thereby extending the range of viruses restricted in vivo by IFITM proteins and suggesting overall that IFITM1 is broadly antiviral and that this antiviral function is associated with cell surface localization.IMPORTANCE Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon-stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is interferon-inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can inhibit infection with a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localize to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo, and Ifitm1^-/- mice were more susceptible to viral lung infection. These data contribute to our understanding of how ISGs prevent viral infections.

Keywords: IFITM1; innate immunity; paramyxovirus; pneumovirus; restriction factor.

FIG 1

IFITM1 restricts replication of a wide range of RNA viruses in vitro. (A) Overexpression of IFITM proteins in Vero cells detected by Western blotting using an antibody to the C-terminal HA tag, including IFITM1 (Vero_M1), IFITM2 (Vero_M2), and IFITM3 (Vero_M3). Detection of β-actin expression was used as a control, and Vero Empty is the non-IFITM expression vector negative control. (B) Localization of different IFITM proteins was detected by confocal microscopy using an antibody to an inserted HA tag (red). Nuclei are stained with DAPI (blue). (C) Analysis of surface expression of HA-tagged IFITM1 by flow cytometry on nonfixed and nonpermeabilized cells. (D) Colocalization of IFITM1 (red) and wheat germ agglutinin (green) was detected by confocal microscopy. Nuclei are stained with DAPI (blue). (E) Transduced Vero cells were seeded in 24-well plates and infected at a range of MOIs with influenza A virus A/Puerto Rico/8/1934 (H1N1 PR8), parainfluenza virus 3 (PIV3), measles virus (rMV-Edt and rMV-EZ), respiratory syncytial virus (rgRSV and RSV-B05), mumps virus (mumps), human metapneumovirus NL/1/00-GFP (rHMPV NL1/1/00), human metapneumovirus NL/1/99-GFP (rHMPV NL/1/99), and Newcastle disease virus (rNDV). At 24 hpi, cells were fixed, and the level of infection of each cell line was measured by flow cytometry. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (by analysis of variance [ANOVA], compared to cells transduced with an empty vector control [n = 3]).

FIG 2

IFITM1 restricts HSV-1 infection. (A) A549 cell lines stably expressing an empty vector, IFITM3, IFITM2, or IFITM1 were generated using lentiviruses. The cell lines were infected with HSV-1/GFP (MOI of 5; n = 3). GFP expression was measured on a Cellomics ArrayScan instrument at 7 hpi and normalized to infection levels in untransduced A549 cells. (B) Transduced A549 cells were infected with HSV-1/GFP at an MOI of 0.01. Cells were harvested at 44 hpi, and GFP expression was detected by flow cytometry. (C) MRC-5 cells were treated with IFN-α2a, siRNA targeting IFITM1, or nontargeting siRNA or mock treated. Total RNA was extracted, and the expression of IFITM1 was measured. Data are presented as percentages of expression relative to the mock-treated cells ± standard deviations (SD) (n = 3). (D) Treated MRC-5 cells were infected with HSV-1/GFP at an MOI of 0.5 for 7 h, and GFP expression was measured on a Cellomics ArrayScan instrument (means ± SD). Significance was determined by ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

IFITM1 domains necessary for membrane localization and virus restriction. (A) Twenty mutant human IFITM1 proteins were designed by mutating sequential blocks of 6 amino acids to alanine from the N to the C terminus of the protein. (B) A selection of these proteins with alanine blocks in the CIL domain were overexpressed in Vero cells using lentiviral constructs and puromycin selection. Expression of the HA-tagged protein was detected by Western blotting. (C) Localization of mutant protein expression was compared to that of wild-type human IFITM1. HA-tagged proteins are shown in green (anti-HA-488), and LAMP1 expression is shown in red. (D) Analysis of surface expression of HA-tagged CIL mutants of IFITM1 by flow cytometry on nonfixed and nonpermeabilized cells. (E) Representative plot showing relative surface expression of CIL mutants. (F) Vero cells were also infected with influenza virus, measles virus (rMV), or mumps virus at an MOI of 1, and the level of infection of each cell line was measured by fluorescence microscopy at 24 hpi (Cellomics ArrayScan). (G) Mutant IFITM1 proteins were also overexpressed in A549 cells. Cells were infected with rgRSV (MOI of 0.8) for 24 h prior to analysis of infectivity by flow cytometry (n = 3). (H) A total of 93 single nucleotide polymorphisms (SNPs) in the IFITM1 gene were identified. UTR, untranslated region. (I) Location of these SNPs in the human IFITM1 protein marked in red. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (by ANOVA, with significance relative to wild-type IFITM1 [n = 3]).

FIG 4

Ifitm1^−/− mice increase RSV but not mCMV infection. Homozygous knockouts and wild-type mice were intranasally infected with 5 × 10⁵ PFU of RSV-A2. (A) Weight loss was measured over the course of 7 days. (B) RSV viral load was measured by quantitative RT-PCR for the RSV L gene at day 4 after infection. (C and D) Cells in airways (C) and lungs (D) after infection. BAL, bronchoalveolar lavage fluid. (E and F) Lungs were homogenized and centrifuged, and the supernatant was collected for IL-6 (E) and IL-1β (F) analyses 4 days after infection. Mean values are shown (n ≥ 5) (A and B). Points represent data for individual animals (C to E). (G and H) WT and Ifitm1^−/− mice were infected with mCMV, weight loss was measured throughout (G), and after 4 days, the virus load in spleen and liver was measured by a plaque assay (H). (I) IL-6 concentrations in spleens and livers of mCMV-infected WT and Ifitm1^−/− mice 4 days after infection. (J and K) BMDM cells (J) and MEFs (K) were infected with mCMV. (L) Ifitm1 was quantified in lung, liver, and spleen of BALB/c mice (n = 5). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (by ANOVA [A and L] or a t test [B to K]).