Article Figures & Data
Figures
- Figure S1. Schematic overview of IFITM3 variants analyzed in this study.
- Figure 1. Endogenous human IFITM3 restricts CHIKV infection.
(A) Immunoblot of indicated HeLa cell lysates using indicated antibodies. (B) Flow cytometry analysis of interferon-induced transmembrane expression in permeabilized HeLa cells. (C) Immunofluorescence microscopy analysis of indicated HeLa cell lines/clones upon mock treatment and treatment with 5,000 IU/ml IFN-α for 48 h (scale bar = 20 µm). (D) HeLa cells were infected with EGFP-CHIKV after a 6 h IFN-α pre-treatment duration at indicated MOIs. The percentage of EGFP-positive infected cells was quantified 24 h postinfection by flow cytometry. (E) HeLa cells were infected with MAYV-GFP at indicated MOIs The percentage of EGFP-positive infected cells was quantified 24 h postinfection by flow cytometry. (F) HeLa cells were infected with Influenza A virus at indicated MOIs. 24 h postinfection, cell-associated viral HA mRNA was quantified by quantitative RT-PCR. SNP, single nucleotide polymorphism; MFI, mean fluorescence intensity; MOI, multiplicity of infection.
Source data are available for this figure.
Source Data for Figure 1[LSA-2020-00909_SdataF1.zip]
- Figure 2. Ectopic expression of human interferon-induced transmembrane (IFITM)-HA proteins restricts alphaviral infection by interfering with glycoprotein-mediated entry.
HEK293T cell lines stably expressing HA-tagged IFITM proteins were generated via retroviral transduction and puromycin selection. (A) Protein expression was evaluated by immunoblotting using indicated antibodies. (B) Cells were immunostained with an anti-HA antibody and analyzed by flow cytometry, measuring IFITM surface levels in membrane-intact cells and total expression in permeabilized cells. Mean fluorescence intensity is inset for each condition. (C) Immunofluorescence microscopy of surface and intracellular immunostaining of heterologously expressed IFITM proteins using anti-HA antibody (scale bar = 20 µm). (D) IFITM-HA expressing cells were infected with EGFP-CHIKV of increasing MOIs for 24 h, and the percentage of EGFP-positive cells was quantified by flow cytometry. (E) IFITM-HA expressing cells were infected with EGFP-CHIKV or MAYV strain TRVL15537. 24 h postinfection, supernatant was collected and plaque assays were performed on Vero E6 cells to quantify infectious virus progeny. (F) Cell lines were inoculated for 48 h with lentiviral firefly luciferase-expressing reporter pseudoparticles decorated with glycoproteins of murine leukemia virus, Ebola virus, or different CHIKV lineages. Luciferase activity in transduced cells was quantified luminometrically and normalized to the vector control cell line.
- Figure S2. HeLa cells were infected with EGFP-CHIKV after 6 h IFN-α pre-treatment duration and with EGFP-MAYV, at indicated MOIs.
The percentage of EGFP-positive cells was quantified 24 h postinfection by flow cytometry. Shown are dot plots from one representative experiment.
- Figure S3. Antiviral activity of human IFITM proteins.
(A) Interferon-induced transmembrane-HA expressing cells were infected with EGFP-CHIKV of increasing MOIs for 24 h, and the percentage of EGFP-positive cells was quantified by flow cytometry. Shown are dot plots from one representative experiment. (B) interferon-induced transmembrane-HA expressing cells were infected with EGFP-CHIKV of increasing MOIs for 24 h, and cells were analyzed by fluorescence microscopy.
- Figure 3. Cell-to-cell transmission of and direct infection by CHIKV differ in efficiency, but share susceptibility to interferon-induced transmembrane-mediated restriction.
Confluent HEK293T cells were infected with EGFP-CHIKV or EGFP-HSV-1 for 6 h. Subsequently, cells were overlaid with 0.8% agarose or left untreated. (A, B) EGFP-positive cells were measured 1 and 2 d postinfection via flow cytometry and (B) microscopic analysis of CHIKV-infected cells was performed 24 h postinfection (scale bar = 500 µm). (C) Confluent HEK293T cells ectopically expressing interferon-induced transmembrane-HA proteins were infected with EGFP-CHIKV for 6 h followed by the application of a 0.8% agarose overlay where indicated. 24 h postinfection, EGFP-positive cells were measured via flow cytometry.
- Figure S4. Antivital activity of human IFITM proteins in the context of cell-to-cell transmission.
(A) EGFP-positive cells were quantified 1 and 2 d postinfection with EGFP-HSV-1 and EGFP-CHIKV via flow cytometry. (B) Confluent HEK293T cells ectopically expressing interferon-induced transmembrane-HA proteins were infected with EGFP-CHIKV for 6 h followed by the application of a 0.8% agarose overlay where indicated. 24 h postinfection, EGFP-positive cells were quantified via flow cytometry. Shown are dot plots from one representative experiment.
- Figure 4. Reduction of cell surface expression of antiviral interferon-induced transmembrane (IFITM) proteins in CHIKV-infected cells.
(A) Indicated HEK293T cells were infected with EGFP-CHIKV (MOI 10) for 24 h and were immunostained with an anti-HA antibody for flow cytometry. (B) Quantification of IFITM-HA expression in EGFP-positive relative to EGFP-negative cells after infection with EGFP-CHIKV for 24 h. (C) Immunoblot analysis of IFITM-HA expression in cells infected with EGFP-CHIKV at the indicated MOIs and quantitative analysis thereof. (D) Indicated HEK293T cell lines were infected with EGFP-CHIKV (MOI 10) for 24 h. Permeabilized cells were immunostained with an anti-HA antibody and analyzed microscopically (scale bar = 20 µm). Arrowheads indicate EGFP-positive cells with IFITM-HA expression (red) or lack thereof. (E) Indicated cell lines were transfected individually with full-length EGFP-CHIKV RNA and HIV-1 EGFP DNA and were immunostained with an anti-HA antibody. Mean fluorescence intensitys of IFITM-HA EGFP-positive cells were determined via flow cytometry and normalized to the mean fluorescence intensity of EGFP-negative cells. (F) Quantification of IFITM3-HA surface level 24 h after individual transfection of HEK293T IFITM-3-HA cells with full length EGFP-CHIKV mRNA or RNA encoding the non-structural proteins 1–4 and EGFP.
Source data are available for this figure.
Source Data for Figure 4[LSA-2020-00909_SdataF1.zip]
- Figure S5. Indicated cell lines were transfected individually with full-length EGFP-CHIKV RNA and HIV-1 EGFP DNA and were immunostained with an anti-HA antibody.
Shown are dot plots from one representative experiment.
- Figure 5. Posttranscriptional reduction of endogenous IFITM3 expression in CHIKV-infected cells.
(A) HeLa cells were infected with EGFP-CHIKV (MOI 100) for 24 h, permeabilized, and immunostained for IFITM3 before microscopic analysis. Dotted lines indicate the border of EGFP-positive cells (scale bar = 50 µm). (B) Quantification of microscopic images of infected HeLa cells. IFITM3 mean fluorescence intensity (MFI) was determined using ImageJ and is plotted against EGFP MFI (mock cells n = 50; CHIKV-infected cells n = 80). (C) HeLa cells were infected with EGFP-CHIKV and EGFP-MAYV (MOI 10) for 24 h. Subsequently, permeabilized cells were stained for IFITM3, ISG15, MX1, or BST-2 and MFI values were normalized to EGFP-negative cells. (D) HeLa cells were infected with EGFP-CHIKV or EGFP-MAYV (MOI 10) and 24 h postinfection, IFITM3, and IFIT1 mRNA levels were measured by quantitative RT-PCR.
Source data are available for this figure.
Source Data for Figure 5[LSA-2020-00909_SdataF1.zip]
- Figure S6. HeLa cells were infected with EGFP-CHIKV (MOI 10) for 24 h.
Subsequently, permeabilized cells were stained for IFITM3, ISG15, MX1, or BST-2. Shown are dot plots from one representative experiment.
- Figure 6. Reduction of virion infectivity through expression of IFITM3 in virus-producing cells.
(A) Immunoblot analysis of lysates and concentrated supernatants of 293T and Hela cells transfected with full-length EGFP-CHIKV mRNA or pBR-NL4.3-GFP plasmid at 24 h posttransfection. (B) Viral titers produced by cells transfected in (A) normalized to the relative amount of GFP-positive cells. Viral titers were determined by titration and flow cytometric analysis of supernatants on 293T cells (CHIKV) or Tzm-bl cells (HIV), respectively. (C) Relative specific infectivity of produced virions as calculated by the infectivity per abundance of viral capsid.
Source data are available for this figure.
Source Data for Figure 6[LSA-2020-00909_SdataF1.zip]
Tables
IFITM3 clone Type of gene edit Gene edit (nt position refers to NM_021034.3) Genomic DNA sequence Guide RNA target (PAM) KO +1 frameshift Insertion of A between nts 84 and 85 CCTGTCA[+A]ACAGTGGCCAG GGGGGCTGGCCACTGTTGAC(AGG) Δexon1 Deletion Deletion of 381 nts post nt 36 CGACCGCCGCTGGTCTT [deletion of 381 nt] 1:CGACCGCCGCTGGTCTTCGC(TGG) TCCCGTGTGTGCCCACG 2:CGTGGGCACACACGGGACAG(AGG) SNP rs12252-C T to C transition T89C CCTGTCAACAGCGGCCAGCCCCC guide:GGGGGCTGGCCACTGTTGAC(AGG) +HDR-Donor: CTGGACACCATGAATCACACTGTCCAAACCTTCTTCTCTCCTGT CAACAGCGGCCAGCCCCCCAACTATGAGATGCTCAAGGAGGAGCAC Δ1st ATG Deletion Deletion of 31 nts post nt 36 CGACCGCCGCTGGTCTT [deletion of 31 nt] 1:CGACCGCCGCTGGTCTTCGC(TGG) 2:TGACAGGAGAGAAGAAGGTT(TGG) CTTCTTCTCTCCTGTCAA nt, nucleotide.
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