Figure 1.
Epitope-tagged FL IFITM3 and Δ21 IFITM3 restrict IAVs.
Naïve A549 cells and A549 cells transduced with tet-inducible c-myc-tagged (A), flag-tagged (B) FL IFITM3 or Δ21 IFITM3 were treated with or without 1000 ng/ml doxycycline (** 600 ng/ml doxycycline). Two days later, cells were infected with MLV-GFP pseudotyped with the indicated viral entry glycoproteins. Infected cells were harvested, fixed with formaldehyde, and analyzed by flow cytometry 48 hours after infection. The relative infectivity was determined as the percentage of GFP positive cells normalized to that of naïve A549 cells. (C) The same aliquots of cells used in (A) or (B) were analyzed by western blotting. The expression of IFITM3 isoforms was measured using the indicated antibodies. The expression of β–tubulin was used as our loading controls. The results are the averages of three independent infection replicates. The error bars indicate standard deviations. Each panel represents at least two sets of experiments with similar results. * indicates statistical significance (p<0.03 by Student's t test) as compared with naïve cells.
Figure 2.
Native FL IFITM3 and Δ21 IFITM3 restrict IAVs.
(A) A549 cells transduced with the vector alone or with tet-inducible native FL IFITM3 or Δ21 IFITM3 were treated with the indicated concentrations of doxycycline or interferon-β. Two days later, cells were infected by MLV-GFP pseudotyped with the indicated viral entry glycoproteins. Infected cells were harvested, fixed with formaldehyde, and analyzed by flow cytometry 48 hours after infection. The relative infectivity was determined as the percentage of GFP positive cells normalized to that of vector-transduced A549 cells. (B) The same aliquots of cells used in (A) were incubated with infectious influenza A/PR/8/34 (H1N1) virus at an M.O.I. of 1 or with influenza A/England/42/72 (H3N2) virus at an M.O.I of 0.5. Infected cells were labeled with anti-H1 or anti-H3 antibodies and with an PE-conjugated secondary antibody. Cells were analyzed by flow cytometry 16 hours after infection and the relative infectivity was determined as the percentage of PE positive cells normalized to that of vector-transduced A549 cells. (C) Experiments were similar to that in (B) except that cells were incubated with infectious influenza A/Virginia/ATCC3/2009 (H1N1) virus at an M.O.I. of 1. (D) The same aliquots of cells used in (A) or (B) were analyzed by western blotting. The expression of IFITM3 isoforms was measured using the indicated antibodies. The results are the averages of three independent infection replicates. The error bars indicate standard deviations. Each panel represents at least two sets of experiments with similar results. * indicates statistical significance (p<0.03 by Student's t test) as compared with vector-transduced cells.
Figure 3.
Y20A IFITM3 restricts IAV entry.
A549 (A), 293T (C), or Vero E6 (E) cells transduced with vector alone or to express FL IFITM3, Δ21 IFITM3, or Y20A IFITM3 were incubated with MLV-GFP pseudotyped with the indicated viral entry glycoproteins. Infected cells were harvested, fixed with formaldehyde, and analyzed by flow cytometry 48 hours after infection. The relative infectivity was determined as the percentage of GFP positive cells normalized to that of vector-transduced A549 cells. Expression of the indicated IFITM3 variants in A549 (B), 293T (D), or Vero E6 (F) cells were detected by western blotting using the indicated antibodies. The results are the averages of three independent infection replicates. The error bars indicate standard deviations. Each panel represents at least two sets of experiments with similar results. * indicates statistical significance (p<0.03 by Student's t test) as compared with vector-transduced cells.
Figure 4.
Δ21 IFITM3 and Y20A IFITM3 have boarder subcellular distributions.
A549 cells expressing native FL IFITM3, Δ21 IFITM3, or Y20A IFITM3 were fixed, permeabilized, and labeled with anti-LAMP2 (green) and anti-IFITM2/3 (red) and DAPI (blue) antibodies. DAPI was used as nuclear counterstain. Cells were imaged by confocal microscopy. Each panel represents at least two sets of experiments with similar results.
Figure 5.
Epitope-tagged IFITM3 isoforms have altered subcellular distributions.
A549 cells expressing the indicated c-myc- (A) or flag-tagged (B) IFITM3 isoforms were fixed, permeabilized, and labeled with anti-LAMP2 (green) and anti-IFITM2/3 (red) and DAPI (blue) antibodies. Cells were then imaged by confocal microscopy. Each panel represents at least two sets of experiments with similar results.