Fig 1.
IFITM3 is ubiquitinated by NEDD4.
A) Alignment of IFITM3 N-terminal amino acids from various species. Bold and underlined text highlights the conserved PPxY motif. B) Mouse embryonic fibroblasts (MEFs) were stimulated overnight with IFN-α (160 units/mL) to ensure production of IFITM3, and imaged by fluorescent confocal microscopy with staining for endogenous IFITM3, NEDD4, LAMP1, and nuclei (DAPI). Images were taken with a 60x objective and 2.5x zoom. Pseudocolored merged images in different staining combinations are shown. C-E), HEK293T cells were co-transfected with plasmids expressing IFITM3 and epitope tagged ubiquitin ligases, NEDD4, CLB-B, SMURF1 and SMURF2, as indicated. Cell lysates were immunoprecipited with anti-myc resin, and examined by Western blotting with anti-myc and anti-ubiquitin (Ub) antibodies. Western blots of cell lysates with anti-HA (C) or anti-FLAG (D,E) antibodies were performed to confirm expression of the ubiquitin ligases. Anti-GAPDH Western blotting was performed to confirm comparable protein loading.
Fig 2.
The IFITM3 PPxY motif is required for ubiquitination by NEDD4.
A-C) HEK293T cells were co-transfected with plasmids expressing myc-hIFITM3 or FLAG-NEDD4 as indicated. A-B) Cell lysates were immunoprecipitated with anti-myc resin, and examined by Western blotting with anti-myc and anti-ubiquitin (Ub). Western blotting of cell lysate with anti-FLAG antibodies was performed to confirm expression of NEDD4. Western blotting with anti-GAPDH antibodies was performed to confirm comparable protein loading. C) Cell lysates were immunoprecipitated with anti-myc or anti-FLAG resin, and co-immunoprecipitation was examined by Western blotting with both anti-myc and anti-FLAG antibodies for each immunoprecipitate. Western blots of cell lysates with anti-myc and anti-FLAG antibodies were performed to confirm expression of IFITM3 and NEDD4, respectively. Anti-GAPDH Western blotting was performed to confirm comparable protein loading.
Fig 3.
NEDD4 catalytic activity is required for IFITM3 ubiquitination.
HEK293T cells were transfected with the indicated mouse or human IFITM3 constructs and were co-transfected with plasmids expressing HA-NEDD4 or a catalytically inactive HA-NEDD4-C867A mutant. IFITM3 was immunoprecipitated with anti-myc resin and subjected to anti-myc and anti-ubiquitin (Ub) Western blotting. Cell lysates were probed with anti-NEDD4 antibodies to confirm expression of NEDD4 constructs. Anti-GAPDH staining served as a protein loading control.
Fig 4.
NEDD4 ubiquitinates IFITM3 in vitro.
HA-hIFITM3 was added to reactions containing NEDD4-compatible E1 and E2 ubiquitin ligases, ubiquitin (WT or mutants in which only K48 or K63 were not mutated), and reaction buffer containing ATP in the presence or absence of NEDD4. The reaction was allowed to proceed for 1 h at 37°C, and IFITM3 was re-immunoprecipitated and subjected to Western blotting with anti-ubiquitin (Ub) and anti-HA antibodies. IgG H.C. indicates detection of the heavy chain of the immunoglobulin used for immunoprecipitation.
Fig 5.
NEDD4 knockout decreases IFITM3 ubiquitination and protects cells from virus infection.
A) Cell lysates from NEDD4 WT and KO MEFs were subjected to anti-IFITM3, anti-NEDD4, and anti-actin immunoblotting to evaluate NEDD4 levels in each cell line and the effect of NEDD4 on endogenous IFITM3 levels. Retroviral reconstitution of the indicated cells with an empty retrovirus control or retrovirus expressing NEDD4 is denoted by + Vector and + NEDD4, respectively. B) 2 mg of protein from NEDD4 WT and KO cell lysates were immunoprecipitated for endogenous IFITM3 and examined by Western blotting with anti-ubiquitin and anti-IFITM3 antibodies. C) The indicated cell lines were infected for 24 h with influenza A virus (IAV) PR8 and X-31 strains at an MOI of 5. Cells were then fixed and stained with anti-influenza virus NP to measure the percentage of cells infected using flow cytometry. D) NEDD4 WT and KO MEFs were infected with influenza virus 2011 isolates (IAV or influenza B virus (IBV)) or Sendai virus (SeV) at an MOI of 5 for 24 h, or were infected with VSV G-pseudotyped retrovirus (VSV G-pseudo) for 48 h. Cells were then fixed and stained with anti-influenza NP in the case of influenza virus infections or were examined for GFP positivity in the case of VSV G-pseudo or SeV to measure the percentage of cells infected using flow cytometry. C,D) Non-infected samples were used as a baseline for gating of infected cells. Results shown are representative of at least three independent experiments, each performed with triplicate samples. The average percent infection of WT NEDD4 cells was set to 1 for the calculation of relative percent infection. Error bars represent standard deviation of triplicate samples. * Indicates a p-value less than 0.001 calculated by Student’s t-test in comparison to values for NEDD4 WT cells.
Fig 6.
NEDD4 regulates cellular susceptibility to influenza virus infection by controlling IFITM3 levels.
A,B) NEDD4 WT and KO MEFs were transfected for 24 h with control siRNA (siCont) or siRNA targeting IFITM3 (siIFITM3). A) Cells were collected just prior to infection for confirmation of IFITM3 knockdown by anti-IFITM3 Western blotting, with anti-actin blotting serving as a protein loading control. B) Following siRNA treatment, cells were infected with influenza virus strain PR8 at an MOI of 10 for 24 h. Cells were then fixed and stained with anti-influenza virus NP to measure the percentage of cells infected using flow cytometry. Results shown are representative of three independent experiments, each with samples run in triplicate. Error bars represent standard deviation. * Indicates p-value less than 0.001 calculated with Student’s t-test for comparison of samples denoted by horizontal lines.
Fig 7.
NEDD4 knockdown in human lung cells increases IFITM3 levels and resistance to influenza virus infection.
A-C) A549 cells were transfected for 48 h with control siRNA (siControl) or siRNA targeting human NEDD4 (siNEDD4). A) Cells were collected just prior to infection for confirmation of NEDD4 knockdown by anti-NEDD4 Western blotting, with anti-actin blotting serving as a loading control. Anti-IFITM3 blotting demonstrates an increase in IFITM3 upon NEDD4 knockdown. B,C) Following siRNA treatment, cells were infected with influenza virus strain PR8 at an MOI of 2.5 for 6 h. Cells were then fixed and stained with anti-influenza virus NP to measure the percentage of cells infected using flow cytometry. Results shown are representative of three independent experiments, with samples run in triplicate. Error bars represent standard deviation of triplicate samples. * Indicates a p-value less than 0.0001 calculated with Student’s t-test. D) NCI-H358 and NCI-H2009 cells were transfected for 48 h with siControl or siNEDD4. Cell lysates were subjected to immunoblotting with anti-NEDD4 to confirm NEDD4 knockdown, anti-IFITM3 to demonstrate increase in endogenous IFITM3 upon NEDD4 knockdown, and anti-GAPDH as a loading control. E) A549 cells were treated with equal volumes Dimethyl Sulfoxide (DMSO) as a control, MG132 (10 μM), Chloroquine (10 μM), Bafilomycin A1 (1 μM), or Leupeptin (100 μM) for 24 h. Cells were also treated with IFN-α (100 units/mL) for comparison. Cell lysates were subjected to anti-IFITM3 immunoblotting to evaluate endogenous IFITM3 levels with each treatment. Western blotting with anti-GAPDH served as a loading control.