E3 Ubiquitin Ligase NEDD4 Promotes Influenza Virus Infection by Decreasing Levels of the Antiviral Protein IFITM3
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.