Fig. 4.

NLRX1 interacts with PB1-F2. (A) NLRX1 expressing construct was cotransfected with either empty vector or HA-tagged PB1-F2 in HEK293T cells; 48 h later, cells were lysed and immunoprecipitated proteins were blotted for either HA-PB1-F2 or NLRX1 as indicated. The two lower gels show expression of PB1-F2 and NLRX1 in whole cell extracts (WCEs). (B) Confocal microscopy analysis demonstrating colocalization of NLRX1 and PB1-F2. A549 cells were transiently transfected with myc-tagged PB1-F2 expressing vector or empty vector; 24 h later, cells were fixed, and yellow regions are the areas of NLRX1 and PB1-F2 colocalization. Nuclei were stained using DAPI (blue). (C) HEK293T cells were infected with 5 MOI of IAV or IAV ΔPB1-F2. Cell lysates were collected 24 h pi to perform immunoprecipitation using either isotype-antibody control, anti-NLRX1 antibodies, or anti-PB1-F2 polyclonal serum. Samples were analyzed by Western blot. WCEs (Input) from infected cells served as a control to detect NLRX1 and viral PB1-F2 protein. Actin was used as a loading control. (D) PB1-F2 specifically interacts with NLRX1 but not other mitochondrial proteins. HEK293T cells were infected either with parental IAV or IAV ΔPB1-F2 strain, and pull-down assay was performed. PB1-F2 interacted with NLRX1 but not other mitochondrial proteins, Tom20, AIF, and Cox IV (Left). WCEs (Input) from infected cells served as a control to detect NLRX1 and other mitochondrial proteins (Right). (E) Direct interaction between PB1-F2 and NLRX1 proteins. Recombinant GST–PB1-F2 protein was incubated with the mitochondrial extract and immunoprecipitated complex was analyzed by immunoblot. Coomassie gel served as a control to demonstrate the presence of GST–PB1-F2 (37 kDa) and GST control (26 kDa) proteins. (F) A549 lung epithelial cells were infected with 1 MOI of IAV or IAV ΔPB1-F2. Endogenous interaction in infected cells was evaluated by coimmunoprecipitation and immunoblot with anti NLRX1. The two bottom gels show expression of NLRX1 and PB1-F2 in whole cell extracts. Actin served as a loading control in this experiment. (G) Confocal microscopy analysis demonstrating colocalization of endogenous NLRX1 and PB1-F2. A549 cells were infected (MOI of 5) with IAV (Top and Middle) or with IAV Δ PB1-F2 (Lower); 24 h after infection, cells were stained with the mitochondrial tracker CMXRos (red), fixed, and then stained for NLRX1 and PB1-F2. IAV-infected cells costained with NLRX1-specific rabbit polyclonal antibody (green), PB1-F2–specific mouse polyclonal serum (blue), and mitochondria (red). White regions are areas of NLRX1 and PB1-F2 colocalization on the mitochondria. (H) WT and Nlrx1^−/− deficient BMDMs were infected with 5 MOI of IAV; 16 h later, cells were collected and mitochondria lysates were prepared. Coimmunoprecipitation assay was performed using isotype control, anti-NLRX1 antibodies and anti–PB1-F2 polyclonal serum. WCEs (Input) and mitochondrial fractions were analyzed via Western blot to visualize NLRX1, viral PB1-F2, and mitochondrial VDAC1 proteins. Data are representative of two to three independent experiments.