Influenza Virus PB1-F2 Protein Induces Cell Death through Mitochondrial ANT3 and VDAC1
Figure 5
PB1-F2 Protein Interacts with ANT3 and VDAC1
(A) Cellular proteins pulled down with GST-PB1-F2 were separated by 12% SDS-PAGE and silver-stained. The asterisks mark the protein bands that are unique to the GST-PB1-F2 lanes (~36 kDa, 55 kDa, and 80 kDa). Lanes 4 and 5 represent the results of two separate pulldown experiments.
(B) PB1-F2 specifically interacts with ANT3 and VDAC1 but not other outer and inner mitochondrial membrane proteins. Interaction of PB1-F2 with ANT3 and VDAC1 was confirmed in 293T cells by coimmunoprecipitation with transfected HA-tagged ANT3 and flag-tagged VDAC1 (top five images). Tom20, COXIV, and TIM 44 proteins were used as the mitochondrial coimmunoprecipitation controls.
(C) Transfected ANT3 and VDAC1 localize to mitochondria. 293T cells were transfected with Flag-tagged ANT3 and VDAC1 for 24 h and were subsequently fractionated to generate the cytosolic and mitochondrial fractions. Tom20 protein served as a control for the mitochondrial fraction. Each sample was processed in triplicate (lanes 1, 2, and 3, ANT3; lanes 4, 5, and 6, VDAC1).
(D) PB1-F2 directly interacts with ANT3 and VDAC1. 35S-labeled ANT3 and VDAC1 were expressed in vitro using a rabbit reticulocyte lysate system and subjected to pulldown with either 5 μg GST (left lane) or GST-PB1-F2 (right lane). Proteins were separated by 12% SDS-PAGE and visualized by autoradiography.
(E) PB1-F2 expressed during viral infection interacts with ANT3 and VDAC1. 293T cells were transfected with Flag-tagged ANT3 and VDAC1 proteins and 24 h later were infected with wild-type PR8 virus (PR8) or the virus knocked out for PB1-F2 expression (del) at an MOI of 2. 15 h after infection, the cells were collected and the immunoprecipitation with anti-PB1-F2 polyclonal serum was performed. Influenza virus NP was used as a control to show equal levels of infection between the samples. WB, Western blot.