Influenza Virus Induces Cholesterol-Enriched Endocytic Recycling Compartments for Budozone Formation via Cell Cycle-Independent Centrosome Maturation
Fig 6
The pericentrosomal ERC is important for the enrichment of vRNP and cholesterol.
(A and B) Association of vRNP with Rab11a in YB-1 KD cells. At 48 h post transfection with either non-targeting or YB-1 siRNA, HeLa cells constitutively expressing FLAG-Rab11a were infected with influenza virus at MOI of 10. At 8 h post infection, cells were subjected to immunoprecipitation assays with either non-specific IgG or anti-FLAG antibody (panel A) and GST pull-down assays using GST-RBD (panel B) as descried in Fig 2, respectively. Co-precipitated proteins were analyzed by western blotting with anti-PB1, anti-NP, and anti-Rab11a antibodies. (C) Accumulation of cholesterol in ERC in infected cells. At 48 h post transfection with either non-targeting or YB-1 siRNA, HeLa cells were infected with influenza virus at MOI of 10. At 6 h post infection, cells were pulse-labeled with 100 μg/ml of transferrin conjugated with Alexa 568 (red) for 30 min at 37°C, followed by incubation without Alexa 568-labeled transferrin for 30 min. After fixing in 4% PFA, cells were incubated with 200 μg/ml filipin to visualize cholesterol (green). In panel C, quantitative determination of the each signal (cholesterol, green; transferrin, red) was performed along the white dashed arrows shown within the panel C, enlarged panels. Y-axis indicates the normalized pixel intensity.