Fig. 2

BinCARD1 positively regulates the nuclear import of the vRNP complex and newly synthesized NP. A The effect of BinCARD siRNA1 treatment on the internalization of IAV was analyzed. BinCARD siRNA1- or scrambled siRNA-treated A549 cells were infected with WSN (H1N1) virus (MOI = 5) on ice at 4 °C for 1 h, incubated at 37 °C for 30 min to allow viral internalization, and washed with ice-cold PBS (pH = 1.3) to remove uninternalized viral particles. Cell lysates were subjected to western blotting with a mouse anti-NP mAb to detect the amount of internalized virus particles. B The effect of BinCARD siRNA1 treatment on the uncoating process of IAV was analyzed. BinCARD siRNA1-, scrambled siRNA- or vATPase siRNA-treated A549 cells were treated with CHX to inhibit protein synthesis and were then infected with WSN (H1N1) (MOI = 50) virus. At 1.5 h p.i., the infected cells were stained with a rabbit anti-M1 pAb and Alexa Fluor 633 goat anti-rabbit IgG (H + L) (red) and visualized by confocal microscopy. C The effect of BinCARD siRNA1 treatment on the cellular localization of NP during IAV infection was determined by confocal microscopy. BinCARD siRNA1- or scrambled siRNA-treated A549 cells were infected with WSN (H1N1) (MOI = 5) virus. At 2, 3, 4 and 5 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H + L) (red). The nuclei were stained with DAPI. D Quantitative analysis of NP localization in virus-infected BinCARD siRNA1-treated cells. On the basis of the confocal microscopy images presented in C, the localization of NP (indicative of vRNP localization) upon the appearance of its nuclear localization was divided into the following categories: weak nuclear localization, strong nuclear localization, simultaneous localization at the boundary between the nucleus and the cytoplasm, or predominant cytoplasmic localization. The data shown are derived from 100 cells visualized by confocal microscopy with a 40X objective lens. E The effect of BinCARD1 knockout on the nuclear import of the vRNP complex was determined through a cell fractionation experiment. BinCARD_KO A549 cells and A549 control cells were infected with WSN (H1N1) (MOI = 5) virus. At 3 h p.i., the cells were separated into nuclear (N) and cytoplasmic (C) fractions. The proteins in each fraction were subjected to western blotting with a mouse anti-NP mAb. Densitometric analysis of the western blots was performed with ImageJ software. Lamin B1 and GAPDH were used as the loading controls for the nuclear and cytoplasmic fractions, respectively. F The effect of BinCARD1 knockout on the nuclear import of NP was assessed by confocal microscopy. BinCARD_KO A549 cells and A549 control cells were transfected with a vector to express WSNNP. At 20 h p.t., the cells were stained with a mouse anti-NP mAb and a rabbit anti-BinCARD pAb, incubated with Alexa Fluor 633 goat anti-mouse IgG (H + L) (red) and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (green), and visualized by confocal microscopy