Figure 4.

PI4P on IBs Was Generated by PI4KB

(A) HeLa cells were transfected with plasmids encoding N-Myc (0.1 μg), HA-P (0.4 μg), and FLAG-PI4KB (0.5 μg) for 24 h and analyzed for colocalization of FLAG-PI4KB and IBs. The fluorescence intensity profile of IBs (green) and PI4KB (red) was measured along the line drawn on a 2× zoom panel by Leica Application Suite Advanced Fluorescence Lite.

(B–D) Effects of PI4KB knockdown on HPIV3 replication. HeLa cells were infected with HPIV3 (MOI = 0.1) and transfected with shRNA targeting PI4KB (3 μg) for 24 h. The HPIV3 protein HN was analyzed via WB (B), extracellular viral production was analyzed via plaque assay (C), and HPIV3 RNA was analyzed by qPCR (D).

(E) HeLa cells were transfected with the indicated plasmids for 24 h and analyzed for colocalization of endogenous PI4KB and IBs.

(F) HeLa cells stably expressing GFP-P were infected with HPIV3 (MOI = 0.1) for up to 36 h and analyzed for colocalization of endogenous PI4KB and IBs. Yellow arrows indicate representative colocalization of PI4KB and IBs.

(G and H) HeLa cells were transfected with N-Myc (0.5 μg) and HA-P (2 μg) for 24 h, and the ER was extracted to detect PI4KB in RER when expressing IBs (N and P) or non-IBs (N_L478A and P) via WB. Representative images are shown (G). Also shown is quantitative analysis of the PI4KB content in RER when expressing IBs (N and P) or non-IBs (N_L478A and P) (H).

Images are representative of experiments carried out at least three times. Error bars, mean ± SD of three experiments (n = 3). Student’s t test; ^★p < 0.05, ^★★p < 0.01, ^★★★p < 0.001; ns, non-significant. Scale bars, 10 μm. See also Figure S4.