Figure 5.

PI4KB Facilitates HPIV3 Replication

(A) PIK93 reduced PI4P on IBs. HeLa cells were treated with PIK93 (2 μM and 4 μM) for 1 h and transfected with plasmids encoding N-Myc (0.1 μg) and HA-P (0.4 μg) for 24 h. PI4P on IBs was analyzed by immunofluorescence.

(B–D) Effects of the PI4KB-specific inhibitor PIK93 on HPIV3 replication. HeLa cells were treated with PIK93 up to 4 μM for 1 h and infected with HPIV3 (MOI = 0.1) for 36 h. Cell lysates were harvested, and 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–G) HeLa cells were infected with HPIV3 (MOI = 0.1) and transfected with the indicated plasmids. The HPIV3 protein HN was analyzed via WB (E), extracellular viral production was analyzed via plaque assay (F), and HPIV3 RNA was analyzed via qPCR (G).

(H and I) PI4KB increases minigenome reporter activity.

(H) The effect of PI4KB on minigenome reporter RNA synthesis. vTF7-3-infected HeLa cells were transfected with pGEM4-N (125 ng), pGEM4-P (62.5 ng), pGEM4-L (100 ng), pOCUS-HPIV3-MG (50 ng), and increasing amounts of plasmid encoding PI4KB (200 ng, 400 ng, 800 ng, and 1600 ng). Relative luciferase activity was measured according to the manufacturer’s instructions. Expression of N, P, and PI4KB was detected via WB, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

(I) PI4KB has no effect on vTF7-3. Plasmids encoding Renilla luciferase (5 ng) and plasmids encoding N, P, L, and the minigenome were transfected alone or together with FLAG-PI4KB (1600 ng) in vTF7-3 infected HeLa cells to exclude nonspecific effects of PI4KB on vTF7-3. Relative luciferase activity was measured, and expression of PI4KB was detected via WB.

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. Scale bars, 10 μm. See also Figure S5.