Inhibition of SG formation facilitates HPIV3 replication.

(A) HeLa-GFP-G3BP cells were mock-infected or infected with HPIV3 (MOI = 1) for 24 h. HPIV3 -vRNA (purple) and +vRNA (red) were detected via RNA-FISH. Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (B-E) Wild type or G3BP-deficient HeLa cells were infected with HPIV3 (MOI = 1) for 24 h. (B) Cells were immunostained for HPIV3 (purple), TIA-1 (green), and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (C) The percentage of cells containing SGs was quantified in three independent experiments. (D) Cell lysates were analyzed via western blot using anti-HN, anti-G3BP, and anti-GAPDH antibodies. (E) The supernatants were collected for a plaque assay to determine the viral titer. (F-I) HeLa cells were transfected with an empty plasmid or plasmids encoding eIF2α or the nonphosphorylatable mutant, eIF2α-S51A, for 24 h, then infected with HPIV3 (MOI = 1) for additional 24 h. (F) Cells were immunostained for HPIV3 (purple), G3BP (green), and HA tag (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (G) The percentage of cells containing SGs was quantified in three independent experiments. (H) Cell lysates were analyzed via western blot using anti-HN, anti-HA, and anti-GAPDH antibodies. (I) The supernatants were collected for a plaque assay to determine the viral titer. Data are represented as means ±SD. Student’s t test: * P<0.05, ** P<0.01, *** P<0.001, ns = not significant.