The inhibitory effect of HPIV3 IBs on SG formation is associated with the viral RNAs-holding capacity of the IBs.

(A and B) HeLa cells were co-transfected with plasmids encoding GFP-P and N-Myc, GFP-P and NΔN10-Myc, or GFP-P^RSV and Myc-N^RSV for 24 h, then infected with HPIV3 for another 24 h. HPIV3 (A) +vRNAs (red) and (B)–vRNAs (red) were detected via RNA-FISH. The white scale bar corresponds to 10 μm. (C) HPIV3 mini-genome replicon system assay was performed and cell lysates were analyzed via western blot using anti-Myc and anti-GAPDH antibodies. (D and E) HeLa cells were transfected with an empty plasmid or co-transfected with plasmids encoding GFP-P and N-Myc, GFP-P and NΔN10-Myc, or GFP-P^RSV and Myc-N^RSV for 24 h, then infected with HPIV3 for another 24 h. (D) Cells were immunostained for HPIV3 (purple) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (E) The percentage of cells containing IBs or SGs was quantified in three independent experiments. Cell lysates were analyzed via western blot using anti-HN, anti-Myc, anti-GFP and anti-GAPDH antibodies. Data are represented as means ±SD. Student’s t test: * P<0.05, ** P<0.01, *** P<0.001, ns = not significant.