Figure 2.

HPIV3 IBs Are Surrounded by a Fragmented ER Membrane, and IB Formation Depends on the ER

(A) HeLa cells were transfected with vector (a), N_L478A-Myc and HA-P (b), or N-Myc and HA-P (c) for 24 h or infected with HPIV3 (MOI = 0.1) for 36 h and analyzed via electron microscopy to assess the ultrastructure of the ER and IBs. Black arrows indicate the fragmented ER membrane. The black spheroid structure indicates an IB.

(B) Surface projections of a three-dimensional reconstruction with accumulations of fragmented ER membranes around IBs. HeLa cells expressing GFP-tagged P were mock-treated or infected with HPIV3 (MOI = 0.1), transfected with mcherry-Calnexin for 36 h, and analyzed via Zeiss Airyscan Super-resolution microscopy. Representative Airyscan images were acquired without pixel saturation. Boxed regions were magnified and processed to a three-dimensional surface reconstruction.

(C) HeLa cells were transfected with the indicated plasmids for 24 h. Cell lysates were harvested and treated with a hypotonic solution for 20 min and broken with a Dounce homogenizer. The homogenate was left untreated or treated with Triton X-100, trypsin, or Triton X-100 plus trypsin and analyzed via WB.

(D) HeLa cells are treated in the way described above, and the contents of N and P protein were analyzed to determine quantity by Quantity one software.

(E–G) Effects of the ER stress inducer Tu on IB formation. HeLa cells were transfected with N-Myc and HA-P for 12 h, treated with Tu (2 μg/mL) for 6 h, and analyzed for the location of Calnexin and the size and number of IBs. Representative images are shown (E). Also shown are quantitative analysis of the average number of IBs per cell treated with DMSO or Tu (F; n = 3, 50 cells were counted) and quantitative analysis of the average diameter of IBs treated with DMSO or Tu (G; n = 3, 100 IBs were counted).

(H–J) Effects of the ER stress inducers Tg and Tu on HPIV3 replication. HeLa cells were infected with HPIV3 (MOI = 0.1) for 12 h and treated with Tg (1 μM) for 3 h or Tu (2 μg/mL) for 6 h. The HPIV3 protein HN was analyzed via western blot (WB) (H), extracellular viral production was analyzed via plaque assay (I), and HPIV3 RNA was analyzed by qPCR (J).

(K–M) The IB components N and P were rich in rough ER. HeLa cells were transfected with plasmids encoding N-Myc (0.5 μg) and HA-P (2 μg) or N_L478A-Myc (0.5 μg) and HA-P (2 μg) for 24 h, and the RER was extracted to detect IB composition. Representative images are shown (K). Also shown is quantitative analysis of the N or N_L478A and P content in rough endoplasmic reticulum (RER) when expressing IBs (N and P) or non-IBs (N_L478A and P) (L and M).

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 in (A), 500 nm; scale bars in (B), 3 μm; All other scale bars, 10 μm. See also Figure S2.