FIG 4.

PRRSV particles are transported to IFITM3-containing endosomes or lysosomes. (A) Marc-145-IFITM3-flag cells transfected with pcDNA3.1-Rab5-GFP, -Rab7, and -LAMP1-YFP plasmids for 48 h were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Cells were labeled with anti-IFITM3 and -Rab7 antibodies followed by Alexa Fluor 594-conjugated goat anti-mouse IgG (H&L) antibody for IFITM3 (red) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H&L) antibody for Rab7 (green), and cell nuclei were counterstained using DAPI. Rab5 and LAMP1 (green) were observed directly using a confocal microscope. (B) PRRSV were allowed to bind to prechilled Marc-145-IFITM3-flag cells for 1 h on ice, unbound virions were washed, and cells were warmed to 37°C for 45 min. IFITM3 was stained using an anti-IFITM3 antibody and the corresponding fluorescent second antibody (blue), and PRRSV was stained using an anti-N antibody and a fluorescent second antibody (red). (C) Marc-145-IFITM3-flag cells were infected with 0.1 MOI of PRRSV for 12 h, and then cells were transfected with pcDNA3.1-Rab5-GFP, -Rab7, and -LAMP1-YFP plasmids for 36 h and fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Cells were labeled as described in Fig. 4A. Scale bar, 10 μM. PRRSV, porcine reproductive and respiratory syndrome virus; IFITM, interferon-induced transmembrane.