Figure 2

Overexpression of GBP1 inhibits PRRSV replication by disrupting the stress fibers of the actin cytoskeleton. A Marc-145-Vector or Marc-145-GBP1-flag cells were visualized using rabbit anti-flag pAb and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H&L) antibodies, and actin was visualized using Alexa Fluor 546-labeled phalloidin antibody. As the control, Marc-145-Vector cells were treated with 2 μM cytochalasin D for 12 h, and actin was visualized using labeled phalloidin. B The growth curves of cells treatment with 0, 0.5, 1.0, 2.0 μM of cytochalasin were shown. Cells for each treatment were seeded at a concentration of 1 × 10⁴ cells/well and split daily for 4 consecutive days and cells were counted to determine cell number. C–E Marc-145 cells infected with 0.1 MOI of PRRSV were treated with 0, 0.5, 1.0, 2.0 μM cytochalasin D from 1 hpi onwards. At 36 hpi, cells were collected to determine the expression of ORF7 mRNA, progeny viral copies in the supernatant and N protein via quantitative PCR and Western blotting, respectively. F Marc-145 cells were infected with GFP-PRRSV (0.1 MOI). When ~60% of the cells were GFP positive, cells were sorted and used as donor cells to co-culture with Marc-145-RFP cells (red, target cells) for 2 h. Target cells were sorted and cultured with or without 2.0 μM cytochalasin D, and transmission was monitored by the fraction of GFP⁺RFP⁺ target cells at 36 hpi. G GFP-PRRSV-positive Marc-145 cells were sorted and used as donor cells for co-culture with Marc-145-GBP1-RFP cells for 2 h. Target cells were sorted and cultured, and transmission was monitored by the fraction of GFP⁺RFP⁺ target cells at 36 hpi. GBP1 guanylate-binding protein 1; PRRSV porcine reproductive and respiratory syndrome virus; hpi h post-infection; ORF open reading frame; GFP green fluorescent protein; RFP red fluorescent protein.