Figure 5

ISG15 inhibits autophagy, which is required for CSFV replication. CMV, CMV-ISG15, shN, and shISG15 cells were infected with CSFV (MOI = 1) for 12 h and 24 h. LC3 protein (A, D) was analyzed by Western blot, and LC3 puncta (B, E) in PAMs were observed by confocal immunofluorescence microscopy. Cells were fixed and subjected to IFA by immunostaining with anti-LC3 antibody. Representative cells are shown. Scale bar = 10 μm. The number of LC3 puncta per cell was determined by image analysis. C Transmission electron microscopy images. CMV and CMV-ISG15 cells were mock-infected or infected with CSFV (MOI = 1) for 24 h and studied by electron microscopy. Representative images of CMV cells (a), CSFV-infected CMV cells (b), magnified view of autophagosome-like vesicles (c), CMV-ISG15 cells (d), and CSFV-infected CMV-ISG15 cells (e) are shown. Scale bars = 1 μm (a, b, d, and e) and 200 nm (c). Black arrows indicate the structures with the characteristics of autophagosomes. F, G ShN and shISG15 cells were pretreated with 3MA for 4 h after 1 h of virus adsorption, after which the cells were provided medium with 3MA. After 24 h, CSFV replication was quantified by RT-qPCR and IFA, respectively. CMV and CMV-ISG15 cells (H and I) or shN and shISG15 cells (J, K) were treated with DMSO or rapamycin. Expression of LC3 protein (H, J) and LC3 puncta (I, K) in PAMs were quantified by image analysis. Data (B, E, F, G, I, and K) represent the mean ± SD of three independent experiments and were measured in technical duplicate. Comparisons between groups were calculated using Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001