Fig. 1.

PFV infection promotes the accumulation of autophagosomes. a BHK-21 cells were infected with PFV to analyze LC3 protein expression by western blotting. After 1.5 h of virus absorption at 37 °C, the cells were further cultured in maintenance medium. The cells were infected with mock or PFV at an MOI of 0.5. After PFV infection, cell samples were harvested at 0, 12, 24, 48, 72 and 96 hpi, and cell extracts were blotted with anti-LC3 and anti-Tas antibodies. b GFP-LC3 dots were visualized via confocal microscopy. BHK-21 cells were transfected with GFP-LC3 plasmids for 24 h, followed by PFV infection (MOI = 0.5) or mock infection for 24 h, and the GFP-LC3 aggregates in the cells were assessed via confocal microscopy. Scale bars 10 μm. The graph shows the quantification of autophagosomes by calculating the average number of dots in 20 cells. c Autophagic vacuoles were detected via TEM. BHK-21 cells infected with PFV at an MOI of 0.5 were processed and analyzed at 24 hpi for the accumulation of autophagosomes via transmission electron microscopy. Black frame indicated representative autophagosomes and arrow indicated representative PFV capsid structures during PFV infection. Scale bars 500 and 100 nm. d BHK-21 cells were infected with UV-inactivated PFV, and the inactivated virus infectivity was confirmed by examining the viral structure protein Gag via western blot. Before infection, PFV were radiated by UV for 0, 0.5, 1.0, 1.5 and 2.0 h respectively. Mock-infected supernatant was also radiated with UV for 2.0 h before infecting cells. Then cells were infected with these UV-inactivated mock supernatant or PFV supernatant for 24 h. e BHK-21 cells were inoculated with normal PFV or UV-inactivated PFV (MOI = 0.5) for 24 h. Before infection, PFV and Mock supernatant were treated with UV for 1.5 h. Then, the cell samples were processed and blotted with anti-LC3 antibody. Quantitation of protein levels from the western blot by using Quantity one software (Bio-Rad); all data are representative of three independent experiments with triplicate samples. Significance was analyzed with a two-tailed Student’s t test. ^ns P > 0.05, ***P < 0.001