Skip to main content
. 2019 Jan 11;529:160–168. doi: 10.1016/j.virol.2019.01.009

Fig. 3.

Fig. 3

Infection of PK-15 cells by PSV is inhibited by disruption of caveolae/raft-dependent endocytic mechanisms. (A) Inhibition of CTB uptake into PK-15 cells was determined by nystatin or MβCD. Pretreating cells with 10 μM nystatin or 2 mM MβCD resulted in near-complete blockade of CTB uptake. Bars, 30 µm. (B) PK-15 cells were mock-treated or pre-treated with different concentrations of nystatin or MβCD, followed by infection with PSV at a MOI of 2 in the continued presence of the drugs or inhibitors were post treated after virus added for 1 h. At 8 h post-infection, cells were processed for western blot and levels of virus VP1 protein synthesis were detected using anti-VP1 antibody. Quantification of the bands corresponding to VP1 was corrected with α-tubulin and then normalized to mock-treated cells (set as 1). (C) After pretreated with preconcerted inhibitors, PK-15 cells were infected with PSV at 4 °C for 1 h and then at 37 °C for 1 h. The infected cells were lysed to determine viral RNA copy number by RT-qPCR. (D) Virus titers were determined by TCID50 of samples prepared at 8 h post-infection from PK-15 cells infected with PSV in the presence of different concentrations of nystatin or MβCD. (E) The silencing efficiency of siCav1 was determined by qRT-PCR using the 2-ΔΔCT method. (F) PK-15 cells were transfected with siCav1 or siRNA negative control (siNC) for 24 h and then infected with PSV (MOI = 2) for 8 h to virus propagation. Viral VP1 protein synthesis levels were estimated by western blot. (G) The effect of wild-type caveolin 1 (Cav1 WT) and dominant negative mutant of caveolin 1 (Cav1 DN) on PSV infection was determined by western blot. Data are presented as means ± SD from at least two independent experiments. *P < 0.05, **P < 0.01 compared to the mock-treated cells. (H) PSV particles colocalized with caveolin. PK-15 cells were transfected with EGFP-tagged caveolin before incubation with PSV. The enlarged box indicates PSV (red) that were swallowed into the vesicle structure, which bears caveolin (green) on the outer surface; colocalized signals were widely observed using confocal microscopy.