FIG 4.

PDCoV entry into IPI-2I cells depends on dynamin. (A) Effect of dynasore on PDCoV binding and entry. IPI-2I cells were pretreated with increasing subtoxic doses as described in above Fig. 3A, and lysed cells were subjected to determine viral RNA copy numbers by RT-qPCR. (B and C) Dynasore inhibited PDCoV and VSV-GFP infection. Cells were pretreated with increasing subtoxic doses of dynasore or DMSO at 37°C for 1 h and then inoculated with PDCoV (B) or VSV-GFP (C) at 37°C for 6 h, followed by determination of viral infection by RNA copy numbers and viral protein expression levels by RT-qPCR and Western blot analysis. (D) Viral titer detection for PDCoV in the medium from cells treated as described in panel B. (E) IFA for the effect of dynasore on PDCoV infection. Cells were pretreated as described in panels B, followed by IFA. Bar, 100 μm. Relative fluorescence intensity is quantified by Image‐Pro Plus software as shown in panel E on the right. (F) CCK-8-based cell viability assay for dynasore as described in Materials and Methods. (G and H) Inhibitory effect of the dynamin DN construct on PDCoV entry (G) and infection (H) was determined via RT-qPCR and confocal microscopy. Cells transfected with plasmid constructs encoding Flag-tagged WT and DN dynamin were infected with PDCoV (MOI of 5). At 1 and 6 hpi at 37°C, cells were harvested and subjected to RT-qPCR and IFA, respectively. Bar, 10 μm. (I and J) Dynamin knockdown inhibited PDCoV entry (I) and infection (J). Si-dynamin- or siCtrl-transfected cells were infected with PDCoV (MOI of 5). At 1 and 6 hpi at 37°C, the cells were lysed to determine the viral RNA copy numbers via RT-qPCR. Target protein expression was quantitatively estimated by ImageJ software and presented as the density value relative to that of the β-actin. The presented results represent the means and standard deviations of data from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.