FIG 9.

The chemical depletion of PtdIns(3)P in avian cells inhibits IBDV replication and reduces its infectivity. (A) Treatment of infected avian cells with Vps34-IN1 impairs IBDV infection. QM7 cells were either mock infected or infected with IBDV at an MOI of 1 PFU/cell and at 2 h p.i. were treated with DMSO or 1 μM Vps34-IN1. At 24 h p.i., cells were fixed, permeabilized, stained with antibodies against VP3 (white) and analyzed by spinning-disc confocal microscopy. Panels a to d show representative images of infected cells. Images are merged z-stacks. The images are representative of three independent experiments. Bars, 10 μm. The percentages of IBDV-infected cells were calculated by scoring 100 cells per condition, and the results are presented in the graph. Data are means and SD. **, P < 0.05. (B) Intracellular accumulation of VP2 and VP3 proteins in QM7 cells depleted of PtdIns(3)P. QM7 cells were either mock infected or infected and treated as for panel A and at 24 h p.i. were processed by Western blotting using the corresponding anti-VP2 and VP3 antibodies described in Materials and Methods. The Western blots and the data shown in the normalized bar graph correspond to an experiment representing three independent trials. Data are means and SD. ***, P < 0.01. (C) QM7 cells were either mock infected or infected and treated as for panel A. At 24 h p.i., the supernatants were collected for extracellular virus titration, and the cellular pellets were processed for intracellular viral titration as described in Materials and Methods. The image shows a representative plaque assay results from three independent trials. Normalized extra- and intracellular viral titers are shown in the graphs. The crude viral titers are shown above the bars. Error bars show SD. ***, P < 0.01; **, P < 0.05.