Infectious Bursal Disease Virus VP5 Polypeptide: A Phosphoinositide-Binding Protein Required for Efficient Cell-to-Cell Virus Dissemination
Fig 7
VP5 C-terminal ablation affects the IBDV plaque size phenotype.
A. Plaque assays. QM7 cell monolayers were infected with the VP5 knockout mutant (KO) virus, the wild type (WT) virus and mutant IBDV viruses lacking 3 (Δ3CT), 10 (Δ10CT), and 14 (Δ14CT) VP5 C-terminal residues, respectively. After infection, monolayers were covered with semisolid medium. After three days, the medium was removed and virus-induced plaques were detected by immunostaining using serum specifically recognizing the VP2 IBDV capsid polypeptide. B. Statistical analysis. The statistical analysis was carried on images from immnunostained monolayers using the GraphPad Prism software 4.0. A Kruskal-Wallis test was performed followed by Dunn’s multiple comparison tests between lysis plaque areas detected in monolayers infected with the different mutant viruses and those generated by the wild type virus. Presented data correspond to three independent experiments totalizing 750 plaques for each virus. Significant differences (p<0.001) between KO, Δ10CT, Δ14CT and WT viruses are indicated with asterisks (***). C. Extracellular virus yields. QM7 cells were infected with the VP5 knockout mutant (white columns), wild type (black) and mutants viruses VP5Δ3CT (light grey), VP5Δ10CT (medium grey), and Δ14CT (dark grey), at an MOI of 3 PFU/cell. At 8, 16 and 24 h p.i. cultures were harvested and subjected to centrifugation at 15,000xg for 15 min to eliminate cells and large debris. The corresponding supernatants were used to determine extracellular virus titres. Presented data correspond to three independent experiments.