FIG 4.

Inducible expression of MDV ICP27 rescues vΔ54 deficient replication. (A) QTR-ICP27 cell lines were treated with 1 μg/ml Dox (+Dox) or left untreated (no Dox) for 24 h and then fixed. Anti-V5 mAb was used to identify V5-tagged ICP27 protein, and cells were counterstained with Hoechst 33342 (blue). Scale bars represent 200 nm. QTR-ICP27A showed no basal (no Dox) or inducible (+Dox) ICP27 expression, while QTR-ICP27B and -ICP27C had no basal expression but had induced ICP27 expression. (B) QTR-ICP27 cell lines were used to test the ability of inducible expression of ICP27 to rescue cell-to-cell spread of vΔ54. All three cell lines were transfected with rRLORF4mRFP or rΔ54 BAC DNA in duplicate. After 1 day, one well was treated with 1 μg/ml of Dox every other day for 7 days. Cells were fixed and plaques were detected using polyclonal anti-MDV chicken sera plus secondary anti-chicken IgG Alexa Fluor 568. Scale bars represent 200 nm. (C) Plaque size assays showing the mean plaque area produced by rRLORF4mRFP and rΔ54 in Dox-treated QTR-ICP27A, -ICP27B, and -ICP27C at 7 dpi. Ten plaques were measured for each group, and box plots with statistical results are shown. The fold change in plaque sizes generated for rRLORF4mRFP and rΔ54 in QTR-ICP27B and -ICP27C cells compared to QTR-ICP27A is shown in each box.