Fig 7.
(A) Detection of ORFV073 in virions. OFTu cells were infected with OV-IA82Δ073 or OV-IA82RV073Flag and harvested at 90–95% cytopathic effect. Supernatants and pellets from infected cultures were purified for extracellular enveloped virus (EEV) and intracellular mature virus (IMV) as described in Material and Methods. Whole cell lysates (10 μg) from mock and OV-IA82RV073Flag infected cells (MOI = 10) (24 h p.i) and purified OV-IA82Δ073 and OV-IA82RV073Flag virion proteins (10 μg) were resolved by SDS-PAGE, blotted to the nitrocellulose membrane and probed with antibodies against flag or structural protein ORFV086. # and ## denote 30 kDa and a doublet 40 kDa ORFV073 bands in EEV fraction, respectively. (B) Effect of translation inhibition on ORFV073-mediated block of NF-κB-p65 nuclear translocation. OFTu cells were pre-treated with the protein synthesis inhibitor cycloheximide (CHX) (50 μg/ml) for 30 min and then mock infected or infected with OV-IA82, OV-IA82Δ073 or OV-IA82RV073Flag (MOI = 10) in presence of CHX (50 μg/ml). Cells were fixed at 1 h p.i., incubated with antibody against NF-κB-p65, stained with Alexa Fluor 488-labeled antibody and DAPI, and examined by confocal microscopy. Cells (n = approximately 300) expressing nuclear NF-κB-p65 in each group were counted and results depicted as mean percentage of cells containing nuclear NF-κB-p65 over two independent experiments (*, P<0.05). Result shown in (A) are representative of six independent experiments.