FIG 2.

Phosphorylation status of VP8 and growth characteristics of viruses in MDBK cells. (A) Analysis of VP8 proteins by immunoprecipitation. MDBK cells were infected with WT BoHV-1, BoHV-1-YVP8, BoHV-1-YmVP8, and BoHV-1-RVP8 at an MOI of 1 and labeled with [³²P]orthophosphate. Cell lysates were collected at 20 hpi and used for VP8 purification by incubation with anti-VP8 polyclonal antibody and protein G Sepharose. (Top) The samples were separated by SDS-PAGE and exposed to Imaging Screen K. (Bottom) The gels were stained with ProtoBlue Safe to indicate the amount of protein loading. The relative difference of each sample from WT VP8 is shown as a percentage at the top. (B) Titration of viruses. MDBK cells were infected with viruses at an MOI of 1. The supernatant and cells were collected at 24 hpi. Viruses from infected cells and supernatants were quantified by plaque titration on MDBK cell monolayers. The data were analyzed by two-tailed t test. The statistical significance of the difference between the values is shown; **, P ≤ 0.01. (C to F) Single-step growth curve of viruses. MDBK cells were infected with viruses at an MOI of 1, and the cell culture medium and cells were harvested separately at the indicated time points. The titer of infectious virus progeny in each sample was determined by plaque assay on MDBK cells. The error bars indicate standard deviations.