FIG 4.
Inhibition of DNA replication with AraC does not arrest alternative wrapping events. (A and B) MDBK cells were infected at an MOI of 5 with BoHV-1 in the absence or presence of 100 μg/ml AraC. (A) The level of BoHV-1 genomic DNA was measured at 16 h by semiquantitative PCR using primers specific for UL47. (B) Synthesis of the late VP8 protein was measured by Western blotting of samples harvested at the indicated times. Un, uninfected. (C to I) MDBK cells were infected at an MOI of 5 with BoHV-1 in the presence of 100 μg/ml AraC. Samples were fixed and processed at 16 h, before processing for transmission electron microscopy. (C and D) Incomplete budding through the INM in the direction of the perinuclear space. (E) Several sealed vesicles are shown in the perinuclear space, with the arrow indicating a vesicle containing a B capsid. (F and G) Curved cytoplasmic tubules within the cytoplasm, frequently in association with electron-dense aggregates of material, indicated by the black arrows. (H and I) Extracellular space immediately adjacent to the plasma membrane, showing electron-dense particles, without a capsid cargo. Bars = 200 nm (C to G and I) and 2 μm (H). (J) MDBK cells were infected at an MOI of 5 with BoHV-1 in the absence (−AraC) or presence (+AraC) of 100 μg/ml AraC. Total virus was harvested at the indicated times and titrated on MDBK cells. Data from three biological replicates under each condition are shown.