FIG. 5.

The carboxy-terminal ORF 50Δ1 construct produces a stable protein which binds to the ORF 50 response elements. (a) OMK cells were seeded at 10⁶ cells per 35-mm-diameter petri dish and washed in labelling medium. Controls remained untransfected (lane 1) or were transfected with 2 μg of pBK50Δ1 (lane 2), pBK50 (lane 3) or infected with HVS (lane 4). The cells were incubated in labelling medium, harvested, and then lysed after 24 h. For each immunoprecipitation, 20 μl of the anti-ORF 50 polyclonal antibody was incubated with protein A-Sepharose beads for 16 h at 4°C. Immunoprecipitations were then performed with each cell lysate, using the anti-ORF 50 antibody. Beads were then pelleted, washed, and resuspended in Laemmli buffer; precipitated polypeptides were resolved on an SDS–12% polyacrylamide gel and analyzed by autoradiography. (b) Gel retardation assays were performed as previously described (53). Briefly, the ORF 50 response elements contained in a set of oligonucleotides were annealed and radiolabelled. These were incubated with nuclear extracts of untransfected OMK cells (lane 1) or cells transfected pBK50b (lane 2) and pBK50Δ1 (lane 3). The protein-nucleic acid complexes (indicated by arrow) were separated on a 5% polyacrylamide gel, run in 1% TBE buffer, and detected by autoradiography.