FIG. 5.

LMP1 expression during the lytic cycle. Western blot analysis of LMP1 expression in EBV lytic cycle-positive P3HR1-c16 cells was performed. Cells were cotransfected with 8 μg of a BZLF1 expression plasmid to induce the EBV lytic cycle and with 3 μg of a rat CD2 surface antigen expression plasmid to allow transfected cells to be positively selected by immunomagnetic separation. At 24 h following transfection, viable cells were labeled with OX34 (an anti-rat CD2 MAb) followed by FITC-conjugated anti-IgG; then they were immunomagnetically sorted with anti-FITC MACS microbeads. BZLF1-transfected cells were positively sorted on the basis of rat CD2 expression, while the unbound flowthrough cells were taken to be untransfected, latent cells. Cell samples were analyzed by Western immunoblotting. When probed with EE human serum (top panel), the blot showed staining of BZLF1, early antigens, and late antigens in the positively sorted cells and not in the untransfected, flowthrough cells. Reprobing the blot for LMP1 with MAbs CS.1-4 (lower panel) demonstrated induced expression of LMP1 in the positively sorted, lytically infected cells.