Pulse-chase experiments and characterization of poxvirus recombinants. (A) Pulse-chase and immunoprecipitation analysis were used to determine T½ of CMV-pp65 protein expressed by pp65-VV, UbRpp65-VV, and UbMpp65-VV. Hu TK^- cells infected with VV expressing CMV pp65, UbRpp65, and UbMpp65 were pulsed with ³⁵S-Met and ³⁵S-Cys for 30 minutes, and chased with 10x unlabeled Met and Cys for 0, 0.25, 1, 5, and 20 hours. Cell lysates were immunoprecipitated by anti-pp65 mAb (103-28) and anti–β-gal mAb and analyzed by SDS-PAGE. Lanes 1 to 5 represent VV-pp65. Lanes 6 to 9 represent UbRpp65-VV. Lanes 10 to 13 represent UbMpp65-VV. (B) Western blot detection of pp65, pp150, and IE4 expressed by pp65/pp150-MVA, UbRpp65-MVA, UbMpp65-MVA, IE4-MVA, and UbRIE4-MVA. In lanes 1, 8, and 9 are AD169 CMV cell lysates (Microbix Biosystems, Toronto, ON, Canada) for detection of pp65, pp150, and IE4 and uninfected BHK cell lysates in lanes 2, 7, and 10. Cell lysates from pp65/pp150-MVA–infected BHK-21 cells (lanes 3, 6), UbRpp65-MVA–infected BHK-21 cells (lane 4), UbMpp65-MVA–infected BHK-21 cells (lane 5), IE4-MVA–infected BHK-21 cells (lane 11), and UbRIE4-MVA–infected BHK-21 cells (lane 12) are shown. All lanes were loaded with the same amount of protein as determined by Braford method. pp65 protein expression was detected using mAb (103-28), pp150 expression was detected using mAb (1.XPA 36), and IE1 exon 4 expression was detected using mAb (p63-27).