Fig 5.
Characterization of US2-11-competent and US2-11-negative HCMV strains, expressing an EYFP-tagged version of pp71. (A) Schematic representation of the viral mutants. The US2-11 gene region in Ad169/EYFP-pp71 (32) or wt HCMV (RV-HB15) was deleted and replaced by a kanamycin resistance gene, using BAC recombineering. The resulting mutants were designated AD169/EYFP-pp71_ΔUS2-11 or HCMV_ΔUS2-11. (B) Immunoblot analysis of cell lysates of HFF infected with the indicated viruses at an MOI of 1 for 4 days. Lysates from 1 × 105 cells were applied to each lane. Antibodies directed against pp71 or β-actin were used for detection. (C) Quantification of the pp71 or EYFP-pp71 bands from panel B, using the Odyssey analysis system. Signals from individual bands were normalized to β-actin in each case (RFI, relative fluorescence intensity). (D) Immunoblot analysis of the expression levels of IE1 in HFF infected with the different virus strains under CX/AcD blocking conditions at 125 genomes per cell. Lysates from 2 × 105 cells were applied to each lane. (E) Quantification of the IE1 bands from panel D, using the Odyssey analysis system. Signals from individual bands were normalized to β-actin in each case.