FIG. 7.

Construction of the recombinant HCMV (AD-GFP/UL117) expressing the functional GFP-tagged pUL117. (A) The diagram illustrates the UL119-UL115 genomic region of AD-GFP/UL117. The GFP coding sequence (gray triangle) was fused in frame to the N terminus of the UL117 ORF. As a result, the transcript encoding the GFP/UL117 fusion protein (indicated as the second line) was expressed under the control of the endogenous UL117 promoter, whereas the UL117.5 transcript (indicated as the third line) remains unaltered. (B) Detection of the GFP/UL117 fusion protein. HF were either mock infected or infected with AD-GFP/UL117 at a multiplicity of infection (moi) of 10 PFU/cell. At 96 h postinfection, cell lysates were analyzed by Western blotting using antibodies (α-) specific to GFP or UL117. The antibody to the viral IE1 protein or β-actin was used as the infection control or the loading control, respectively. (C) AD-GFP/UL117 replicated indistinguishably from wild-type virus. HF were infected with AD-GFP/UL117 or wild-type virus at a multiplicity of infection of 0.01 PFU/cell, culture medium was collected at indicated times, and yields of cell-free virus were determined by plaque assay.