FIG. 3.

Replication of recombinant viruses containing IE86 mutations. (A) HFF cells were transfected, in triplicate, with Towne, Q548A, Q548R, or Rev Q548R BAC DNA. Medium was changed on transfected cells every 4 days, and Q548R BAC-transfected cells were split 1:2 at day 14 to promote replication. Total DNA was harvested at 1, 5, 9, 13, 21, and 28 days posttransfection. Real-time PCR with primer/probe sets to detect HCMV gB DNA and cellular 18S rRNA genes was performed. BAC DNA replication was measured based on the amount of HCMV gB DNA, normalized to cellular 18S rRNA genes, relative to Towne-transfected cells at 1 day posttransfection. (B) HFF cells were infected, in triplicate, with equal DNA input of Towne, Q548A, or Q548R RV. Cells and supernatant were harvested at 1, 5, and 10 days postinfection, and triplicate samples were pooled and stored. A plaque assay was performed, in triplicate, with serial dilutions of stored virus, and plaques were counted at 10 to 14 days postinfection for Towne and Q548A RV-infected cells or 14 to 21 days postinfection for Q548R RV-infected cells.