FIG. 3.
Quantitation with real-time RT-PCR of YFP-containing transcripts from recombinant HCMV viruses. (A) The recombinant virus BADsubUL21.5 was constructed by replacing the UL21.5 transcription unit with a cassette containing the kanamycin resistance (kan) and β-galactosidase (lacZ) genes. The cassette was replaced with the UL21.5 gene fused with the YFP gene in BADinUL21.5YFP and with the YFP gene and the polyadenylation signal (pA) from simian virus 40 virus in BADsubUL21.5YFP. (B) Total RNA was isolated from fibroblasts infected for 72 h and cell-free virus for both HCMV recombinant strains BADinUL21.5YFP and BADsubUL21.5YFP. Quantitative RT-PCR was completed with primers specific to RNAs containing YFP and T/IRL 7. The results are presented as the ratio of the CT value obtained from the virion RNA sample to the CT value obtained from RNA isolated from infected cells. The ratios were normalized to the CT ratio for T/IRL 7 and are presented as the inverse value. The data are presented as the standard deviation from two experiments.