Figure 6

Deletion mutations in putative viral promoter elements reveal reduction in transcriptional output in cell culture and decreased recovery of infectious virus in vivo . (A) Diagram of the genomic location of intron locus promoter region deletions. (B) Multi-step growth analysis of replication of all three recombinant viruses in comparison to WT MCMV. Mouse fibroblasts were infected (MOI = 0.05) and culture supernatants were collected every 24 hours and titrated by plaque assay. Graph represents three biological replicates. (C, D, and E) Quantification of intron locus RNAs in cells infected with promoter mutant viruses. Mouse fibroblasts were infected (MOI = 1.0) and total RNA was harvested 48 hours post infection. Intron locus transcript levels are quantified relative to WT MCMV transcript levels by qRT-PCR. The primer probes sets used are the same as shown in Figure 4A. Graphs represent three biological replicates. (F and G) Three-month-old female BALB/c mice were infected with an i.p. dose of 5 x 10⁵ PFU with the indicated viruses. At 14 days post infection, animals were euthanized and tissues collected for analysis of infectious virus yield (F) and viral genome number (G). (F) Salivary gland homogenates were analyzed by plaque assay on mouse fibroblasts. p values represent the Student’s T Test result between WT MCMV infected cells or mice and cells or mice infected with the given recombinant viral mutant for each transcript analyzed (*p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001). WT MCMV = WT; MCMVdel 20 = 20DEL; MCMVdel 100 = 100DEL; MCMVdel135 = 135DEL; MCMVdel HP = HPM; MCMVdel SD = SDM.