FIG. 5.

Inhibition of the proteasome partially restores IE2-86 protein levels. Human fibroblasts were infected with HCMV Towne at an MOI of 5. At 24 h p.i., Roscovitine or DMSO was added to the cultures. At 42 h p.i., growth medium was replenished and Roscovitine (20 μM) (+), Lactacysin (10 μM) (+), or DMSO (−) was added. At 48 h p.i., cells were harvested and samples were processed for RNA analysis and Western blotting as described in Materials and Methods. (A) The levels of viral transcripts were determined by quantitative real-time RT-PCR as described in Materials and Methods using probes specific for IE2-86 and pp150. The percentage of mRNA was calculated relative to the quantity in untreated, infected cells at 48 h p.i. (48V), which was set to 100%. Bars represent the average values of two independent experiments, while the diamond and circle symbols correspond to the values from each experiment (experiments [Expt.] 1 and 2). (B) Protein levels were determined by Western blotting using antibodies to IE2-86, pp150, and actin. The diamond and circle symbols denote the corresponding Western blot for the RNA analyses shown in panel A.