FIG. 4.

Q548R mutant IE86 protein is able to negatively autoregulate expression from the MIE promoter. (A) HFF cells were transfected, in triplicate, with WT, H446A/H452A, or Q548R recombinant BAC DNA. Total RNA was harvested at 48 h posttransfection and converted to cDNA by reverse transcription. Real-time PCR was performed with primer/probe sets to detect HCMV MIE cDNA and cellular 18S complementary rRNA genes. MIE RNA transcription was measured based on the amount of HCMV MIE cDNA, normalized to cellular 18S complementary rRNA genes, relative to WT. (B) HFF cells were transfected with WT, H446A/H452A, or Q548R recombinant BAC DNA. Total protein was harvested at 24 h posttransfection. Western blot analysis was performed using antibodies to detect viral MIE proteins and cellular β-tubulin. In both figures, the H446A/H452A BAC contains mutations to the putative zinc finger of IE86, which is known to be defective in autoregulation of the MIE promoter (data not shown).