FIG. 4.

Cdk9 kinase activity is not a requirement for its recruitment to the viral transcriptosomes. G₀-synchronized cells were released into G₁ and infected with HCMV Towne (V; MOI of 5) or mock (M) supernatant. Infections took place in the presence of Roscovitine (Rosco; 16 μM), Flavopiridol (Flavo; 50 nM), DRB (15 μM), or control dimethylsulfoxide (DMSO). Cells were harvested at 8 h p.i. Total cell lysates from equal numbers of cells were subjected to Western blotting using H5 (which detects pSer2-RNAP IIo), anti-cdk9, and CH16.0 (which detects IE1/2). β-actin protein levels were checked as a loading control. Cells were fixed for IFA at 8 h p.i. and stained with antibodies specific for IE2 and cdk9, followed by fluorescein isothiocyanate- and Cy3-conjugated isotype-specific secondary antibodies, which were used for IFA. Nuclei were stained with Hoechst dye. The white arrow in each panel indicates a region of colocalization, although more are present. All of the images represent confocal optical 0.2-μm sections at a magnification of ×1,000 under conditions of oil immersion.