Fig. 5.

Mode of in vitro trans-stimulation: vCDK/pUL97 can increase the activity of human CDK7 significantly. (A) 293T cells were transiently transfected with plasmids encoding pUL97-Flag or with an empty vector control. Cells were lysed 2 d post-transfection, and pUL97-Flag and/or CDK7 were immunoprecipitated using the indicated antibodies; a mouse Fc fragment served as a negative control. Dynabeads-bound proteins were eluted in 150 µl enzyme buffer. 25 µl of eluted Dynabeads were denatured in 25 µl 2x loading buffer and subjected to SDS-PAGE and Wb analysis. The quantity of immunoprecipitated pUL97-Flag and CDK7 was determined densitometrically, measuring the mean expression levels derived from two different Wbs. Values of CDK7 and pUL97-Flag obtained from the double-immunoprecipitated sample (lane 2) were normalized to those obtained from the single-immunoprecipitated sample of CDK7 (lane 1) and pUL97-Flag (lane 3). (B) Samples based on the immunoprecipitation of CDK7, pUL97-Flag, CDK7 plus pUL97-Flag, as well as Fc control samples, were subjected to a qSox-IVKA (using the CDK7-specific sensor peptide AQT0459). The background signal of the Fc control was subtracted from kinase signals. The resulting kinase-specific signals were multiplied by the normalization factors from (A), to compensate for different quantities of the kinases in the IVKA reactions. Additionally, the kinase signal of single-immunoprecipitated pUL97-Flag was subtracted from double-immunoprecipitated CDK7 plus pUL97-Flag, to obtain a corrected CDK7-specific kinase signal. All kinase signals were then normalized to the kinase signal derived from CDK7 single immunoprecipitation. Mean values ± SD from three independent biological replicates are given, as derived from measurements in triplicates. (C) The concentration of 0.35 µM of pUL97-specific inhibitor maribavir (MBV; corresponds to mean antiviral EC₅₀ value) or DMSO as solvent control was added to CDK7 + pUL97-Flag samples and CDK7-specific kinase activity was measured using a qSox-IVKA (using the CDK7-specific sensor peptide AQT0459). Mean values ± SD are given, as derived from one representative biological replicate, with measurements in triplicates. Statistical analysis was performed using an ordinary two-way ANOVA: ***, p < 0.001; *, p < 0.05; n.s., not significant.