Fig 2: Cell cycle inhibitors have variable effects on BKPyV-induced G2/M phase cell cycle arrest. (A) The cell cycle status of RPTE cells was determined under a number of different experimental conditions. RPTE cells were infected with BKPyV (MOI of 3) or mock infected and then subjected to CDK4/6 inhibition (1 µM PD0332991), CDK1/2 inhibition (20 µM Roscovitine), or CDK1 inhibition (5 µM RO-3306) at 24 hpi; cells were subsequently collected for analysis at 48 hpi. Collected cells were stained with propidium iodide (PI) and analyzed by flow cytometry (n = 3). Error bars represent standard deviations. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (two-sample t test for changes in proportion of cells in G1). (B) Histograms of PI staining for each experimental condition of a single experiment are shown. (C) Cell viability tests. RPTE cells were treated with 1 µM PD0332991, 20 µM Roscovitine, or 5 µM RO-3306 for 24 h and then subjected to a trypan blue exclusion assay according to the manufacturer’s protocol (n = 3).

Fig 3: In vitro assembly XL-IP assay demonstrating the HCMV core NEC–CDK interaction and its sensitivity to CDK inhibitors. The 293T cells were cultivated in 10 cm petri dishes and used for transient transfection. Cells were harvested at 2 d p.t. In vitro assembly assay was performed by addition of 3 µM of the CDK inhibitors R25 or CDK2 Inh II to lysates during the assembly process of pUL50-V5, pUL53-His and CDK1-HA. DMSO treatment was used as a reference control. (A) Input samples of total lysates were collected before DSS addition in order to monitor the expression levels of the individual proteins by Wb. (B) Core NEC and associated protein kinases of cross-linked lysates were specifically detected by Wb using the indicated antibodies. (C) The interactions of core NEC–CDK were demonstrated by coimmunoprecipitation and Wb detection using the respective tag-specific antibodies (green frames, precipitation control of CDK1; orange frames, pUL53–CDK1 interaction; red frames, inhibition of pUL53–CDK1 interaction under R25 or CDK2 Inh II treatment; blue frames, R25-/CDK2 Inh II-mediated reduction of CDK2 association). HMW/LMW, high/low molecular weight marker.

Fig 4: Inhibitory effect of kinase inhibitors on the association of CDK1 and CDK2 with oligomeric HCMV core NEC. HFFs were seeded in 6-well plates, used for HCMV AD169 UL50-HA infection at an MOI of 1.5 and treated with R25 at 3 d p.i. Cells were harvested at 5 d p.i. and cross-link was performed by addition of different concentrations of DSS. (A) Lysate controls were kept before DSS addition and expression levels of the individual proteins were monitored by Wb. (B) The associated protein kinases of cross-linked lysates were additionally detected by Wb analysis (see oligomeric forms marked by arrow-heads). (C) Quantitation of the band intensities was performed by densitometry (AIDA Image Analyzer v.4.23 software). All determinations were made in quadruplicate by using two identical Wbs (one of each shown under (B)). The mean values of CDK band intensity were corrected against the highest band intensity under DMSO control treatment. Statistical significance was calculated by Student’s t-test (* p = 0.05; ** p = 0.01).

Fig 5: Inhibitory effect of kinase inhibitors on the association of CDK1 with oligomeric HCMV core NEC. (A) Proteins derived from total cellular lysates. (B) Proteins derived from coimmunoprecipitates. HFFs were seeded in 6-well plates, used for HCMV AD169 UL50-HA infection at an MOI of 1.5 and treated with the mixture of R25 and MBV at 3 d p.i. Cells were harvested at 5 d p.i. and cross-link was performed by addition of different concentrations of DSS. Core NEC oligomers and their associated protein kinases could be illustrated by Wb analysis. Different order of core NEC oligomers could be demonstrated by coimmunoprecipitation and Wb detection using the respective tag-specific antibodies.