Fig. 1. CHPKs target key regulators of cellular replication and transcription.

a Experimental setup. SILAC heavy and light labeled HEK-293T cells were transfected with constructs encoding HA-tagged versions of human herpesviral kinases or control vectors in triplicates (n = 3). Samples were subjected to anti-HA affinity purification followed by mass-spectrometry. b Candidate selection. Candidate interaction partners were distinguished from background binders based on a combination of the t-test p-value of three replicates and the mean of the SILAC ratios. P-value cut-off at 0.05 and fold-change cut-off at FDR = 1%. A volcano plot for the HHV6-U69 kinase is exemplarily shown. c Cross-comparison of kinase interaction partners. All candidate interaction partners were compared for co-enrichment across the indicated kinases of α-, β-, and γ-HHVs. Enrichment profiles were clustered and selected sets of clusters were subjected to GO enrichment (inlet and brackets). d Cyclin–CDK complexes co-purify with β-herpesviral kinases. Depicted are the SILAC ratios of individual replicates (n = 3) for prey proteins CDK1, CCNB1, CCNA2, and CDK2 across AP–MS samples of the indicated kinases. e Validation of Cyclin A–CDK binding to β-herpesviral kinases. Whole cell extracts from transfected 293T cells were subjected to Cyclin A co-IP and analyzed by immunoblotting for the presence of Cyclin A, CDK2, and HA-tagged kinases. The immunoblots are representative of three independent experiments with similar results. f Alignment of putative Cyclin A binding sites in human and rodent β-herpesvirus kinases. Basic residues are highlighted in blue, bulky hydrophobic residues in pink. g Schematic of human herpesviral kinases. Shown are the location of the conserved catalytic domains and the relative positions of RXL/Cy motifs (pink) and predicted or experimentally validated nuclear localization signals (NLS) (gray) within the less-conserved N-terminal parts of the proteins.