Abstract
Herpesviruses encode conserved protein kinases (CHPKs) to stimulate phosphorylation-sensitive processes during infection. How CHPKs bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) CHPKs. We find that CHPKs can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a signature of β-herpesvirus kinases. Cyclin A is recruited via RXL motifs that overlap with nuclear localization signals (NLS) in the non-catalytic N termini. This architecture is conserved in HHV6, HHV7 and rodent cytomegaloviruses. Cyclin A binding competes with NLS function, enabling dynamic changes in CHPK localization and substrate phosphorylation. The cytomegalovirus kinase M97 sequesters Cyclin A in the cytosol, which is essential for viral inhibition of cellular replication. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.
Highlights
Background binderCandidate interactor−log[10] (t-test pvalue) Intensity Vector control L H LHA-tagged kinase b HHV6-U69 3 U690 −2 0 2 4 6 log[2] SILAC ratio HA-IP transfected/vector control c log[2] SILAC ratio HA-IP transfected/vector controlAffinity purification mass spectrometry (AP-MS) α β γ
Taking mouse cytomegalovirus (MCMV, murid herpesvirus 1 (MuHV-1)) as a model system, we show that during infection the stoichiometric formation of cytoplasmic v-cyclindependent kinase (CDK) and Cyclin A assemblies causes a global shift in substrate phosphorylation and the viral shutoff of host DNA synthesis
To discriminate candidate interactors from background binders, they were required to fall below a p-value of 0.05 and meet a SILAC fold-change cut-off at 1% false discovery rate (FDR). (Fig. 1b, Supplementary Fig. 2b, c)
Summary
CHPKs target key regulators of transcription and replication. Systems-level approaches have provided important insights into the molecular functions of CHPKs. To identify CHPK interaction partners, we transfected SILAC (stable isotope labeling of amino acids in cell culture) heavy and light labeled HEK-293T cells with HA-tagged CHPKs of seven different human herpesviruses (HHVs) or vector controls We performed these experiments in triplicates, including label-swaps (Fig. 1a), and subjected the samples to HA affinity purification (HA-AP) and shotgun proteomics. We quantified about 1500–2000 proteins in individual samples (Supplementary Fig. 1d) and classified 135 proteins as candidate interaction partners to at least one kinase (Supplementary Data 1) This list includes several previously found CHPK interactors and substrates, such as SAMHD1 We found the chaperonin containing TCP1 and the kinase maturation complex (CDC37, HSP90) to co-purify with all kinases analyzed This confirms the previous observation that CHPKs interact with the same set of cellular proteins that assist in folding and maturation of cellular kinases[32,33]. Our interactome of human CHPKs provides a rich resource and suggests a Transfection
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