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.

疱疹病毒编码保守蛋白激酶 (CHPK) 以在感染期间刺激磷酸化敏感过程。CHPKs 如何与细胞因子结合以及这如何影响它们的调节功能尚不清楚。在这里，我们使用定量蛋白质组学来确定人类疱疹病毒 (HHV) CHPKs 的细胞相互作用伙伴。我们发现 CHPKs 可以针对转录和复制的关键调节因子。与细胞周期蛋白 A 和相关因子的相互作用被确定为 β-疱疹病毒激酶的特征。细胞周期蛋白 A 是通过 RXL 基序募集的，这些基序与非催化 N 末端的核定位信号 (NLS) 重叠。这种结构在 HHV6、HHV7 和啮齿动物巨细胞病毒中是保守的。细胞周期蛋白 A 结合与 NLS 功能竞争，使 CHPK 定位和底物磷酸化发生动态变化。巨细胞病毒激酶 M97 将细胞质中的细胞周期蛋白 A 隔离，这对于病毒抑制细胞复制至关重要。我们的数据突出了细胞周期蛋白和病毒激酶之间微调和生理上重要的相互作用。