Upon cell entry, herpesviruses deliver a multitude of premade virion proteins to their hosts. The interplay between these incoming proteins and cell-specific regulatory factors dictates the outcome of infections at the cellular level. Here, we report a unique type of virion-host cell interaction that is essential for the cell cycle and differentiation state-dependent onset of human cytomegalovirus (HCMV) lytic gene expression. The major tegument 150-kDa phosphoprotein (pp150) of HCMV binds to cyclin A2 via a functional RXL/Cy motif resulting in its cyclin A2-dependent phosphorylation. Alanine substitution of the RXL/Cy motif prevents this interaction and allows the virus to fully escape the cyclin-dependent kinase (CDK)-mediated block of immediate early (IE) gene expression in S/G2 phase that normally restricts the onset of the HCMV replication cycle to G0/G1. Furthermore, the cyclin A2-CDKpp150 axis is also involved in the establishment of HCMV quiescence in NTera2 cells, showing the importance of this molecular switch for differentiation state-dependent regulation of IE gene expression. Consistent with the known nucleocapsid-binding function of pp150, its RXL/Cy-dependent phosphorylation affects gene expression of the parental virion only, suggesting a cis-acting, virus particle-associated mechanism of control. The pp150 homologs of other primate and mammalian CMVs lack an RXL/Cy motif and accordingly even the nearest relative of HCMV, chimpanzee CMV, starts its lytic cycle in a cell cycle-independent manner. Thus, HCMV has evolved a molecular sensor for cyclin A2-CDK activity to restrict its IE gene expression program as a unique level of selflimitation and adaptation to its human host.H erpesviruses are highly adapted to their hosts. This is due to a long coevolutionary process and based on an intricate molecular virus-host interaction network. Interestingly, a significant fraction of herpesviral gene products is already present in the very first phase of infection, after virus entry but before the onset of de novo viral gene expression. These factors originate from the tegument, a characteristic, protein-rich layer within herpesvirus particles, occupying the space between nucleocapsid and virus envelope. Tegument proteins serve a number of important functions. They are involved in the cytoplasmic transport of nucleocapsids, nuclear delivery of viral genomes, immune evasion, and transactivation of immediate early (IE) gene transcription (1). Importantly, they also provide a unique opportunity for the virus to flexibly respond to different cell types and states immediately after entry and set the course for a productive or nonproductive infection at the earliest possible stage.A compelling example for a tegument-derived sensor of the host cell milieu is the 71-kDa phosphoprotein (pp71, also referred to as the UL82 gene product "pUL82") of human cytomegalovirus (HCMV, human herpesvirus 5), a widespread human pathogen. Permissiveness for HCMV depends on the cellular differentiation state (2) and is intimately l...

Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.

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.

Generally, the antagonism between host restriction factors and viral countermeasures decides on cellular permissiveness or resistance to virus infection. Human cytomegalovirus (HCMV) has evolved an additional level of self-imposed restriction by the viral tegument protein pp150. Depending on a cyclin A-binding motif, pp150 prevents the onset of viral gene expression in the S/G2 cell cycle phase of otherwise fully permissive cells. Here we address the physiological relevance of this restriction during productive HCMV infection by employing a cyclin A-binding deficient pp150 mutant virus. One consequence of unrestricted viral gene expression in S/G2 was the induction of a G2/M arrest. G2-arrested but not mitotic cells supported viral replication. Cyclin A destabilization by the viral gene product pUL21a was required to maintain the virus-permissive G2-arrest. An HCMV double-point mutant where both pp150 and pUL21a are disabled in cyclin A interaction forced mitotic entry of the majority of infected cells, with a severe negative impact on cell viability and virus growth. Thus, pp150 and pUL21a functionally cooperate, together building a cell cycle synchronization strategy of cyclin A targeting and avoidance that is essential for productive HCMV infection.

Herpesviruses encode conserved protein kinases to stimulate phosphorylation-sensitive processes during infection. How these kinases 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) kinases. We find that these kinases can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a specific signature of β-herpesvirus kinases. Cyclin A is recruited via RXL-motifs that overlap with nuclear localization signals (NLS) and locate in the non-catalytic N-terminal regions. This architecture is conserved for viral kinases of HHV6, HHV7 and rodent CMVs. Docking to Cyclin A competes with NLS function, enabling dynamic changes in kinase localization and substrate phosphorylation. The viral kinase redirects Cyclin A to the cytosol, which is essential for the inhibition of cellular DNA replication during infection. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.

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