Functional Relevance of the Interaction between Human Cyclins and the Cytomegalovirus-Encoded CDK-Like Protein Kinase pUL97.

The replication of human cytomegalovirus (HCMV) is characterized by a complex network of virus-host interaction. This involves the regulatory viral protein kinase pUL97, which represents a viral cyclin-dependent kinase ortholog (vCDK) combining typical structural and functional features of host CDKs. Notably, pUL97 interacts with the three human cyclin types T1, H and B1, whereby the binding region of cyclin T1 and the region conferring oligomerization of pUL97 were both assigned to amino acids 231-280. Here, we addressed the question of whether recombinant HCMVs harboring deletions in this region were impaired in cyclin interaction, kinase functionality or viral replication. To this end, recombinant HCMVs were generated by traceless BACmid mutagenesis and were phenotypically characterized using a methodological platform based on qPCR, coimmunoprecipitation, in vitro kinase assay (IVKA), Phos-tag Western blot and confocal imaging analysis. Combined data illustrate the following: (i) infection kinetics of all three recombinant HCMVs, i.e., ORF-UL97 ∆231-255, ∆256-280 and ∆231-280, showed impaired replication efficiency compared to the wild type, amongst which the largest deletion exhibited the most pronounced defect; (ii) specifically, this mutant ∆231-280 showed a loss of interaction with cyclin T1, as demonstrated by CoIP and confocal imaging; (iii) IVKA and Phos-tag analyses revealed strongly affected kinase activity for ∆231-280, with strong impairment of both autophosphorylation and substrate phosphorylation, but less pronounced impairments for ∆231-255 and ∆256-280; and (iv) a bioinformatic assessment of the pUL97-cyclin T1 complex led to the refinement of our current binding model. Thus, the results provide initial evidence for the functional importance of the pUL97-cyclin interaction concerning kinase activity and viral replication fitness.

The Cytomegalovirus Protein Kinase pUL97:Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting.

Human cytomegalovirus (HCMV) expresses a variety of viral regulatory proteins that undergo close interaction with host factors including viral-cellular multiprotein complexes. The HCMV protein kinase pUL97 represents a viral cyclin-dependent kinase ortholog (vCDK) that determines the efficiency of HCMV replication via phosphorylation of viral and cellular substrates. A hierarchy of functional importance of individual pUL97-mediated phosphorylation events has been discussed; however, the most pronounced pUL97-dependent phenotype could be assigned to viral nuclear egress, as illustrated by deletion of the UL97 gene or pharmacological pUL97 inhibition. Despite earlier data pointing to a cyclin-independent functionality, experimental evidence increasingly emphasized the role of pUL97-cyclin complexes. Consequently, the knowledge about pUL97 involvement in host interaction, viral nuclear egress and additional replicative steps led to the postulation of pUL97 as an antiviral target. Indeed, validation experiments in vitro and in vivo confirmed the sustainability of this approach. Consequently, current investigations of pUL97 in antiviral treatment go beyond the known pUL97-mediated ganciclovir prodrug activation and henceforward include pUL97-specific kinase inhibitors. Among a number of interesting small molecules analyzed in experimental and preclinical stages, maribavir is presently investigated in clinical studies and, in the near future, might represent a first kinase inhibitor applied in the field of antiviral therapy.

Phosphosite Analysis of the Cytomegaloviral mRNA Export Factor pUL69 Reveals Serines with Critical Importance for Recruitment of Cellular Proteins Pin1 and UAP56/URH49.

Human cytomegalovirus (HCMV) encodes the viral mRNA export factor pUL69, which facilitates the cytoplasmic accumulation of mRNA via interaction with the cellular RNA helicase UAP56 or URH49. We reported previously that pUL69 is phosphorylated by cellular CDKs and the viral CDK-like kinase pUL97. Here, we set out to identify phosphorylation sites within pUL69 and to characterize their importance. Mass spectrometry-based phosphosite mapping of pUL69 identified 10 serine/threonine residues as phosphoacceptors. Surprisingly, only a few of these sites localized to the N terminus of pUL69, which could be due to the presence of additional posttranslational modifications, like arginine methylation. As an alternative approach, pUL69 mutants with substitutions of putative phosphosites were analyzed by Phos-tag SDS-PAGE. This demonstrated that serines S46 and S49 serve as targets for phosphorylation by pUL97. Furthermore, we provide evidence that phosphorylation of these serines mediates cis/trans isomerization by the prolyl isomerase Pin1, thus forming a functional Pin1 binding motif. Surprisingly, while abrogation of the Pin1 motif did not affect the replication of recombinant cytomegaloviruses, mutation of serines next to the interaction site for UAP56/URH49 strongly decreased viral replication. This was correlated with a loss of UAP56/URH49 recruitment. Intriguingly, the critical serines S13 and S15 were located within a sequence resembling the UAP56 binding motif (UBM) of cellular mRNA adaptor proteins like REF and UIF. We propose that betaherpesviral mRNA export factors have evolved an extended UAP56/URH49 recognition sequence harboring phosphorylation sites to increase their binding affinities. This may serve as a strategy to successfully compete with cellular mRNA adaptor proteins for binding to UAP56/URH49.IMPORTANCE The multifunctional regulatory protein pUL69 of human cytomegalovirus acts as a viral RNA export factor with a critical role in efficient replication. Here, we identify serine/threonine phosphorylation sites for cellular and viral kinases within pUL69. We demonstrate that the pUL97/CDK phosphosites within alpha-helix 2 of pUL69 are crucial for its cis/trans isomerization by the cellular protein Pin1. Thus, we identified pUL69 as the first HCMV-encoded protein that is phosphorylated by cellular and viral serine/threonine kinases in order to serve as a substrate for Pin1. Furthermore, our study revealed that betaherpesviral mRNA export proteins contain extended binding motifs for the cellular mRNA adaptor proteins UAP56/URH49 harboring phosphorylated serines that are critical for efficient viral replication. Knowledge of the phosphorylation sites of pUL69 and the processes regulated by these posttranslational modifications is important in order to develop antiviral strategies based on a specific interference with pUL69 phosphorylation.

Patient-Derived Cytomegaloviruses with Different Ganciclovir Sensitivities from UL97 Mutation Retain Their Replication Efficiency and Some Kinase Activity In Vitro.

Mutations in the cytomegalovirus UL97 kinase gene contribute to antiviral resistance. Mutations A594S and G598D from two clinical isolates were analyzed, and bacterial artificial chromosome (BAC)-engineered A594S recombinant cytomegalovirus exhibited a ganciclovir-resistant phenotype on plaque reduction. Viral replication was comparable to that of the wild type. Cell-based kinase activity and autophosphorylation of ectopically expressed proteins showed that mutants retained some kinase activity. This study showed that patient-derived cytomegalovirus with different ganciclovir sensitivities retained replication efficiency and exhibited some kinase activity in vitro.

Human cytomegaloviral multifunctional protein kinase pUL97 impairs zebrafish embryonic development and increases mortality.

Cytomegalovirus is a worldwide-distributed human pathogen, which is the leading cause of congenital virus infection, affecting 0.5 to 2% of live births. To date, it is largely unclear which molecular mechanisms underlie the symptomatic outcomes. This is mainly due to species specificity and limited homology among cytomegalovirus genomes. As it is not possible to infect model organisms with human cytomegalovirus, the aim of this study was to develop a heterologous system allowing in the future the elucidation of the pathological role of individual viral proteins. As a model organism the zebrafish has been chosen due to its ease of manipulation and characterization as well as its large offspring. As cytomegalovirus model protein, pUL97 was characterized because it is multiply involved in virus-host interaction. Here, we show in zebrafish embryos, that (i) pUL97 can be expressed in zebrafish, (ii) increasing pUL97 expression levels quantitatively correlate with both minor and major pathological defects, (iii) pUL97 expression impairs cell cycle progression and induces cell death, (iv) active pUL97, but not an inactive mutant, induces excess mortality, and (v) co-administration of a pUL97 inhibitor reduces embryonic pathology. Collectively, these data indicate the suitability of zebrafish to elucidate the pathological role of human cytomegaloviral proteins.

Cyclins B1, T1, and H differ in their molecular mode of interaction with cytomegalovirus protein kinase pUL97.

Human cytomegalovirus (HCMV) is a common ß-herpesvirus causing life-long latent infections. HCMV replication interferes with cell cycle regulation in host cells because the HCMV-encoded cyclin-dependent kinase (CDK) ortholog pUL97 extensively phosphorylates the checkpoint regulator retinoblastoma protein. pUL97 also interacts with cyclins B1, T1, and H, and recent findings have strongly suggested that these interactions influence pUL97 substrate recognition. Interestingly, here we detected profound mechanistic differences among these pUL97-cyclin interactions. Our study revealed the following. (i) pUL97 interacts with cyclins B1 and H in a manner dependent on pUL97 activity and HCMV-specific cyclin modulation, respectively. (ii) The phosphorylated state of both proteins is an important determinant of the pUL97-cyclin B1 interaction. (iii) Activated phospho-Thr-315 cyclin H is up-regulated during HCMV replication. (iv) Thr-315 phosphorylation is independent of intracellular pUL97 or CDK7 activity. (v) pUL97-mediated in vitro phosphorylation is detectable for cyclin B1 but not H. (vi) Mutual transphosphorylation between pUL97 and CDK7 is not detectable, and an MS-based phosphosite analysis indicated that pUL97 might unexpectedly not be phosphorylated in its T-loop. (vii) The binary complexes pUL97-cyclin H and CDK7-cyclin H as well as the ternary complex pUL97-cyclin-H-CDK7 are detectable in an assembly-based CoIP approach. (viii) pUL97 self-interaction can be bridged by the transcriptional cyclins T1 or H but not by the classical cell cycle-regulating B1 cyclin. Combined, our findings unravel a number of cyclin type-specific differences in pUL97 interactions and suggest a multifaceted regulatory impact of cyclins on HCMV replication.

Human cytomegalovirus utilises cellular dual-specificity tyrosine phosphorylation-regulated kinases during placental replication.

INTRODUCTION: Congenital cytomegalovirus (HCMV) infection may cause significant fetal malformation and in severe cases fetal and neonatal death. Fetal injury may be caused indirectly by the placental response to infection. Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) have recently been identified as critical kinases for HCMV replication. In this study we provide first evidence that DYRK1A and DYRK1B are utilised during HCMV placental replication. METHODS: DYRK expression was investigated in AD169- and Merlin-infected TEV-1 trophoblast cells, ex vivo placental explants and naturally infected clinical placentae by immunofluorescence, western blot, co-immunoprecipitation and RT-qPCR. RESULTS: HCMV-infected placental cells showed accumulation and re-localisation of DYRK1A and DYRK1B protein to areas of cytoplasmic virion assembly complexes and nuclear viral replication compartments, respectively. This accumulation was a result of upregulated DYRK1A/B protein expression with HCMV inducing up to a 5.3-fold increase in DYRK1A and up to a 4.7-fold increase in DYRK1B protein, relative to mock-infected TEV-1 cells (p < 0.0001). Increased DYRK protein expression was correlated with DYRK1A/B mRNA upregulation, with HCMV-infected cells showing up to a 3.7-fold increase and 2.9-fold increase in DYRK1A and DYRK1B mRNA levels respectively (p < 0.05). Protein-protein interactions were detected between DYRK1A/1B complexes and HCMV immediate early IE2p86, early pp65 and pUL44 and late pp150 proteins. Treatment of HCMV-infected TEV-1 cells and placental explants with DYRK inhibitors significantly inhibited HCMV replication (p < 0.05) indicating these cellular kinases are required during HCMV placental replication. CONCLUSION: HCMV modulates cellular DYRKs during placental replication which may have implications for congenital HCMV pathogenesis and represent promising antiviral targets.

Dynamic regulatory interaction between cytomegalovirus major tegument protein pp65 and protein kinase pUL97 in intracellular compartments, dense bodies and virions.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen of considerable clinical importance. Understanding the processes that are important for viral replication is essential for the development of therapeutic strategies against HCMV infection. The HCMV-encoded protein kinase pUL97 is an important multifunctional regulator of viral replication. Several viral and cellular proteins are phosphorylated by pUL97. The phosphoprotein pp65 is one important substrate of pUL97. It is the most abundant tegument protein of HCMV virions, mediating the upload of other virion constituents and contributing to particle integrity. Further to that, it interferes with host innate immune defences, thereby enabling efficient viral replication. By applying different approaches, we characterized the pp65-pUL97 interaction in various compartments. Specifically, the pUL97 interaction domain of pp65 was defined (282-415). A putative cyclin bridge that enhances pUL97-pp65 interaction was identified. The impact of pUL97 mutation on virion and dense body morphogenesis was addressed using pUL97 mutant viruses. Alterations in the proteome of viral particles were seen, especially with mutant viruses expressing cytoplasmic variants of pUL97. On the basis of these data we postulate a so far poorly recognized functional relationship between pp65 and pUL97, and present a refined model of pp65-pUL97 interaction.

Facile access to potent antiviral quinazoline heterocycles with fluorescence properties via merging metal-free domino reactions.

Most of the known approved drugs comprise functionalized heterocyclic compounds as subunits. Among them, non-fluorescent quinazolines with four different substitution patterns are found in a variety of clinically used pharmaceuticals, while 4,5,7,8-substituted quinazolines and those displaying their own specific fluorescence, favourable for cellular uptake visualization, have not been described so far. Here we report the development of a one-pot synthetic strategy to access these 4,5,7,8-substituted quinazolines, which are fluorescent and feature strong antiviral properties (EC50 down to 0.6±0.1 µM) against human cytomegalovirus (HCMV). Merging multistep domino processes in one-pot under fully metal-free conditions leads to sustainable, maximum efficient and high-yielding organic synthesis. Furthermore, generation of artesunic acid-quinazoline hybrids and their application against HCMV (EC50 down to 0.1±0.0 µM) is demonstrated. Fluorescence of new antiviral hybrids and quinazolines has potential applications in molecular imaging in drug development and mechanistic studies, avoiding requirement of linkage to external fluorescent markers.

Inhibitors of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) exert a strong anti-herpesviral activity.

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of (val)ganciclovir therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy of antiviral treatment is based on the exploitation of cell-directed signaling, e. g. pathways with a known relevance for carcinogenesis and tumor drug development. Here we describe a principle for putative antiviral drugs based on targeting dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). DYRKs constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins, including regulators of the cell cycle, e.g. DYRK1B can induce cell cycle arrest, a critical step for the regulation of HCMV replication. Here we provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK inhibitors (SC84227, SC97202 and SC97208, Harmine and AZ-191). Using established replication assays for laboratory and clinically relevant strains of HCMV, concentration-dependent profiles of inhibition were obtained. Mean inhibitory concentrations (EC50) of 0.98 ± 0.08 µM/SC84227, 0.60 ± 0.02 µM/SC97202, 6.26 ± 1.64 µM/SC97208, 0.71 ± 0.019 µM/Harmine and 0.63 ± 0.23 µM/AZ-191 were determined with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover, rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a similarly high sensitivity to these compounds. Thus, we conclude that DYRK signaling represents a promising target pathway for the development of novel anti-herpesviral strategies.

Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities.

The Interaction between Cyclin B1 and Cytomegalovirus Protein Kinase pUL97 is Determined by an Active Kinase Domain.

Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain.