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.

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.

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.