The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins

The human cytomegalovirus (HCMV)-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK) ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231–280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins.

[1]  T. Hunter,et al.  The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. , 1988, Science.

[2]  M. Winkler,et al.  UL69 of human cytomegalovirus, an open reading frame with homology to ICP27 of herpes simplex virus, encodes a transactivator of gene expression , 1994, Journal of virology.

[3]  J. Milton,et al.  Identification of multiple cyclin subunits of human P-TEFb. , 1998, Genes & development.

[4]  B. Roizman,et al.  Cellular Elongation Factor 1δ Is Modified in Cells Infected with Representative Alpha-, Beta-, or Gammaherpesviruses , 1999, Journal of Virology.

[5]  D. Coen,et al.  A Recombinant Human Cytomegalovirus with a Large Deletion in UL97 Has a Severe Replication Deficiency , 1999, Journal of Virology.

[6]  J. Ashby.  References and Notes , 1999 .

[7]  M. Marschall,et al.  Recombinant Green Fluorescent Protein-Expressing Human Cytomegalovirus as a Tool for Screening Antiviral Agents , 2000, Antimicrobial Agents and Chemotherapy.

[8]  A. Giordano,et al.  Distinct regions of cyclinT1 are required for binding to CDK9 and for recruitment to the HIV‐1 Tat/TAR complex , 2001, Journal of cellular biochemistry. Supplement.

[9]  M. Stein-Gerlach,et al.  Inhibitors of human cytomegalovirus replication drastically reduce the activity of the viral protein kinase pUL97. , 2001, The Journal of general virology.

[10]  M. Stein-Gerlach,et al.  Direct targeting of human cytomegalovirus protein kinase pUL97 by kinase inhibitors is a novel principle for antiviral therapy. , 2002, The Journal of general virology.

[11]  Daniel Romaker,et al.  The protein kinase pUL97 of human cytomegalovirus interacts with and phosphorylates the DNA polymerase processivity factor pUL44. , 2003, Virology.

[12]  D. Coen,et al.  Phosphorylation of the RNA polymerase II carboxyl-terminal domain in human cytomegalovirus-infected cells and in vitro by the viral UL97 protein kinase. , 2004, Virology.

[13]  Judy Y. Yen,et al.  Cyclin-Dependent Kinase Activity Is Required at Early Times for Accurate Processing and Accumulation of the Human Cytomegalovirus UL122-123 and UL37 Immediate-Early Transcripts and at Later Times for Virus Production , 2004, Journal of Virology.

[14]  Human Cytomegalovirus Infection Induces Specific Hyperphosphorylation of the Carboxyl-Terminal Domain of the Large Subunit of RNA Polymerase II That Is Associated with Changes in the Abundance, Activity, and Localization of cdk9 and cdk7 , 2005, Journal of Virology.

[15]  M. Marschall,et al.  Cellular p32 Recruits Cytomegalovirus Kinase pUL97 to Redistribute the Nuclear Lamina* , 2005, Journal of Biological Chemistry.

[16]  L. Schang,et al.  Five Years of Progress on Cyclin-Dependent Kinases and other Cellular Proteins as Potential Targets for Antiviral Drugs , 2006, Antiviral chemistry & chemotherapy.

[17]  Cyclin-Dependent Kinase Activity Is Required for Efficient Expression and Posttranslational Modification of Human Cytomegalovirus Proteins and for Production of Extracellular Particles , 2006, Journal of Virology.

[18]  Daniel Romaker,et al.  Analysis of the structure-activity relationship of four herpesviral UL97 subfamily protein kinases reveals partial but not full functional conservation. , 2006, Journal of medicinal chemistry.

[19]  L. Schang.  First demonstration of the effectiveness of inhibitors of cellular protein kinases in antiviral therapy , 2006, Expert review of anti-infective therapy.

[20]  Jens Milbradt,et al.  Cytomegaloviral proteins pUL 50 and pUL 53 are associated with the nuclear lamina and interact with cellular protein kinase C , 2007 .

[21]  Jens Milbradt,et al.  Cytomegaloviral proteins pUL50 and pUL53 are associated with the nuclear lamina and interact with cellular protein kinase C. , 2007, The Journal of general virology.

[22]  M. Marschall,et al.  Mapping of a self-interaction domain of the cytomegalovirus protein kinase pUL97. , 2007, The Journal of general virology.

[23]  A. J. Kapasi,et al.  Inhibition of the Cyclin-Dependent Kinases at the Beginning of Human Cytomegalovirus Infection Specifically Alters the Levels and Localization of the RNA Polymerase II Carboxyl-Terminal Domain Kinases cdk9 and cdk7 at the Viral Transcriptosome , 2007, Journal of Virology.

[24]  S. Chou,et al.  Viral and Cell Cycle–Regulated Kinases in Cytomegalovirus-Induced Pseudomitosis and Replication , 2007, PLoS pathogens.

[25]  S. Chou.  Cytomegalovirus UL97 mutations in the era of ganciclovir and maribavir , 2008, Reviews in medical virology.

[26]  D. Coen,et al.  Phosphorylation of Retinoblastoma Protein by Viral Protein with Cyclin-Dependent Kinase Function , 2008, Science.

[27]  Richard D. Smith,et al.  Human Cytomegalovirus UL97 Kinase Activity Is Required for the Hyperphosphorylation of Retinoblastoma Protein and Inhibits the Formation of Nuclear Aggresomes , 2008, Journal of Virology.

[28]  Jens Milbradt,et al.  Cytomegaloviral protein kinase pUL97 interacts with the nuclear mRNA export factor pUL69 to modulate its intranuclear localization and activity. , 2009, The Journal of general virology.

[29]  M. Prichard.  Function of human cytomegalovirus UL97 kinase in viral infection and its inhibition by maribavir , 2009, Reviews in medical virology.

[30]  A. Schreiber,et al.  Antiviral treatment of cytomegalovirus infection and resistant strains. , 2009, Expert opinion on pharmacotherapy.

[31]  M. Marschall,et al.  Molecular targets for antiviral therapy of cytomegalovirus infections. , 2009, Future microbiology.

[32]  M. Marschall,et al.  Cyclin-dependent Kinases Phosphorylate the Cytomegalovirus RNA Export Protein pUL69 and Modulate Its Nuclear Localization and Activity* , 2009, Journal of Biological Chemistry.

[33]  U. Wolfrum,et al.  Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells. , 2010, The Journal of general virology.

[34]  R. Kalejta,et al.  Cyclin-Dependent Kinase-Like Function Is Shared by the Beta- and Gamma- Subset of the Conserved Herpesvirus Protein Kinases , 2010, PLoS pathogens.

[35]  P. Tomasec,et al.  Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication. , 2010, The Journal of clinical investigation.

[36]  T. Tahirov,et al.  Crystal structure of HIV-1 Tat complexed with human P-TEFb , 2010, Nature.

[37]  Heinrich Sticht,et al.  Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation. , 2011, The Journal of general virology.

[38]  Jens Milbradt,et al.  Regulatory roles of protein kinases in cytomegalovirus replication. , 2011, Advances in virus research.

[39]  B. Klebl,et al.  Recruitment of cyclin-dependent kinase 9 to nuclear compartments during cytomegalovirus late replication: importance of an interaction between viral pUL69 and cyclin T1. , 2011, The Journal of general virology.

[40]  Richard J. Edwards,et al.  ELM—the database of eukaryotic linear motifs , 2011, Nucleic Acids Res..

[41]  T. Wittenberg,et al.  Nuclear import of isoforms of the cytomegalovirus kinase pUL97 is mediated by differential activity of NLS1 and NLS2 both acting through classical importin-α binding. , 2012, The Journal of general virology.

[42]  R. Baumgartner,et al.  Profiling of the kinome of cytomegalovirus-infected cells reveals the functional importance of host kinases Aurora A, ABL and AMPK. , 2013, Antiviral research.

[43]  S. Chou,et al.  Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility , 2014, Journal of Virology.