title: ORF36 protein kinase of Kaposi's sarcoma herpesvirus activates the c-Jun N-terminal kinase signaling pathway.
abstract: alpha-, beta-, and gamma-Herpesviruses encode putative viral protein kinases. The herpes simplex virus UL13, varicella-zoster virus ORF47, and Epstein-Barr virus BGLF4 genes all show protein kinase domains in their protein sequences. Mutational analysis of these herpesviruses demonstrated that the viral kinase is important for optimal virus growth. Previous studies have shown that ORF36 of Kaposi's sarcoma herpesvirus (KSHV) has protein kinase activity and is autophosphorylated on serine. The gene for ORF36 is expressed during lytic growth of the virus and has been classified as a late gene. Inspection of the ORF36 sequence indicated potential motifs that could be involved in activation of cellular transcription factors. To analyze the function of ORF36, the cDNA for this viral gene was tagged with the FLAG epitope and inserted into an expression vector for mammalian cells. Transfection experiments in 293T and SLK cells demonstrated that expression of ORF36 resulted in phosphorylation of the c-Jun N-terminal kinase. Autophosphorylation of ORF36 is important for JNK activation because a mutation in the predicted catalytic domain of ORF36 blocked its ability to phosphorylate JNK. Western blot analysis, using phosphospecific antibodies, revealed that mitogen-activated kinases MKK4 and MKK7 were phosphorylated by ORF36 but not by the kinase-negative mutant. Binding experiments in transfected cells also demonstrated that both the wild type and kinase-negative mutant of ORF36 form a complex with JNK, MKK4, and MKK7. In addition, using a tetracycline-inducible Rta BCBL-1 cell line (TREx BCBL1-Rta), JNK was phosphorylated during lytic replication, and inhibition of JNK activation blocked late viral gene expression but not early viral gene expression. In summary, these studies demonstrate that KSHV ORF36 activates the JNK pathway; thus this cell signaling pathway may function in the KSHV life cycle by regulating viral and/or cellular transcription.
authors.affiliation: Center for Comparative Medicine, University of California, Davis, California 95616, USA.
authors.forename: M Sabry
authors.initials: MS
authors.lastname: Hamza
authors.forename: Richard A
authors.initials: RA
authors.lastname: Reyes
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Izumiya
authors.forename: Ronald
authors.initials: R
authors.lastname: Wisdom
authors.forename: Hsing-Jien
authors.initials: HJ
authors.lastname: Kung
authors.forename: Paul A
authors.initials: PA
authors.lastname: Luciw
mesh_terms: D015153:Blotting, Western; D002384:Catalysis; D020134:Catalytic Domain; D002455:Cell Division; D002460:Cell Line; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D019288:Herpesvirus 8, Human / Q000201:enzymology*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D008239:Lysine / Q000737:chemistry; D048670:MAP Kinase Kinase 4; D048688:MAP Kinase Kinase 7; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation; D016366:Open Reading Frames; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics*; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D012694:Serine; D011494:Protein Kinases; C492430:ORF36 protein kinase, Kaposi's sarcoma herpesvirus; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D048688:MAP Kinase Kinase 7; C482559:MAP2K4 protein, human; C482569:MAP2K7 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D008239:Lysine