title: Phosphorylation of Kaposi's sarcoma-associated herpesvirus processivity factor ORF59 by a viral kinase modulates its ability to associate with RTA and oriLyt.
abstract: ORF59 of Kaposi's sarcoma-associated herpesvirus (KSHV) plays an essential role in viral lytic replication by providing DNA processivity activity to the viral DNA polymerase (ORF9). ORF59 forms a homodimer in the cytoplasm and binds and translocates ORF9 into the nucleus, where it secures ORF9 to the origin of lytic DNA replication (oriLyt) in order to synthesize long DNA fragments during replication. ORF59 binds to oriLyt through an immediate early protein, replication and transcription activator (RTA). Here, we show that viral kinase (ORF36) phosphorylates serines between amino acids 376 and 379 of ORF59 and replacement of the Ser378 residue with alanine significantly impairs phosphorylation. Although mutating these serine residues had no effect on binding between ORF59 and ORF9, viral polymerase, or ORF36, the viral kinase, it significantly reduced the ability of ORF59 to bind to RTA. The results for the mutant in which Ser376 to Ser379 were replaced by alanine showed that both Ser378 and Ser379 contribute to binding to RTA. Additionally, the Ser376, Ser378, and Ser379 residues were found to be critical for binding of ORF59 to oriLyt and its processivity function. Ablation of these phosphorylation sites reduced the production of virion particles, suggesting that phosphorylation is critical for ORF59 activity and viral DNA synthesis.
authors.affiliation: Center for Molecular Medicine, Department of Microbiology & Immunology, University of Nevada, Reno, School of Medicine, Reno, Nevada, USA.
authors.forename: Maria E
authors.initials: ME
authors.lastname: McDowell
authors.forename: Pravinkumar
authors.initials: P
authors.lastname: Purushothaman
authors.forename: Cyprian C
authors.initials: CC
authors.lastname: Rossetto
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Pari
authors.forename: Subhash C
authors.initials: SC
authors.lastname: Verma
mesh_terms: D015153:Blotting, Western; D018929:Cell Culture Techniques; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005816:Genetic Complementation Test; D017874:Immediate-Early Proteins / Q000378:metabolism*; D047468:Immunoprecipitation; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D017874:Immediate-Early Proteins; C110348:ORF59 protein, Human herpesvirus 8; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators; D014764:Viral Proteins; D011494:Protein Kinases; C492430:ORF36 protein kinase, Kaposi's sarcoma herpesvirus; D004259:DNA-Directed DNA Polymerase