Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome

• Published in: Proteomics (Weinheim) Vol. 15; no. 12; pp. 2006 - 2022
• Main Authors: Oberstein, Adam, Perlman, David H., Shenk, Thomas, Terry, Laura J.
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 01.06.2015; Wiley Subscription Services, Inc
• Subjects: Blotting, Western; Cancer; Cell biology; Cell cycle; Cells, Cultured; Chromatography, Liquid; Cytomegalovirus; Cytomegalovirus - physiology; Cytomegalovirus Infections - metabolism; Cytomegalovirus Infections - virology; Egress; Fibroblasts; Fibroblasts - metabolism; Fibroblasts - virology; Herpesvirus; Human cytomegalovirus; Humans; Kinases; Lamins; Nuclear pore complex; Peptides; Phosphoproteins - metabolism; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Protein Interaction Domains and Motifs; Protein-serine/threonine kinase; Proteins; Proteome - analysis; Proteomics - methods; Replication; Retina; Retinoblastoma; Retinoblastoma protein; Substrates; Tandem Mass Spectrometry; Titanium dioxide; TREX complex; Viral kinase; Virus Replication; Viruses; Cell biology; TREX complex; Viral kinase; Nuclear pore complex; Herpesvirus; Human cytomegalovirus
• ISSN: 1615-9853; 1615-9861; 1615-9861
• DOI: 10.1002/pmic.201400607

Replication of human cytomegalovirus (HCMV) is regulated in part by cellular kinases and the single viral Ser/Thr kinase, pUL97. The virus‐coded kinase augments the replication of HCMV by enabling nuclear egress and altering cell cycle progression. These roles are accomplished through direct phosphorylation of nuclear lamins and the retinoblastoma protein, respectively. In an effort to identify additional pUL97 substrates, we analyzed the phosphoproteome of SILAC‐labeled human fibroblasts during infection with either wild‐type HCMV or a pUL97 kinase‐dead mutant virus. Phosphopeptides were enriched over a titanium dioxide matrix and analyzed by high‐resolution MS. We identified 157 unambiguous phosphosites from 106 cellular and 17 viral proteins whose phosphorylation required UL97. Analysis of peptides containing these sites allowed the identification of several candidate pUL97 phosphorylation motifs, including a completely novel phosphorylation motif, LxSP. Substrates harboring the LxSP motif were enriched in nucleocytoplasmic transport functions, including a number of components of the nuclear pore complex. These results extend the known functions of pUL97 and suggest that modulation of nuclear pore function may be important during HCMV replication.