Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus

• Published in: Journal of general virology 2017-10, Vol.98 (10), p.2569-2581
• Main Authors: Sonntag, Eric, Milbradt, Jens, Svrlanska, Adriana, Strojan, Hanife, Häge, Sigrun, Kraut, Alexandra, Hesse, Anne-Marie, Amin, Bushra, Sonnewald, Uwe, Couté, Yohann, Marschall, Manfred
• Format: Article
• Language: English
• Subjects: Active Transport, Cell Nucleus; Biochemistry, Molecular Biology; CDC2 Protein Kinase; Cell Nucleus - metabolism; Cyclin-Dependent Kinases - antagonists & inhibitors; Cyclin-Dependent Kinases - metabolism; Cytomegalovirus - metabolism; Genomics; Humans; Life Sciences; Mass Spectrometry; Phosphorylation; Protein Kinase C-alpha - antagonists & inhibitors; Protein Kinase C-alpha - genetics; Protein Kinase C-alpha - metabolism; Viral Proteins - metabolism; Virus Release - physiology
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/jgv.0.000931

Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.