Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 07 Jan 2019 11:15:32 Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus Eric Sonntag, 1 Jens Milbradt, 1, * Adriana Svrlanska, 1 Hanife Strojan, 1 Sigrun H€ age, 1 Alexandra Kraut, 2 Anne-Marie Hesse, 2 Bushra Amin, 3 † Uwe Sonnewald, 3 Yohann Cout e 2 and Manfred Marschall 1, * Abstract 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 PKCa 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. INTRODUCTION Human cytomegalovirus (HCMV) is a globally distributed beta-herpesvirus that causes life-long latent infection in the human host. In the immunocompetent host, HCMV may remain asymptomatic, whereas in immunosuppressed indi- viduals, e.g. transplant recipients, those with tumours and AIDS patients, HCMV infection can lead to severe symp- toms and life-threatening viral pathogenesis. Most seriously, congenital HCMV infection acquired during pregnancy rep- resents a serious risk for the unborn and neonates to develop severe developmental defects and cytomegalovirus inclusion disease. Viral pathogenesis is closely linked with the efficiency of viral replication in individual tissues, a pronounced virulence, and so far insufficiently understood determinants of virus–host interaction. On the molecular level, recent investigations have stressed the importance of multiprotein complexes assembled between virus and host constituents. Such protein complexes appear to represent rate-limiting determinants of cytomegalovirus replication. We and others have concentrated on the study of the HCMV-specific nuclear egress complex (NEC). The nuclear envelope represents a physical barrier separating the nucleus from the cytoplasm. HCMV genomic replication starts in the host cell nucleus, where preformed capsids are packaged and exported to the cytoplasm for further virion matura- tion. The nucleo–cytoplasmic transition of capsids is a Received 16 June 2017; Accepted 4 September 2017 Author affiliations: 1 Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), Erlangen, Germany; 2 Universit e Grenoble Alpes, CEA, INSERM, BIG-BGE, F-38000 Grenoble, France; 3 Department of Biology, Institute for Biochemistry, FAU, Erlangen, Germany. *Correspondence: Jens Milbradt, jens.milbradt@fau.de; Manfred Marschall, manfred.marschall@fau.de Keywords: human cytomegalovirus; nuclear egress; core NEC; protein-protein interaction; cellular protein kinases; phosphorylation. Abbreviations: CDK, cyclin-dependent kinase; CoIP, coimmunoprecipitation; cVAC, cytoplasmic virion assembly complex; E, early; EBV, Epstein- Barr virus; EC50, 50 % effective concentration; FDR, false discovery rate; GCV, ganciclovir; GSK3, glycogen synthase kinase 3; HCMV, human cytomegalovirus; HSV-1, herpes simplex virus type 1; HSV-2, herpes simplex virus type 2; IE, immediate early; IgHC, immunoglobulin heavy chain; IgLC, immunoglobulin light chain; lambda PP, lambda phosphatase; IP, immunoprecipitation; IVKA, in vitro kinase assay; KO, knock-out; L, late; LC, liquid chromatography; LiCl, lithium chrloride; MCMV, murine cytomegalovirus; MCP, major capsid protein; NEC, nuclear egress complex; Phos-tag, phosphate affinity SDS-PAGE; p.i., post-infection; PKC, protein kinase C; p.t., post-transfection; siRNA, small interfer- ing RNA. †Present address: Department of Chemistry, University of Pittsburgh, Pittsburgh 15260, PA, USA. One supplementary table and six supplementary figures are available with the online Supplementary Material. RESEARCH ARTICLE Sonntag et al., Journal of General Virology 2017;98:2569–2581 DOI 10.1099/jgv.0.000931 000931 ã 2017 The Authors 2569