1887

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. 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.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000931
2017-10-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/98/10/2569.html?itemId=/content/journal/jgv/10.1099/jgv.0.000931&mimeType=html&fmt=ahah

References

  1. Marschall M, Marzi A, Aus dem Siepen P, Jochmann R, Kalmer M et al. Cellular p32 recruits cytomegalovirus kinase pUL97 to redistribute the nuclear lamina. J Biol Chem 2005; 280:33357–33367 [View Article][PubMed]
    [Google Scholar]
  2. Milbradt J, Hutterer C, Bahsi H, Wagner S, Sonntag E et al. The prolyl isomerase pin1 promotes the herpesvirus-induced phosphorylation-dependent disassembly of the nuclear lamina required for nucleocytoplasmic egress. PLoS Pathog 2016; 12:e1005825 [View Article][PubMed]
    [Google Scholar]
  3. Milbradt J, Kraut A, Hutterer C, Sonntag E, Schmeiser C et al. Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus. Mol Cell Proteomics 2014; 13:2132–2146 [View Article][PubMed]
    [Google Scholar]
  4. Mou F, Forest T, Baines JD. US3 of herpes simplex virus type 1 encodes a promiscuous protein kinase that phosphorylates and alters localization of lamin A/C in infected cells. J Virol 2007; 81:6459–6470 [View Article][PubMed]
    [Google Scholar]
  5. Mou F, Wills E, Baines JD. Phosphorylation of the UL31 protein of herpes simplex virus 1 by the US3-encoded kinase regulates localization of the nuclear envelopment complex and egress of nucleocapsids. J Virol 2009; 83:5181–5191 [View Article][PubMed]
    [Google Scholar]
  6. Cano-Monreal GL, Wylie KM, Cao F, Tavis JE, Morrison LA. Herpes simplex virus 2 UL13 protein kinase disrupts nuclear lamins. Virology 2009; 392:137–147 [View Article][PubMed]
    [Google Scholar]
  7. Morris JB, Hofemeister H, O'Hare P. Herpes simplex virus infection induces phosphorylation and delocalization of emerin, a key inner nuclear membrane protein. J Virol 2007; 81:4429–4437 [View Article][PubMed]
    [Google Scholar]
  8. Sharma M, Bender BJ, Kamil JP, Lye MF, Pesola JM et al. Human cytomegalovirus UL97 phosphorylates the viral nuclear egress complex. J Virol 2015; 89:523–534 [View Article][PubMed]
    [Google Scholar]
  9. Kato A, Yamamoto M, Ohno T, Kodaira H, Nishiyama Y et al. Identification of proteins phosphorylated directly by the Us3 protein kinase encoded by herpes simplex virus 1. J Virol 2005; 79:9325–9331 [View Article][PubMed]
    [Google Scholar]
  10. Kato A, Yamamoto M, Ohno T, Tanaka M, Sata T et al. Herpes simplex virus 1-encoded protein kinase UL13 phosphorylates viral Us3 protein kinase and regulates nuclear localization of viral envelopment factors UL34 and UL31. J Virol 2006; 80:1476–1486 [View Article][PubMed]
    [Google Scholar]
  11. Park R, Baines JD. Herpes simplex virus type 1 infection induces activation and recruitment of protein kinase C to the nuclear membrane and increased phosphorylation of lamin B. J Virol 2006; 80:494–504 [View Article][PubMed]
    [Google Scholar]
  12. Leach NR, Roller RJ. Significance of host cell kinases in herpes simplex virus type 1 egress and lamin-associated protein disassembly from the nuclear lamina. Virology 2010; 406:127–137 [View Article][PubMed]
    [Google Scholar]
  13. Lee CP, Huang YH, Lin SF, Chang Y, Chang YH et al. Epstein-barr virus BGLF4 kinase induces disassembly of the nuclear lamina to facilitate virion production. J Virol 2008; 82:11913–11926 [View Article][PubMed]
    [Google Scholar]
  14. Muranyi W, Haas J, Wagner M, Krohne G, Koszinowski UH. Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina. Science 2002; 297:854–857 [View Article][PubMed]
    [Google Scholar]
  15. Hamirally S, Kamil JP, Ndassa-Colday YM, Lin AJ, Jahng WJ et al. Viral mimicry of Cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress. PLoS Pathog 2009; 5:e1000275 [View Article][PubMed]
    [Google Scholar]
  16. Sonntag E, Hamilton ST, Bahsi H, Wagner S, Jonjic S et al. Cytomegalovirus pUL50 is the multi-interacting determinant of the core nuclear egress complex (NEC) that recruits cellular accessory NEC components. J Gen Virol 2016; 97:1676–1685 [View Article][PubMed]
    [Google Scholar]
  17. Marschall M, Muller YA, Diewald B, Sticht H, Milbradt J. The human cytomegalovirus nuclear egress complex unites multiple functions: recruitment of effectors, nuclear envelope rearrangement, and docking to nuclear capsids. Rev Med Virol 2017; 27:e1934 [View Article][PubMed]
    [Google Scholar]
  18. Milbradt J, Auerochs S, Marschall M. Cytomegaloviral proteins pUL50 and pUL53 are associated with the nuclear lamina and interact with cellular protein kinase C. J Gen Virol 2007; 88:2642–2650 [View Article][PubMed]
    [Google Scholar]
  19. Milbradt J, Auerochs S, Sticht H, Marschall M. Cytomegaloviral proteins that associate with the nuclear lamina: components of a postulated nuclear egress complex. J Gen Virol 2009; 90:579–590 [View Article][PubMed]
    [Google Scholar]
  20. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 2014; 343:84–87 [View Article][PubMed]
    [Google Scholar]
  21. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013; 8:2281–2308 [View Article][PubMed]
    [Google Scholar]
  22. Rosse C, Linch M, Kermorgant S, Cameron AJ, Boeckeler K et al. PKC and the control of localized signal dynamics. Nat Rev Mol Cell Biol 2010; 11:103–112 [View Article][PubMed]
    [Google Scholar]
  23. Hertel L, Chou S, Mocarski ES. Viral and cell cycle-regulated kinases in cytomegalovirus-induced pseudomitosis and replication. PLoS Pathog 2007; 3:e6 [View Article][PubMed]
    [Google Scholar]
  24. Sharma M, Kamil JP, Coughlin M, Reim NI, Coen DM. Human cytomegalovirus UL50 and UL53 recruit viral protein kinase UL97, not protein kinase C, for disruption of nuclear lamina and nuclear egress in infected cells. J Virol 2014; 88:249–262 [View Article][PubMed]
    [Google Scholar]
  25. Sanchez V, Spector DH. Cyclin-dependent kinase activity is required for efficient expression and posttranslational modification of human cytomegalovirus proteins and for production of extracellular particles. J Virol 2006; 80:5886–5896 [View Article][PubMed]
    [Google Scholar]
  26. Bigley TM, Reitsma JM, Terhune SS. Antagonistic relationship between human cytomegalovirus pUL27 and pUL97 activities during Infection. J Virol 2015; 89:10230–10246 [View Article][PubMed]
    [Google Scholar]
  27. van den Heuvel S, Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science 1993; 262:2050–2054 [View Article][PubMed]
    [Google Scholar]
  28. Schregel V, Auerochs S, Jochmann R, Maurer K, Stamminger T et al. Mapping of a self-interaction domain of the cytomegalovirus protein kinase pUL97. J Gen Virol 2007; 88:395–404 [View Article][PubMed]
    [Google Scholar]
  29. Kinoshita E, Kinoshita-Kikuta E, Takiyama K, Koike T. Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics 2006; 5:749–757 [View Article][PubMed]
    [Google Scholar]
  30. Marschall M, Freitag M, Weiler S, Sorg G, Stamminger T. Recombinant green fluorescent protein-expressing human cytomegalovirus as a tool for screening antiviral agents. Antimicrob Agents Chemother 2000; 44:1588–1597 [View Article][PubMed]
    [Google Scholar]
  31. Schmeiser C, Borst E, Sticht H, Marschall M, Milbradt J. The cytomegalovirus egress proteins pUL50 and pUL53 are translocated to the nuclear envelope through two distinct modes of nuclear import. J Gen Virol 2013; 94:2056–2069 [View Article][PubMed]
    [Google Scholar]
  32. Hutterer C, Eickhoff J, Milbradt J, Korn K, Zeitträger I et al. A novel CDK7 inhibitor of the pyrazolotriazine class exerts broad-spectrum antiviral activity at nanomolar concentrations. Antimicrob Agents Chemother 2015; 59:2062–2071 [View Article][PubMed]
    [Google Scholar]
  33. Lorz K, Hofmann H, Berndt A, Tavalai N, Mueller R et al. Deletion of open reading frame UL26 from the human cytomegalovirus genome results in reduced viral growth, which involves impaired stability of viral particles. J Virol 2006; 80:5423–5434 [View Article][PubMed]
    [Google Scholar]
  34. Rechter S, Scott GM, Eickhoff J, Zielke K, Auerochs S et al. Cyclin-dependent Kinases phosphorylate the cytomegalovirus RNA export protein pUL69 and modulate its nuclear localization and activity. J Biol Chem 2009; 284:8605–8613 [View Article][PubMed]
    [Google Scholar]
  35. Feichtinger S, Stamminger T, Müller R, Graf L, Klebl B et al. Recruitment of cyclin-dependent kinase 9 to nuclear compartments during cytomegalovirus late replication: importance of an interaction between viral pUL69 and cyclin T1. J Gen Virol 2011; 92:1519–1531 [View Article][PubMed]
    [Google Scholar]
  36. Trofe J, Pote L, Wade E, Blumberg E, Bloom RD. Maribavir: a novel antiviral agent with activity against cytomegalovirus. Ann Pharmacother 2008; 42:1447–1457 [View Article][PubMed]
    [Google Scholar]
  37. Chou S, Hakki M, Villano S. Effects on maribavir susceptibility of cytomegalovirus UL97 kinase ATP binding region mutations detected after drug exposure in vitro and in vivo . Antiviral Res 2012; 95:88–92 [View Article][PubMed]
    [Google Scholar]
  38. Engholm-Keller K, Hansen TA, Palmisano G, Larsen MR. Multidimensional strategy for sensitive phosphoproteomics incorporating protein prefractionation combined with SIMAC, HILIC, and TiO2 chromatography applied to proximal EGF signaling. J Proteome Res 2011; 10:5383–5397 [View Article][PubMed]
    [Google Scholar]
  39. Savitski MM, Lemeer S, Boesche M, Lang M, Mathieson T et al. Confident phosphorylation site localization using the Mascot Delta Score. Mol Cell Proteomics 2011; 10:M110.003830 [View Article][PubMed]
    [Google Scholar]
  40. Amin B, Maurer A, Voelter W, Melms A, Kalbacher H. New poteintial serum biomarkers in multiple sclerosis identified by proteomic strategies. Curr Med Chem 2014; 21:1544–1556 [View Article][PubMed]
    [Google Scholar]
  41. Maurer A, Zeyher C, Amin B, Kalbacher H. A periodate-cleavable linker for functional proteomics under slightly acidic conditions: application for the analysis of intracellular aspartic proteases. J Proteome Res 2013; 12:199–207 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000931
Loading
/content/journal/jgv/10.1099/jgv.0.000931
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error