1887

Abstract

Nuclear egress of herpesvirus capsids through the nuclear envelope is mediated by the multimeric nuclear egress complex (NEC). The human cytomegalovirus (HCMV) core NEC is defined by an interaction between the membrane-anchored pUL50 and its nuclear co-factor pUL53, tightly associated through heterodimeric corecruitment to the nuclear envelope. Cellular proteins, such as p32/gC1qR, emerin and protein kinase C (PKC), are recruited by direct interaction with pUL50 for the multimeric extension of the NEC. As a functionally important event, the recruitment of both viral and cellular protein kinases leads to site-specific lamin phosphorylation and nuclear lamina disassembly. In this study, interaction domains within pUL50 for its binding partners were defined by co-immunoprecipitation. The interaction domain for pUL53 is located within the pUL50 N-terminus (residues 10–169), interaction domains for p32/gC1qR (100–358) and PKC (100–280) overlap in the central part of pUL50, and the interaction domain for emerin is located in the C-terminus (265–397). Moreover, expression and formation of core NEC proteins at the nuclear rim were consistently detected in cells permissive for productive HCMV replication, including two trophoblast-cell lines. Importantly, regular nuclear-rim formation of the core NEC was blocked by inhibition of cyclin-dependent kinase (CDK) activity. In relation to the recently published crystal structure of the HCMV core NEC, our findings result in a refined view of NEC assembly. In particular, we suggest that CDKs may play an important regulatory role in NEC formation during HCMV replication.

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2016-07-01
2024-03-29
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References

  1. Adler S. P., Nigro G., Pereira L. 2007; Recent advances in the prevention and treatment of congenital cytomegalovirus infections. Semin Perinatol 31:10–18 [View Article][PubMed]
    [Google Scholar]
  2. Bigley T. M., Reitsma J. M., Terhune S. S. 2015; Antagonistic relationship between human cytomegalovirus pUL27 and pUL97 activities during infection. J Virol 89:10230–10246 [View Article][PubMed]
    [Google Scholar]
  3. Broers J. L., Ramaekers F. C., Bonne G., Yaou R. B., Hutchison C. J. 2006; Nuclear lamins: laminopathies and their role in premature ageing. Physiol Rev 86:967–1008 [View Article][PubMed]
    [Google Scholar]
  4. Brokstad K. A., Kalland K. H., Russell W. C., Matthews D. A. 2001; Mitochondrial protein p32 can accumulate in the nucleus. Biochem Biophys Res Commun 281:1161–1169 [View Article][PubMed]
    [Google Scholar]
  5. Buchkovich N. J., Maguire T. G., Alwine J. C. 2010; Role of the endoplasmic reticulum chaperone BiP, SUN domain proteins, and dynein in altering nuclear morphology during human cytomegalovirus infection. J Virol 84:7005–7017 [View Article][PubMed]
    [Google Scholar]
  6. Chou S., Hakki M., Villano S. 2012; Effects on maribavir susceptibility of cytomegalovirus UL97 kinase ATP binding region mutations detected after drug exposure in vitro and in vivo. Antiviral Res 95:88–92 [View Article][PubMed]
    [Google Scholar]
  7. Dolan A., Cunningham C., Hector R. D., Hassan-Walker A. F., Lee L., Addison C., Dargan D. J., McGeoch D. J., Gatherer D. et al. 2004; Genetic content of wild-type human cytomegalovirus. J Gen Virol 85:1301–1312 [View Article][PubMed]
    [Google Scholar]
  8. Ellsmore V., Reid G. G., Stow N. D. 2003; Detection of human cytomegalovirus DNA replication in non-permissive Vero and 293 cells. J Gen Virol 84:639–645 [View Article][PubMed]
    [Google Scholar]
  9. Feichtinger S., Stamminger T., Müller R., Graf L., Klebl B., Eickhoff J., Marschall M. 2011; 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 92:1519–1531 [View Article][PubMed]
    [Google Scholar]
  10. Gebert S., Schmolke S., Sorg G., Flöss S., Plachter B., Stamminger T. 1997; The UL84 protein of human cytomegalovirus acts as a transdominant inhibitor of immediate-early-mediated transactivation that is able to prevent viral replication. J Virol 71:7048–7060[PubMed]
    [Google Scholar]
  11. Graf L., Webel R., Wagner S., Hamilton S. T., Rawlinson W. D., Sticht H., Marschall M. 2013; The cyclin-dependent kinase ortholog pUL97 of human cytomegalovirus interacts with cyclins. Viruses 5:3213–3230 [View Article][PubMed]
    [Google Scholar]
  12. Grimm K. S., Klupp B. G., Granzow H., Müller F. M., Fuchs W., Mettenleiter T. C. 2012; Analysis of viral and cellular factors influencing herpesvirus-induced nuclear envelope breakdown. J Virol 86:6512–6521 [View Article][PubMed]
    [Google Scholar]
  13. Hamilton S. T., Milbradt J., Marschall M., Rawlinson W. D. 2014; Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts. PLoS One 9:e97231 [View Article][PubMed]
    [Google Scholar]
  14. Hamirally S., Kamil J. P., Ndassa-Colday Y. M., Lin A. J., Jahng W. J., Baek M. C., Noton S., Silva L. A., Simpson-Holley M. et al. 2009; Viral mimicry of Cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress. PLoS Pathog 5:e1000275 [View Article][PubMed]
    [Google Scholar]
  15. Hertel L., Chou S., Mocarski E. S. 2007; Viral and cell cycle-regulated kinases in cytomegalovirus-induced pseudomitosis and replication. PLoS Pathog 3:e6 [View Article][PubMed]
    [Google Scholar]
  16. Hume A. J., Finkel J. S., Kamil J. P., Coen D. M., Culbertson M. R., Kalejta R. F. 2008; Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase function. Science 320:797–799 [View Article][PubMed]
    [Google Scholar]
  17. Hutterer C., Eickhoff J., Milbradt J., Korn K., Zeitträger I., Bahsi H., Wagner S., Zischinsky G., Wolf A. et al. 2015; A novel CDK7 inhibitor of the Pyrazolotriazine class exerts broad-spectrum antiviral activity at nanomolar concentrations. Antimicrob Agents Chemother 59:2062–2071 [View Article]
    [Google Scholar]
  18. Johnson D. C., Baines J. D. 2011; Herpesviruses remodel host membranes for virus egress. Nat Rev Microbiol 9:382–394 [View Article][PubMed]
    [Google Scholar]
  19. Klupp B. G., Granzow H., Mettenleiter T. C. 2011; Nuclear envelope breakdown can substitute for primary envelopment-mediated nuclear egress of herpesviruses. J Virol 85:8285–8292 [View Article][PubMed]
    [Google Scholar]
  20. Krosky P. M., Baek M. C., Coen D. M. 2003; The human cytomegalovirus UL97 protein kinase, an antiviral drug target, is required at the stage of nuclear egress. J Virol 77:905–914 [View Article][PubMed]
    [Google Scholar]
  21. Leach N. R., Roller R. J. 2010; Significance of host cell kinases in herpes simplex virus type 1 egress and lamin-associated protein disassembly from the nuclear lamina. Virology 406:127–137 [View Article][PubMed]
    [Google Scholar]
  22. Lemnitzer F., Raschbichler V., Kolodziejczak D., Israel L., Imhof A., Bailer S. M., Koszinowski U., Ruzsics Z. 2013; Mouse cytomegalovirus egress protein pM50 interacts with cellular endophilin-A2. Cell Microbiol 15:335–351 [View Article][PubMed]
    [Google Scholar]
  23. Lye M. F., Sharma M., El Omari K., Filman D. J., Schuermann J. P., Hogle J. M., Coen D. M. 2015; Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex. EMBO Je201592651
    [Google Scholar]
  24. Marschall M., Marzi A., aus dem Siepen P., Jochmann R., Kalmer M., Auerochs S., Lischka P., Leis M., Stamminger T. 2005; Cellular p32 recruits cytomegalovirus kinase pUL97 to redistribute the nuclear lamina. J Biol Chem 280:33357–33367 [View Article][PubMed]
    [Google Scholar]
  25. Marschall M., Feichtinger S., Milbradt J. 2011; Regulatory roles of protein kinases in cytomegalovirus replication. Adv Virus Res 80:69–101 [View Article][PubMed]
    [Google Scholar]
  26. Mettenleiter T. C., Müller F., Granzow H., Klupp B. G. 2013; The way out: what we know and do not know about herpesvirus nuclear egress. Cell Microbiol 15:170–178 [View Article][PubMed]
    [Google Scholar]
  27. Milbradt J., Auerochs S., Marschall M. 2007; Cytomegaloviral proteins pUL50 and pUL53 are associated with the nuclear lamina and interact with cellular protein kinase C. J Gen Virol 88:2642–2650 [View Article][PubMed]
    [Google Scholar]
  28. Milbradt J., Auerochs S., Sticht H., Marschall M. 2009; Cytomegaloviral proteins that associate with the nuclear lamina: components of a postulated nuclear egress complex. J Gen Virol 90:579–590 [View Article][PubMed]
    [Google Scholar]
  29. Milbradt J., Webel R., Auerochs S., Sticht H., Marschall M. 2010; Novel mode of phosphorylation-triggered reorganization of the nuclear lamina during nuclear egress of human cytomegalovirus. J Biol Chem 285:13979–13989 [View Article][PubMed]
    [Google Scholar]
  30. Milbradt J., Auerochs S., Sevvana M., Muller Y. A., Sticht H., Marschall M. 2012; Specific residues of a conserved domain in the N terminus of the human cytomegalovirus pUL50 protein determine its intranuclear interaction with pUL53. J Biol Chem 287:24004–24016 [View Article][PubMed]
    [Google Scholar]
  31. Milbradt J., Kraut A., Hutterer C., Sonntag E., Schmeiser C., Ferro M., Wagner S., Lenac T., Claus C. et al. 2014; Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus. Mol Cell Proteomics 13:2132–2146 [View Article][PubMed]
    [Google Scholar]
  32. Mocarski E. S., Shenk T., Griffiths P. D., Pass R. F. 2013; Cytomegaloviruses. In Fields Virology pp. 1960–2014 Edited by Knipe D. M., Howley P. M. Philadelphia, PA: Lippincott Williams and Wilkins;
    [Google Scholar]
  33. Muranyi W., Haas J., Wagner M., Krohne G., Koszinowski U. H. 2002; Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina. Science 297:854–857 [View Article][PubMed]
    [Google Scholar]
  34. Ogawa-Goto K., Tanaka K., Gibson W., Moriishi E., Miura Y., Kurata T., Irie S., Sata T. 2003; Microtubule network facilitates nuclear targeting of human cytomegalovirus capsid. J Virol 77:8541–8547 [View Article][PubMed]
    [Google Scholar]
  35. Prichard M. N., Gao N., Jairath S., Mulamba G., Krosky P., Coen D. M., Parker B. O., Pari G. S. 1999; A recombinant human cytomegalovirus with a large deletion in UL97 has a severe replication deficiency. J Virol 73:5663–5670[PubMed]
    [Google Scholar]
  36. Rechter S., Scott G. M., Eickhoff J., Zielke K., Auerochs S., Müller R., Stamminger T., Rawlinson W. D., Marschall M. 2009; Cyclin-dependent kinases phosphorylate the cytomegalovirus RNA export protein pul69 and modulate its nuclear localization and activity. J Biol Chem 284:8605–8613 [View Article][PubMed]
    [Google Scholar]
  37. Sam M. D., Evans B. T., Coen D. M., Hogle J. M. 2009; Biochemical, biophysical, and mutational analyses of subunit interactions of the human cytomegalovirus nuclear egress complex. J Virol 83:2996–3006 [View Article][PubMed]
    [Google Scholar]
  38. Sanchez V., Spector D. H. 2006; Cyclin-dependent kinase activity is required for efficient expression and posttranslational modification of human cytomegalovirus proteins and for production of extracellular particles. J Virol 80:5886–5896 [View Article][PubMed]
    [Google Scholar]
  39. Sandal T., Stapnes C., Kleivdal H., Hedin L., Døskeland S. O. 2002; A novel, extraneuronal role for cyclin-dependent protein kinase 5 (CDK5): modulation of cAMP-induced apoptosis in rat leukemia cells. J Biol Chem 277:20783–20793 [View Article][PubMed]
    [Google Scholar]
  40. Schang L. M., Coccaro E., Lacasse J. J. 2005; Cdk inhibitory nucleoside analogs prevent transcription from viral genomes. Nucleosides Nucleotides Nucleic Acids 24:829–837 [View Article][PubMed]
    [Google Scholar]
  41. Schmeiser C., Borst E., Sticht H., Marschall M., Milbradt J. 2013; The cytomegalovirus egress proteins pUL50 and pUL53 are translocated to the nuclear envelope through two distinct modes of nuclear import. J Gen Virol 94:2056–2069 [View Article][PubMed]
    [Google Scholar]
  42. Schregel V., Auerochs S., Jochmann R., Maurer K., Stamminger T., Marschall M. 2007; Mapping of a self-interaction domain of the cytomegalovirus protein kinase pUL97. J Gen Virol 88:395–404 [View Article][PubMed]
    [Google Scholar]
  43. Sharma M., Bender B. J., Kamil J. P., Lye M. F., Pesola J. M., Reim N. I., Hogle J. M., Coen D. M. 2015; Human cytomegalovirus UL97 phosphorylates the viral nuclear egress complex. J Virol 89:523–534 [View Article][PubMed]
    [Google Scholar]
  44. Soni D. V., Jacobberger J. W. 2004; Inhibition of cdk1 by alsterpaullone and thioflavopiridol correlates with increased transit time from mid G2 through prophase. Cell Cycle 3:349–357 [View Article][PubMed]
    [Google Scholar]
  45. Steingruber M., Socher E., Hutterer C., Webel R., Bergbrede T., Lenac T., Sticht H., Marschall M. 2015; The interaction between cyclin B1 and cytomegalovirus protein kinase pUL97 is determined by an active kinase domain. Viruses 7:4582–4601 [View Article][PubMed]
    [Google Scholar]
  46. Trofe J., Pote L., Wade E., Blumberg E., Bloom R. D. 2008; Maribavir: a novel antiviral agent with activity against cytomegalovirus. Ann Pharmacother 42:1447–1457 [View Article][PubMed]
    [Google Scholar]
  47. Walzer S. A., Egerer-Sieber C., Sticht H., Sevvana M., Hohl K., Milbradt J., Muller Y. A., Marschall M. 2015; Crystal structure of the human cytomegalovirus pul50-pul53 core nuclear egress complex provides insight into a unique assembly scaffold for virus-host protein interactions. J Biol Chem 290:27452–27458 [View Article][PubMed]
    [Google Scholar]
  48. Wolf D. G., Courcelle C. T., Prichard M. N., Mocarski E. S. 2001; Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation. Proc Natl Acad Sci U S A 98:1895–1900 [View Article][PubMed]
    [Google Scholar]
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