1887

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous pathogen of considerable clinical importance. Understanding the processes that are important for viral replication is essential for the development of therapeutic strategies against HCMV infection. The HCMV-encoded protein kinase pUL97 is an important multifunctional regulator of viral replication. Several viral and cellular proteins are phosphorylated by pUL97. The phosphoprotein pp65 is one important substrate of pUL97. It is the most abundant tegument protein of HCMV virions, mediating the upload of other virion constituents and contributing to particle integrity. Further to that, it interferes with host innate immune defences, thereby enabling efficient viral replication. By applying different approaches, we characterized the pp65–pUL97 interaction in various compartments. Specifically, the pUL97 interaction domain of pp65 was defined (282–415). A putative cyclin bridge that enhances pUL97–pp65 interaction was identified. The impact of pUL97 mutation on virion and dense body morphogenesis was addressed using pUL97 mutant viruses. Alterations in the proteome of viral particles were seen, especially with mutant viruses expressing cytoplasmic variants of pUL97. On the basis of these data we postulate a so far poorly recognized functional relationship between pp65 and pUL97, and present a refined model of pp65–pUL97 interaction.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000939
2017-10-12
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/jgv/98/11/2850.html?itemId=/content/journal/jgv/10.1099/jgv.0.000939&mimeType=html&fmt=ahah

References

  1. Mocarski ES, Shenk T, Griffiths PD, Pass RF. Cytomegaloviruses. In Knipe DM, Howley PM. (editors) Fields Virology, 6th ed. Philadelphia: Wolters Kluwer Lippincott Williams & Wilkins; 2013; pp. 1960– 2014
    [Google Scholar]
  2. Gibson W, Bogner E. Morphogenesis of the cytomegalovirus virion and subviral particles. In Reddehase MJ. (editor) Cytomegaloviruses: from molecular pathogenesis to intervention, 2nd ed.vol. 1 Norfolk: Caister Academic Press; 2013; pp. 230– 246
    [Google Scholar]
  3. Mettenleiter TC, Müller F, Granzow H, Klupp BG. The way out: what we know and do not know about herpesvirus nuclear egress. Cell Microbiol 2013; 15: 170– 178 [CrossRef] [PubMed]
    [Google Scholar]
  4. 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 [CrossRef] [PubMed]
    [Google Scholar]
  5. Walzer SA, Egerer-Sieber C, Sticht H, Sevvana M, Hohl K et al. 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 2015; 290: 27452– 27458 [CrossRef] [PubMed]
    [Google Scholar]
  6. Lye MF, Sharma M, El Omari K, Filman DJ, Schuermann JP et al. Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex. Embo J 2015; 34: 2937– 2952 [CrossRef] [PubMed]
    [Google Scholar]
  7. 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 [CrossRef] [PubMed]
    [Google Scholar]
  8. Sanchez V, Greis KD, Sztul E, Britt WJ. Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly. J Virol 2000; 74: 975– 986 [CrossRef] [PubMed]
    [Google Scholar]
  9. Das S, Vasanji A, Pellett PE. Three-dimensional structure of the human cytomegalovirus cytoplasmic virion assembly complex includes a reoriented secretory apparatus. J Virol 2007; 81: 11861– 11869 [CrossRef] [PubMed]
    [Google Scholar]
  10. Schauflinger M, Fischer D, Schreiber A, Chevillotte M, Walther P et al. The tegument protein UL71 of human cytomegalovirus is involved in late envelopment and affects multivesicular bodies. J Virol 2011; 85: 3821– 3832 [CrossRef] [PubMed]
    [Google Scholar]
  11. Schauflinger M, Villinger C, Mertens T, Walther P, von Einem J. Analysis of human cytomegalovirus secondary envelopment by advanced electron microscopy. Cell Microbiol 2013; 15: 305– 314 [CrossRef] [PubMed]
    [Google Scholar]
  12. Das S, Ortiz DA, Gurczynski SJ, Khan F, Pellett PE. Identification of human cytomegalovirus genes important for biogenesis of the cytoplasmic virion assembly complex. J Virol 2014; 88: 9086– 9099 [CrossRef] [PubMed]
    [Google Scholar]
  13. Silva MC, Yu QC, Enquist L, Shenk T. Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids. J Virol 2003; 77: 10594– 10605 [CrossRef] [PubMed]
    [Google Scholar]
  14. Phillips SL, Bresnahan WA. The human cytomegalovirus (HCMV) tegument protein UL94 is essential for secondary envelopment of HCMV virions. J Virol 2012; 86: 2523– 2532 [CrossRef] [PubMed]
    [Google Scholar]
  15. Kalejta RF. Tegument proteins of human cytomegalovirus. Microbiol Mol Biol Rev 2008; 72: 249– 265 [CrossRef] [PubMed]
    [Google Scholar]
  16. Prichard MN. Function of human cytomegalovirus UL97 kinase in viral infection and its inhibition by maribavir. Rev Med Virol 2009; 19: 215– 229 [CrossRef] [PubMed]
    [Google Scholar]
  17. Lee CP, Chen MR. Escape of herpesviruses from the nucleus. Rev Med Virol 2010; 20: 214– 230 [CrossRef] [PubMed]
    [Google Scholar]
  18. Hume AJ, Finkel JS, Kamil JP, Coen DM, Culbertson MR et al. Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase function. Science 2008; 320: 797– 799 [CrossRef] [PubMed]
    [Google Scholar]
  19. Webel R, Milbradt J, Auerochs S, Schregel V, Held C et al. Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation. J Gen Virol 2011; 92: 638– 649 [CrossRef] [PubMed]
    [Google Scholar]
  20. Webel R, Hakki M, Prichard MN, Rawlinson WD, Marschall M et al. Differential properties of cytomegalovirus pUL97 kinase isoforms affect viral replication and maribavir susceptibility. J Virol 2014; 88: 4776– 4785 [CrossRef] [PubMed]
    [Google Scholar]
  21. 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 [CrossRef] [PubMed]
    [Google Scholar]
  22. 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 [CrossRef] [PubMed]
    [Google Scholar]
  23. Wolf DG, Courcelle CT, Prichard MN, Mocarski ES. Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation. Proc Natl Acad Sci USA 2001; 98: 1895– 1900 [CrossRef] [PubMed]
    [Google Scholar]
  24. Marschall M, Feichtinger S, Milbradt J. Regulatory roles of protein kinases in cytomegalovirus replication. Adv Virus Res 2011; 80: 69– 101 [CrossRef] [PubMed]
    [Google Scholar]
  25. Milbradt J, Webel R, Auerochs S, Sticht H, Marschall M. Novel mode of phosphorylation-triggered reorganization of the nuclear lamina during nuclear egress of human cytomegalovirus. J Biol Chem 2010; 285: 13979– 13989 [CrossRef] [PubMed]
    [Google Scholar]
  26. Prichard MN, Gao N, Jairath S, Mulamba G, Krosky P et al. A recombinant human cytomegalovirus with a large deletion in UL97 has a severe replication deficiency. J Virol 1999; 73: 5663– 5670 [PubMed]
    [Google Scholar]
  27. Marschall M, Stein-Gerlach M, Freitag M, Kupfer R, van den Bogaard M et al. Direct targeting of human cytomegalovirus protein kinase pUL97 by kinase inhibitors is a novel principle for antiviral therapy. J Gen Virol 2002; 83: 1013– 1023 [CrossRef] [PubMed]
    [Google Scholar]
  28. Herget T, Freitag M, Morbitzer M, Kupfer R, Stamminger T et al. Novel chemical class of pUL97 protein kinase-specific inhibitors with strong anticytomegaloviral activity. Antimicrob Agents Chemother 2004; 48: 4154– 4162 [CrossRef] [PubMed]
    [Google Scholar]
  29. Hutterer C, Hamilton S, Steingruber M, Zeitträger I, Bahsi H et al. The chemical class of quinazoline compounds provides a core structure for the design of anticytomegaloviral kinase inhibitors. Antiviral Res 2016; 134: 130– 143 [CrossRef] [PubMed]
    [Google Scholar]
  30. Schleiss M, Eickhoff J, Auerochs S, Leis M, Abele S et al. Protein kinase inhibitors of the quinazoline class exert anti-cytomegaloviral activity in vitro and in vivo. Antiviral Res 2008; 79: 49– 61 [CrossRef] [PubMed]
    [Google Scholar]
  31. Irmiere A, Gibson W. Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus. Virology 1983; 130: 118– 133 [CrossRef] [PubMed]
    [Google Scholar]
  32. Varnum SM, Streblow DN, Monroe ME, Smith P, Auberry KJ et al. Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome. J Virol 2004; 78: 10960– 10966 [CrossRef] [PubMed]
    [Google Scholar]
  33. Reyda S, Tenzer S, Navarro P, Gebauer W, Saur M et al. The tegument protein pp65 of human cytomegalovirus acts as an optional scaffold protein that optimizes protein uploading into viral particles. J Virol 2014; 88: 9633– 9646 [CrossRef] [PubMed]
    [Google Scholar]
  34. Schmolke S, Kern HF, Drescher P, Jahn G, Plachter B. The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture. J Virol 1995; 69: 5959– 5968 [PubMed]
    [Google Scholar]
  35. Chevillotte M, Landwehr S, Linta L, Frascaroli G, Lüske A et al. Major tegument protein pp65 of human cytomegalovirus is required for the incorporation of pUL69 and pUL97 into the virus particle and for viral growth in macrophages. J Virol 2009; 83: 2480– 2490 [CrossRef] [PubMed]
    [Google Scholar]
  36. Gilbert MJ, Riddell SR, Plachter B, Greenberg PD. Cytomegalovirus selectively blocks antigen processing and presentation of its immediate-early gene product. Nature 1996; 383: 720– 722 [CrossRef] [PubMed]
    [Google Scholar]
  37. Odeberg J, Plachter B, Brandén L, Söderberg-Nauclér C. Human cytomegalovirus protein pp65 mediates accumulation of HLA-DR in lysosomes and destruction of the HLA-DR alpha-chain. Blood 2003; 101: 4870– 4877 [CrossRef] [PubMed]
    [Google Scholar]
  38. Arnon TI, Achdout H, Levi O, Markel G, Saleh N et al. Inhibition of the NKp30 activating receptor by pp65 of human cytomegalovirus. Nat Immunol 2005; 6: 515– 523 [CrossRef] [PubMed]
    [Google Scholar]
  39. Li T, Chen J, Cristea IM. Human cytomegalovirus tegument protein pUL83 inhibits IFI16-mediated DNA sensing for immune evasion. Cell Host Microbe 2013; 14: 591– 599 [CrossRef] [PubMed]
    [Google Scholar]
  40. Cristea IM, Moorman NJ, Terhune SS, Cuevas CD, O'Keefe ES et al. Human cytomegalovirus pUL83 stimulates activity of the viral immediate-early promoter through its interaction with the cellular IFI16 protein. J Virol 2010; 84: 7803– 7814 [CrossRef] [PubMed]
    [Google Scholar]
  41. Biolatti M, dell'oste V, Pautasso S, von Einem J, Marschall M et al. Regulatory interaction between the cellular restriction factor IFI16 and viral pp65 (pUL83) modulates viral gene expression and IFI16 protein stability. J Virol 2016; 90: 8238– 8250 [CrossRef] [PubMed]
    [Google Scholar]
  42. Kamil JP, Coen DM. Human cytomegalovirus protein kinase UL97 forms a complex with the tegument phosphoprotein pp65. J Virol 2007; 81: 10659– 10668 [CrossRef] [PubMed]
    [Google Scholar]
  43. Gao Y, Colletti K, Pari GS. Identification of human cytomegalovirus UL84 virus- and cell-encoded binding partners by using proteomics analysis. J Virol 2008; 82: 96– 104 [CrossRef] [PubMed]
    [Google Scholar]
  44. Cui Z, Zhang K, Zhang Z, Liu Y, Zhou Y et al. Visualization of the dynamic multimerization of human Cytomegalovirus pp65 in punctuate nuclear foci. Virology 2009; 392: 169– 177 [CrossRef] [PubMed]
    [Google Scholar]
  45. Becke S, Fabre-Mersseman V, Aue S, Auerochs S, Sedmak T et al. Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells. J Gen Virol 2010; 91: 2531– 2541 [CrossRef] [PubMed]
    [Google Scholar]
  46. Oberstein A, Perlman DH, Shenk T, Terry LJ. Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome. Proteomics 2015; 15: 2006– 2022 [CrossRef] [PubMed]
    [Google Scholar]
  47. Büscher N, Paulus C, Nevels M, Tenzer S, Plachter B. The proteome of human cytomegalovirus virions and dense bodies is conserved across different strains. Med Microbiol Immunol 2015; 204: 285– 293 [CrossRef] [PubMed]
    [Google Scholar]
  48. Prichard MN, Britt WJ, Daily SL, Hartline CB, Kern ER. Human cytomegalovirus UL97 Kinase is required for the normal intranuclear distribution of pp65 and virion morphogenesis. J Virol 2005; 79: 15494– 15502 [CrossRef] [PubMed]
    [Google Scholar]
  49. Frankenberg N, Lischka P, Pepperl-Klindworth S, Stamminger T, Plachter B. Nucleocytoplasmic shuttling and CRM1-dependent MHC class I peptide presentation of human cytomegalovirus pp65. Med Microbiol Immunol 2012; 201: 567– 579 [CrossRef] [PubMed]
    [Google Scholar]
  50. Marschall M, Freitag M, Suchy P, Romaker D, Kupfer R et al. The protein kinase pUL97 of human cytomegalovirus interacts with and phosphorylates the DNA polymerase processivity factor pUL44. Virology 2003; 311: 60– 71 [CrossRef] [PubMed]
    [Google Scholar]
  51. Penfold ME, Mocarski ES. Formation of cytomegalovirus DNA replication compartments defined by localization of viral proteins and DNA synthesis. Virology 1997; 239: 46– 61 [CrossRef] [PubMed]
    [Google Scholar]
  52. Graf L, Webel R, Wagner S, Hamilton ST, Rawlinson WD et al. The cyclin-dependent kinase ortholog pUL97 of human cytomegalovirus interacts with cyclins. Viruses 2013; 5: 3213– 3230 [CrossRef] [PubMed]
    [Google Scholar]
  53. Steingruber M, Socher E, Hutterer C, Webel R, Bergbrede T et al. The interaction between cyclin B1 and cytomegalovirus protein kinase pUL97 is determined by an active kinase domain. Viruses 2015; 7: 4582– 4601 [CrossRef] [PubMed]
    [Google Scholar]
  54. Steingruber M, Kraut A, Socher E, Sticht H, Reichel A et al. Proteomic interaction patterns between human cyclins, the cyclin-dependent kinase ortholog pUL97 and additional cytomegalovirus proteins. Viruses 2016; 8: 219 [CrossRef] [PubMed]
    [Google Scholar]
  55. Lowe ED, Tews I, Cheng KY, Brown NR, Gul S et al. Specificity determinants of recruitment peptides bound to phospho-CDK2/cyclin A. Biochemistry 2002; 41: 15625– 15634 [PubMed] [Crossref]
    [Google Scholar]
  56. Distler U, Kuharev J, Navarro P, Levin Y, Schild H et al. Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics. Nat Methods 2014; 11: 167– 170 [CrossRef] [PubMed]
    [Google Scholar]
  57. Webel R, Solbak SM, Held C, Milbradt J, Groß A et al. Nuclear import of isoforms of the cytomegalovirus kinase pUL97 is mediated by differential activity of NLS1 and NLS2 both acting through classical importin-α binding. J Gen Virol 2012; 93: 1756– 1768 [CrossRef] [PubMed]
    [Google Scholar]
  58. Goldberg MD, Honigman A, Weinstein J, Chou S, Taraboulos A et al. Human cytomegalovirus UL97 kinase and nonkinase functions mediate viral cytoplasmic secondary envelopment. J Virol 2011; 85: 3375– 3384 [CrossRef] [PubMed]
    [Google Scholar]
  59. Chou S, Ercolani RJ, Marousek G, Bowlin TL. Cytomegalovirus UL97 kinase catalytic domain mutations that confer multidrug resistance. Antimicrob Agents Chemother 2013; 57: 3375– 3379 [CrossRef] [PubMed]
    [Google Scholar]
  60. Taube R, Lin X, Irwin D, Fujinaga K, Peterlin BM. Interaction between P-TEFb and the C-terminal domain of RNA polymerase II activates transcriptional elongation from sites upstream or downstream of target genes. Mol Cell Biol 2002; 22: 321– 331 [CrossRef] [PubMed]
    [Google Scholar]
  61. Mar EC, Patel PC, Huang ES. Human cytomegalovirus-associated DNA polymerase and protein kinase activities. J Gen Virol 1981; 57: 149– 156 [CrossRef] [PubMed]
    [Google Scholar]
  62. Roby C, Gibson W. Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus. J Virol 1986; 59: 714– 727 [PubMed]
    [Google Scholar]
  63. van Zeijl M, Fairhurst J, Baum EZ, Sun L, Jones TR. The human cytomegalovirus UL97 protein is phosphorylated and a component of virions. Virology 1997; 231: 72– 80 [CrossRef] [PubMed]
    [Google Scholar]
  64. Michelson S, Turowski P, Picard L, Goris J, Landini MP et al. Human cytomegalovirus carries serine/threonine protein phosphatases PP1 and a host-cell derived PP2A. J Virol 1996; 70: 1415– 1423 [PubMed]
    [Google Scholar]
  65. Rieder FJ, Kastner MT, Hartl M, Puchinger MG, Schneider M et al. Human cytomegalovirus phosphoproteins are hypophosphorylated and intrinsically disordered. J Gen Virol 2017; 98: 471– 485 [CrossRef] [PubMed]
    [Google Scholar]
  66. Buerger I, Reefschlaeger J, Bender W, Eckenberg P, Popp A et al. A novel nonnucleoside inhibitor specifically targets cytomegalovirus DNA maturation via the UL89 and UL56 gene products. J Virol 2001; 75: 9077– 9086 [CrossRef] [PubMed]
    [Google Scholar]
  67. Gariano GR, dell'oste V, Bronzini M, Gatti D, Luganini A et al. The intracellular DNA sensor IFI16 gene acts as restriction factor for human cytomegalovirus replication. PLoS Pathog 2012; 8: e1002498 [CrossRef] [PubMed]
    [Google Scholar]
  68. Sinzger C, Schmidt K, Knapp J, Kahl M, Beck R et al. Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome. J Gen Virol 1999; 80: 2867– 2877 [CrossRef] [PubMed]
    [Google Scholar]
  69. Sinzger C, Hahn G, Digel M, Katona R, Sampaio KL et al. Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E. J Gen Virol 2008; 89: 359– 368 [CrossRef] [PubMed]
    [Google Scholar]
  70. Yu D, Smith GA, Enquist LW, Shenk T. Construction of a self-excisable bacterial artificial chromosome containing the human cytomegalovirus genome and mutagenesis of the diploid TRL/IRL13 gene. J Virol 2002; 76: 2316– 2328 [CrossRef] [PubMed]
    [Google Scholar]
  71. Marchini A, Liu H, Zhu H. Human cytomegalovirus with IE-2 (UL122) deleted fails to express early lytic genes. J Virol 2001; 75: 1870– 1878 [CrossRef] [PubMed]
    [Google Scholar]
  72. Borst EM, Hahn G, Koszinowski UH, Messerle M. Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants. J Virol 1999; 73: 8320– 8329 [PubMed]
    [Google Scholar]
  73. Falk CS, Mach M, Schendel DJ, Weiss EH, Hilgert I et al. NK cell activity during human cytomegalovirus infection is dominated by US2-11-mediated HLA class I down-regulation. J Immunol 2002; 169: 3257– 3266 [CrossRef] [PubMed]
    [Google Scholar]
  74. Rowe WP, Hartley JW, Waterman S, Turner HC, Huebner RJ. Cytopathogenic agent resembling human salivary gland virus recovered from tissue cultures of human adenoids. Proc Soc Exp Biol Med 1956; 92: 418– 424 [PubMed] [Crossref]
    [Google Scholar]
  75. 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 [CrossRef] [PubMed]
    [Google Scholar]
  76. Dinkel H, van Roey K, Michael S, Kumar M, Uyar B et al. ELM 2016–data update and new functionality of the eukaryotic linear motif resource. Nucleic Acids Res 2016; 44: D294– D300 [CrossRef] [PubMed]
    [Google Scholar]
  77. Linding R, Russell RB, Neduva V, Gibson TJ. GlobPlot: exploring protein sequences for globularity and disorder. Nucleic Acids Res 2003; 31: 3701– 3708 [CrossRef] [PubMed]
    [Google Scholar]
  78. Dosztányi Z, Csizmok V, Tompa P, Simon I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 2005; 21: 3433– 3434 [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000939
Loading
/content/journal/jgv/10.1099/jgv.0.000939
Loading

Data & Media loading...

Supplementary File 1

PDF

Most Cited This Month

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