1887

Abstract

The nucleocytoplasmic export of cytomegaloviral capsids is regulated by formation of a multi-component nuclear egress complex (NEC), essentially based on viral proteins pUL50 and pUL53. In this study, the generation of recombinant human cytomegaloviruses, expressing tagged versions of pUL50 and pUL53, enabled the investigation of NEC formation in infected primary fibroblasts. For these recombinant viruses, a wild-type-like mode of pUL50–pUL53 interaction and recruitment of both proteins to the nuclear envelope could be demonstrated. Importantly, pUL50 was translocated from an initial cytoplasmic distribution to the nuclear rim, whereas pUL53 accumulated in the nucleus before attaining overall rim colocalization with pUL50. Specified experimental settings illustrated that pUL50 and pUL53 were subject to different pathways of intracellular trafficking. Importantly, a novel nuclear localization signal (NLS) could be identified and functionally verified for pUL53 (amino acids 18–27), whereas no NLS was present in pUL50. Analysis of amino acid replacement mutants further illustrated the differential modes of nuclear import of the two essential viral egress proteins. Taken together, our findings suggest a combination of classical nuclear import (pUL53) and interaction-mediated recruitment (pUL50) as the driving forces for core NEC formation and viral nuclear egress.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.052571-0
2013-09-01
2020-11-24
Loading full text...

Full text loading...

/deliver/fulltext/jgv/94/9/2056.html?itemId=/content/journal/jgv/10.1099/vir.0.052571-0&mimeType=html&fmt=ahah

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 [CrossRef][PubMed]
    [Google Scholar]
  2. Antonin W., Ungricht R., Kutay U. 2011; Traversing the NPC along the pore membrane: targeting of membrane proteins to the INM. Nucleus 2:87–91 [CrossRef][PubMed]
    [Google Scholar]
  3. Beck M., Förster F., Ecke M., Plitzko J. M., Melchior F., Gerisch G., Baumeister W., Medalia O. 2004; Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 306:1387–1390 [CrossRef][PubMed]
    [Google Scholar]
  4. Borgese N., Fasana E. 2011; Targeting pathways of C-tail-anchored proteins. Biochim Biophys Acta 1808:937–946 [CrossRef][PubMed]
    [Google Scholar]
  5. Borgese N., Colombo S., Pedrazzini E. 2003; The tale of tail-anchored proteins: coming from the cytosol and looking for a membrane. J Cell Biol 161:1013–1019 [CrossRef][PubMed]
    [Google Scholar]
  6. Borst E., Messerle M. 2000; Development of a cytomegalovirus vector for somatic gene therapy. Bone Marrow Transplant 25:Suppl 2S80–S82 [CrossRef][PubMed]
    [Google Scholar]
  7. Borst E. M., Benkartek C., Messerle M. 2007; Use of bacterial artificial chromosomes in generating targeted mutations in human and mouse cytomegaloviruses. Curr Protoc Immunol 77:10.32.1–10.32.30[PubMed]
    [Google Scholar]
  8. 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 [CrossRef][PubMed]
    [Google Scholar]
  9. Camozzi D., Pignatelli S., Valvo C., Lattanzi G., Capanni C., Dal Monte P., Landini M. P. 2008; Remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pUL50 and pUL53. J Gen Virol 89:731–740 [CrossRef][PubMed]
    [Google Scholar]
  10. Fuchs W., Klupp B. G., Granzow H., Osterrieder N., Mettenleiter T. C. 2002; The interacting UL31 and UL34 gene products of pseudorabies virus are involved in egress from the host-cell nucleus and represent components of primary enveloped but not mature virions. J Virol 76:364–378 [CrossRef][PubMed]
    [Google Scholar]
  11. Furukawa K., Panté N., Aebi U., Gerace L. 1995; Cloning of a cDNA for lamina-associated polypeptide 2 (LAP2) and identification of regions that specify targeting to the nuclear envelope. EMBO J 14:1626–1636[PubMed]
    [Google Scholar]
  12. Furukawa K., Fritze C. E., Gerace L. 1998; The major nuclear envelope targeting domain of LAP2 coincides with its lamin binding region but is distinct from its chromatin interaction domain. J Biol Chem 273:4213–4219 [CrossRef][PubMed]
    [Google Scholar]
  13. Gonnella R., Farina A., Santarelli R., Raffa S., Feederle R., Bei R., Granato M., Modesti A., Frati L.& other authors ( 2005; Characterization and intracellular localization of the Epstein-Barr virus protein BFLF2: interactions with BFRF1 and with the nuclear lamina. J Virol 79:3713–3727 [CrossRef][PubMed]
    [Google Scholar]
  14. Herget T., Freitag M., Morbitzer M., Kupfer R., Stamminger T., Marschall M. 2004; Novel chemical class of pUL97 protein kinase-specific inhibitors with strong anticytomegaloviral activity. Antimicrob Agents Chemother 48:4154–4162 [CrossRef][PubMed]
    [Google Scholar]
  15. Holmer L., Worman H. J. 2001; Inner nuclear membrane proteins: functions and targeting. Cell Mol Life Sci 58:1741–1747 [CrossRef][PubMed]
    [Google Scholar]
  16. Johnson D. C., Baines J. D. 2011; Herpesviruses remodel host membranes for virus egress. Nat Rev Microbiol 9:382–394 [CrossRef][PubMed]
    [Google Scholar]
  17. Klupp B. G., Granzow H., Mettenleiter T. C. 2000; Primary envelopment of pseudorabies virus at the nuclear membrane requires the UL34 gene product. J Virol 74:10063–10073 [CrossRef][PubMed]
    [Google Scholar]
  18. Klupp B. G., Granzow H., Fuchs W., Keil G. M., Finke S., Mettenleiter T. C. 2007; Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins. Proc Natl Acad Sci U S A 104:7241–7246 [CrossRef][PubMed]
    [Google Scholar]
  19. Kosugi S., Hasebe M., Tomita M., Yanagawa H. 2009; Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc Natl Acad Sci U S A 106:10171–10176 [CrossRef][PubMed]
    [Google Scholar]
  20. Lake C. M., Hutt-Fletcher L. M. 2004; The Epstein-Barr virus BFRF1 and BFLF2 proteins interact and coexpression alters their cellular localization. Virology 320:99–106 [CrossRef][PubMed]
    [Google Scholar]
  21. Lee S. K. 1999; Four consecutive arginine residues at positions 836-839 of EBV gp110 determine intracellular localization of gp110. Virology 264:350–358 [CrossRef][PubMed]
    [Google Scholar]
  22. Lee C. P., Chen M. R. 2010; Escape of herpesviruses from the nucleus. Rev Med Virol 20:214–230 [CrossRef][PubMed]
    [Google Scholar]
  23. Lischka P., Sorg G., Kann M., Winkler M., Stamminger T. 2003; A nonconventional nuclear localization signal within the UL84 protein of human cytomegalovirus mediates nuclear import via the importin alpha/beta pathway. J Virol 77:3734–3748 [CrossRef][PubMed]
    [Google Scholar]
  24. Lötzerich M., Ruzsics Z., Koszinowski U. H. 2006; Functional domains of murine cytomegalovirus nuclear egress protein M53/p38. J Virol 80:73–84 [CrossRef][PubMed]
    [Google Scholar]
  25. Lusk C. P., Blobel G., King M. C. 2007; Highway to the inner nuclear membrane: rules for the road. Nat Rev Mol Cell Biol 8:414–420 [CrossRef][PubMed]
    [Google Scholar]
  26. Marschall M., Helten A., Hechtfischer A., Zach A., Banaschewski C., Hell W., Meier-Ewert H. 1999; The ORF, regulated synthesis, and persistence-specific variation of influenza C viral NS1 protein. Virology 253:208–218 [CrossRef][PubMed]
    [Google Scholar]
  27. Marschall M., Freitag M., Weiler S., Sorg G., Stamminger T. 2000; Recombinant green fluorescent protein-expressing human cytomegalovirus as a tool for screening antiviral agents. Antimicrob Agents Chemother 44:1588–1597 [CrossRef][PubMed]
    [Google Scholar]
  28. 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 [CrossRef][PubMed]
    [Google Scholar]
  29. Marschall M., Feichtinger S., Milbradt J. 2011; Regulatory roles of protein kinases in cytomegalovirus replication. Adv Virus Res 80:69–101 [CrossRef][PubMed]
    [Google Scholar]
  30. Mettenleiter T. C., Klupp B. G., Granzow H. 2009; Herpesvirus assembly: an update. Virus Res 143:222–234 [CrossRef][PubMed]
    [Google Scholar]
  31. Meyer G. A., Radsak K. D. 2000; Identification of a novel signal sequence that targets transmembrane proteins to the nuclear envelope inner membrane. J Biol Chem 275:3857–3866 [CrossRef][PubMed]
    [Google Scholar]
  32. Meyer G., Gicklhorn D., Strive T., Radsak K., Eickmann M. 2002; A three-residue signal confers localization of a reporter protein in the inner nuclear membrane. Biochem Biophys Res Commun 291:966–971 [CrossRef][PubMed]
    [Google Scholar]
  33. 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 [CrossRef][PubMed]
    [Google Scholar]
  34. 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 [CrossRef][PubMed]
    [Google Scholar]
  35. 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 [CrossRef][PubMed]
    [Google Scholar]
  36. 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 [CrossRef][PubMed]
    [Google Scholar]
  37. Mocarski E. S., Shenk T., Pass R. F. 2007; Cytomegaloviruses. In Fields Virology pp. 2701–2772 Edited by Knipe D. M., Howley P. M. Philadelphia: Lippincott Williams & Wilkes;
    [Google Scholar]
  38. Montpetit B., Weis K. 2012; Cell biology. An alternative route for nuclear mRNP export by membrane budding. Science 336:809–810 [CrossRef][PubMed]
    [Google Scholar]
  39. 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 [CrossRef][PubMed]
    [Google Scholar]
  40. Nguyen Ba A. N., Pogoutse A., Provart N., Moses A. M. 2009; NLStradamus: a simple Hidden Markov Model for nuclear localization signal prediction. BMC Bioinformatics 10:202 [CrossRef][PubMed]
    [Google Scholar]
  41. Ohba T., Schirmer E. C., Nishimoto T., Gerace L. 2004; Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore. J Cell Biol 167:1051–1062 [CrossRef][PubMed]
    [Google Scholar]
  42. Ostlund C., Ellenberg J., Hallberg E., Lippincott-Schwartz J., Worman H. J. 1999; Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy protein. J Cell Sci 112:1709–1719[PubMed]
    [Google Scholar]
  43. Ott M., Tascher G., Hassdenteufel S., Zimmermann R., Haas J., Bailer S. M. 2011; Functional characterization of the essential tail anchor of the herpes simplex virus type 1 nuclear egress protein pUL34. J Gen Virol 92:2734–2745 [CrossRef][PubMed]
    [Google Scholar]
  44. Paßvogel L., Trübe P., Schuster F., Klupp B. G., Mettenleiter T. C. 2013; Mapping of sequences in Pseudorabies virus pUL34 that are required for formation and function of the nuclear egress complex. J Virol 87:4475–4485 [CrossRef][PubMed]
    [Google Scholar]
  45. Reynolds A. E., Ryckman B. J., Baines J. D., Zhou Y., Liang L., Roller R. J. 2001; U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids. J Virol 75:8803–8817 [CrossRef][PubMed]
    [Google Scholar]
  46. 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 [CrossRef][PubMed]
    [Google Scholar]
  47. Shikano S., Li M. 2003; Membrane receptor trafficking: evidence of proximal and distal zones conferred by two independent endoplasmic reticulum localization signals. Proc Natl Acad Sci U S A 100:5783–5788 [CrossRef][PubMed]
    [Google Scholar]
  48. Smith S., Blobel G. 1993; The first membrane spanning region of the lamin B receptor is sufficient for sorting to the inner nuclear membrane. J Cell Biol 120:631–637 [CrossRef][PubMed]
    [Google Scholar]
  49. Sorg G., Stamminger T. 1999; Mapping of nuclear localization signals by simultaneous fusion to green fluorescent protein and to beta-galactosidase. Biotechniques 26:858–862[PubMed]
    [Google Scholar]
  50. Soullam B., Worman H. J. 1995; Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane. J Cell Biol 130:15–27 [CrossRef][PubMed]
    [Google Scholar]
  51. Tischer B. K., von Einem J., Kaufer B., Osterrieder N. 2006; Two-step Red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli . Biotechniques 40:191–197 [CrossRef][PubMed]
    [Google Scholar]
  52. Tischer B. K., Smith G. A., Osterrieder N. 2010; En passant mutagenesis: a two step markerless Red recombination system. Methods Mol Biol 634:421–430 [CrossRef][PubMed]
    [Google Scholar]
  53. Turgay Y., Ungricht R., Rothballer A., Kiss A., Csucs G., Horvath P., Kutay U. 2010; A classical NLS and the SUN domain contribute to the targeting of SUN2 to the inner nuclear membrane. EMBO J 29:2262–2275 [CrossRef][PubMed]
    [Google Scholar]
  54. Webel R., Milbradt J., Auerochs S., Schregel V., Held C., Nöbauer K., Razzazi-Fazeli E., Jardin C., Wittenberg T.& other authors ( 2011; Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation. J Gen Virol 92:638–649 [CrossRef][PubMed]
    [Google Scholar]
  55. Webel R., Solbak S. M. Ø., Held C., Milbradt J., Groß A., Eichler J., Wittenberg T., Jardin C., Sticht H.& other authors ( 2012; 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 93:1756–1768 [CrossRef][PubMed]
    [Google Scholar]
  56. Wu W., Lin F., Worman H. J. 2002; Intracellular trafficking of MAN1, an integral protein of the nuclear envelope inner membrane. J Cell Sci 115:1361–1371[PubMed]
    [Google Scholar]
  57. Yamauchi Y., Shiba C., Goshima F., Nawa A., Murata T., Nishiyama Y. 2001; Herpes simplex virus type 2 UL34 protein requires UL31 protein for its relocation to the internal nuclear membrane in transfected cells. J Gen Virol 82:1423–1428[PubMed]
    [Google Scholar]
  58. Zhu H. Y., Yamada H., Jiang Y. M., Yamada M., Nishiyama Y. 1999; Intracellular localization of the UL31 protein of herpes simplex virus type 2. Arch Virol 144:1923–1935 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.052571-0
Loading
/content/journal/jgv/10.1099/vir.0.052571-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited this month Most Cited RSS feed

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