1887

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP–GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present – indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP–GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP–His polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP–GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP–GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP–MCP interaction.

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2014-08-01
2020-01-24
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References

  1. Adamson W. E., McNab D., Preston V. G., Rixon F. J.. ( 2006; ). Mutational analysis of the herpes simplex virus triplex protein VP19C. . J Virol 80:, 1537–1548. [CrossRef] [PubMed]
    [Google Scholar]
  2. Antinone S. E., Shubeita G. T., Coller K. E., Lee J. I., Haverlock-Moyns S., Gross S. P., Smith G. A.. ( 2006; ). The herpesvirus capsid surface protein, VP26, and the majority of the tegument proteins are dispensable for capsid transport toward the nucleus. . J Virol 80:, 5494–5498. [CrossRef] [PubMed]
    [Google Scholar]
  3. Apcarian A., Cunningham A. L., Diefenbach R. J.. ( 2010; ). Identification of binding domains in the herpes simplex virus type 1 small capsid protein pUL35 (VP26). . J Gen Virol 91:, 2659–2663. [CrossRef] [PubMed]
    [Google Scholar]
  4. Borst E. M., Mathys S., Wagner M., Muranyi W., Messerle M.. ( 2001; ). Genetic evidence of an essential role for cytomegalovirus small capsid protein in viral growth. . J Virol 75:, 1450–1458. [CrossRef] [PubMed]
    [Google Scholar]
  5. Bosse J. B., Bauerfeind R., Popilka L., Marcinowski L., Taeglich M., Jung C., Striebinger H., von Einem J., Gaul U.. & other authors ( 2012; ). A beta-herpesvirus with fluorescent capsids to study transport in living cells. . PLoS ONE 7:, e40585. [CrossRef] [PubMed]
    [Google Scholar]
  6. Brown J. C., Newcomb W. W.. ( 2011; ). Herpesvirus capsid assembly: insights from structural analysis. . Curr Opin Virol 1:, 142–149. [CrossRef] [PubMed]
    [Google Scholar]
  7. Cardone G., Heymann J. B., Cheng N., Trus B. L., Steven A. C.. ( 2012; ). Procapsid assembly, maturation, nuclear exit: dynamic steps in the production of infectious herpesvirions. . Adv Exp Med Biol 726:, 423–439. [CrossRef] [PubMed]
    [Google Scholar]
  8. Casaday R. J., Bailey J. R., Kalb S. R., Brignole E. J., Loveland A. N., Cotter R. J., Gibson W.. ( 2004; ). Assembly protein precursor (pUL80.5 homolog) of simian cytomegalovirus is phosphorylated at a glycogen synthase kinase 3 site and its downstream “priming” site: phosphorylation affects interactions of protein with itself and with major capsid protein. . J Virol 78:, 13501–13511. [CrossRef] [PubMed]
    [Google Scholar]
  9. Chackerian B.. ( 2007; ). Virus-like particles: flexible platforms for vaccine development. . Expert Rev Vaccines 6:, 381–390. [CrossRef] [PubMed]
    [Google Scholar]
  10. Chaudhuri V., Sommer M., Rajamani J., Zerboni L., Arvin A. M.. ( 2008; ). Functions of varicella-zoster virus ORF23 capsid protein in viral replication and the pathogenesis of skin infection. . J Virol 82:, 10231–10246. [CrossRef] [PubMed]
    [Google Scholar]
  11. Chen D. H., Jakana J., McNab D., Mitchell J., Zhou Z. H., Dougherty M., Chiu W., Rixon F. J.. ( 2001; ). The pattern of tegument–capsid interaction in the herpes simplex virus type 1 virion is not influenced by the small hexon-associated protein VP26. . J Virol 75:, 11863–11867. [CrossRef] [PubMed]
    [Google Scholar]
  12. Dai X., Yu X., Gong H., Jiang X., Abenes G., Liu H., Shivakoti S., Britt W. J., Zhu H.. & other authors ( 2013; ). The smallest capsid protein mediates binding of the essential tegument protein pp150 to stabilize DNA-containing capsids in human cytomegalovirus. . PLoS Pathog 9:, e1003525. [CrossRef] [PubMed]
    [Google Scholar]
  13. Desai P., Person S.. ( 1998; ). Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid. . J Virol 72:, 7563–7568.[PubMed]
    [Google Scholar]
  14. Desai P., DeLuca N. A., Person S.. ( 1998; ). Herpes simplex virus type 1 VP26 is not essential for replication in cell culture but influences production of infectious virus in the nervous system of infected mice. . Virology 247:, 115–124. [CrossRef] [PubMed]
    [Google Scholar]
  15. Desai P., Akpa J. C., Person S.. ( 2003; ). Residues of VP26 of herpes simplex virus type 1 that are required for its interaction with capsids. . J Virol 77:, 391–404. [CrossRef] [PubMed]
    [Google Scholar]
  16. Desai P. J., Pryce E. N., Henson B. W., Luitweiler E. M., Cothran J.. ( 2012; ). Reconstitution of the Kaposi’s sarcoma-associated herpesvirus nuclear egress complex and formation of nuclear membrane vesicles by coexpression of ORF67 and ORF69 gene products. . J Virol 86:, 594–598. [CrossRef] [PubMed]
    [Google Scholar]
  17. Gao S. J., Deng J. H., Zhou F. C.. ( 2003; ). Productive lytic replication of a recombinant Kaposi’s sarcoma-associated herpesvirus in efficient primary infection of primary human endothelial cells. . J Virol 77:, 9738–9749. [CrossRef] [PubMed]
    [Google Scholar]
  18. Henson B. W., Perkins E. M., Cothran J. E., Desai P.. ( 2009; ). Self-assembly of Epstein–Barr virus capsids. . J Virol 83:, 3877–3890. [CrossRef] [PubMed]
    [Google Scholar]
  19. Homa F. L., Brown J. C.. ( 1997; ). Capsid assembly and DNA packaging in herpes simplex virus. . Rev Med Virol 7:, 107–122. [CrossRef] [PubMed]
    [Google Scholar]
  20. Huang E., Perkins E. M., Desai P.. ( 2007; ). Structural features of the scaffold interaction domain at the N terminus of the major capsid protein (VP5) of herpes simplex virus type 1. . J Virol 81:, 9396–9407. [CrossRef] [PubMed]
    [Google Scholar]
  21. 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]
  22. Krautwald M., Maresch C., Klupp B. G., Fuchs W., Mettenleiter T. C.. ( 2008; ). Deletion or green fluorescent protein tagging of the pUL35 capsid component of pseudorabies virus impairs virus replication in cell culture and neuroinvasion in mice. . J Gen Virol 89:, 1346–1351. [CrossRef] [PubMed]
    [Google Scholar]
  23. Kreitler D., Capuano C. M., Henson B. W., Pryce E. N., Anacker D., McCaffery J. M., Desai P. J.. ( 2012; ). The assembly domain of the small capsid protein of Kaposi’s sarcoma-associated herpesvirus. . J Virol 86:, 11926–11930. [CrossRef] [PubMed]
    [Google Scholar]
  24. Lai L., Britt W. J.. ( 2003; ). The interaction between the major capsid protein and the smallest capsid protein of human cytomegalovirus is dependent on two linear sequences in the smallest capsid protein. . J Virol 77:, 2730–2735. [CrossRef] [PubMed]
    [Google Scholar]
  25. Lander G. C., Evilevitch A., Jeembaeva M., Potter C. S., Carragher B., Johnson J. E.. ( 2008; ). Bacteriophage lambda stabilization by auxiliary protein gpD: timing, location, and mechanism of attachment determined by cryo-EM. . Structure 16:, 1399–1406. [CrossRef] [PubMed]
    [Google Scholar]
  26. Lebrun M., Thelen N., Thiry M., Riva L., Ote I., Condé C., Vandevenne P., Di Valentin E., Bontems S., Sadzot-Delvaux C.. ( 2014; ). Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates. . Virology 454–455:, 311–327. [CrossRef] [PubMed]
    [Google Scholar]
  27. Lee J. H., Vittone V., Diefenbach E., Cunningham A. L., Diefenbach R. J.. ( 2008; ). Identification of structural protein–protein interactions of herpes simplex virus type 1. . Virology 378:, 347–354. [CrossRef] [PubMed]
    [Google Scholar]
  28. Li Q., Shivachandra S. B., Leppla S. H., Rao V. B.. ( 2006; ). Bacteriophage T4 capsid: a unique platform for efficient surface assembly of macromolecular complexes. . J Mol Biol 363:, 577–588. [CrossRef] [PubMed]
    [Google Scholar]
  29. Lo P., Yu X., Atanasov I., Chandran B., Zhou Z. H.. ( 2003; ). Three-dimensional localization of pORF65 in Kaposi’s sarcoma-associated herpesvirus capsid. . J Virol 77:, 4291–4297. [CrossRef] [PubMed]
    [Google Scholar]
  30. Nagel C. H., Döhner K., Binz A., Bauerfeind R., Sodeik B.. ( 2012; ). Improper tagging of the non-essential small capsid protein VP26 impairs nuclear capsid egress of herpes simplex virus. . PLoS ONE 7:, e44177. [CrossRef] [PubMed]
    [Google Scholar]
  31. Nealon K., Newcomb W. W., Pray T. R., Craik C. S., Brown J. C., Kedes D. H.. ( 2001; ). Lytic replication of Kaposi’s sarcoma-associated herpesvirus results in the formation of multiple capsid species: isolation and molecular characterization of A, B, and C capsids from a gammaherpesvirus. . J Virol 75:, 2866–2878. [CrossRef] [PubMed]
    [Google Scholar]
  32. Newcomb W. W., Homa F. L., Thomsen D. R., Booy F. P., Trus B. L., Steven A. C., Spencer J. V., Brown J. C.. ( 1996; ). Assembly of the herpes simplex virus capsid: characterization of intermediates observed during cell-free capsid formation. . J Mol Biol 263:, 432–446. [CrossRef] [PubMed]
    [Google Scholar]
  33. Newcomb W. W., Homa F. L., Thomsen D. R., Trus B. L., Cheng N., Steven A., Booy F., Brown J. C.. ( 1999; ). Assembly of the herpes simplex virus procapsid from purified components and identification of small complexes containing the major capsid and scaffolding proteins. . J Virol 73:, 4239–4250.[PubMed]
    [Google Scholar]
  34. Nicholson P., Addison C., Cross A. M., Kennard J., Preston V. G., Rixon F. J.. ( 1994; ). Localization of the herpes simplex virus type 1 major capsid protein VP5 to the cell nucleus requires the abundant scaffolding protein VP22a. . J Gen Virol 75:, 1091–1099. [CrossRef] [PubMed]
    [Google Scholar]
  35. Okoye M. E., Sexton G. L., Huang E., McCaffery J. M., Desai P.. ( 2006; ). Functional analysis of the triplex proteins (VP19C and VP23) of herpes simplex virus type 1. . J Virol 80:, 929–940. [CrossRef] [PubMed]
    [Google Scholar]
  36. Perkins E. M., McCaffery J. M.. ( 2007; ). Conventional and immunoelectron microscopy of mitochondria. . Methods Mol Biol 372:, 467–483. [CrossRef] [PubMed]
    [Google Scholar]
  37. Perkins E. M., Anacker D., Davis A., Sankar V., Ambinder R. F., Desai P.. ( 2008; ). Small capsid protein pORF65 is essential for assembly of Kaposi’s sarcoma-associated herpesvirus capsids. . J Virol 82:, 7201–7211. [CrossRef] [PubMed]
    [Google Scholar]
  38. Plafker S. M., Gibson W.. ( 1998; ). Cytomegalovirus assembly protein precursor and proteinase precursor contain two nuclear localization signals that mediate their own nuclear translocation and that of the major capsid protein. . J Virol 72:, 7722–7732.[PubMed]
    [Google Scholar]
  39. Qin L., Fokine A., O’Donnell E., Rao V. B., Rossmann M. G.. ( 2010; ). Structure of the small outer capsid protein, Soc: a clamp for stabilizing capsids of T4-like phages. . J Mol Biol 395:, 728–741. [CrossRef] [PubMed]
    [Google Scholar]
  40. Rixon F. J.. ( 1993; ). Structure and assembly of herpesviruses. . Semin Virol 4:, 135–144. [CrossRef]
    [Google Scholar]
  41. Rixon F. J., Addison C., McGregor A., Macnab S. J., Nicholson P., Preston V. G., Tatman J. D.. ( 1996; ). Multiple interactions control the intracellular localization of the herpes simplex virus type 1 capsid proteins. . J Gen Virol 77:, 2251–2260. [CrossRef] [PubMed]
    [Google Scholar]
  42. Rost B., Yachdav G., Liu J.. ( 2004; ). The PredictProtein server. . Nucleic Acids Res 32: (Web Server), W321–W326. [CrossRef] [PubMed]
    [Google Scholar]
  43. Sathish N., Yuan Y.. ( 2010; ). Functional characterization of Kaposi’s sarcoma-associated herpesvirus small capsid protein by bacterial artificial chromosome-based mutagenesis. . Virology 407:, 306–318. [CrossRef] [PubMed]
    [Google Scholar]
  44. Singh P., Nakatani E., Goodlett D. R., Catalano C. E.. ( 2013; ). A pseudo-atomic model for the capsid shell of bacteriophage lambda using chemical cross-linking/mass spectrometry and molecular modeling. . J Mol Biol 425:, 3378–3388. [CrossRef] [PubMed]
    [Google Scholar]
  45. Spencer J. V., Newcomb W. W., Thomsen D. R., Homa F. L., Brown J. C.. ( 1998; ). Assembly of the herpes simplex virus capsid: preformed triplexes bind to the nascent capsid. . J Virol 72:, 3944–3951.[PubMed]
    [Google Scholar]
  46. Steven A. C., Spear P. G.. ( 1996; ). Herpesvirus capsid assembly and envelopment. . In Structural Biology of Viruses, pp. 312–351. Edited by Burnet R., Chiu W., Garcea R... New York:: Oxford University Press;.
    [Google Scholar]
  47. Tatman J. D., Preston V. G., Nicholson P., Elliott R. M., Rixon F. J.. ( 1994; ). Assembly of herpes simplex virus type 1 capsids using a panel of recombinant baculoviruses. . J Gen Virol 75:, 1101–1113. [CrossRef] [PubMed]
    [Google Scholar]
  48. Thomsen D. R., Roof L. L., Homa F. L.. ( 1994; ). Assembly of herpes simplex virus (HSV) intermediate capsids in insect cells infected with recombinant baculoviruses expressing HSV capsid proteins. . J Virol 68:, 2442–2457.[PubMed]
    [Google Scholar]
  49. Trus B. L., Heymann J. B., Nealon K., Cheng N., Newcomb W. W., Brown J. C., Kedes D. H., Steven A. C.. ( 2001; ). Capsid structure of Kaposi’s sarcoma-associated herpesvirus, a gammaherpesvirus, compared to those of an alphaherpesvirus, herpes simplex virus type 1, and a betaherpesvirus, cytomegalovirus. . J Virol 75:, 2879–2890. [CrossRef] [PubMed]
    [Google Scholar]
  50. Walters J. N., Sexton G. L., McCaffery J. M., Desai P.. ( 2003; ). Mutation of single hydrophobic residue I27, L35, F39, L58, L65, L67, or L71 in the N terminus of VP5 abolishes interaction with the scaffold protein and prevents closure of herpes simplex virus type 1 capsid shells. . J Virol 77:, 4043–4059. [CrossRef] [PubMed]
    [Google Scholar]
  51. Wildy P., Russell W. C., Horne R. W.. ( 1960; ). The morphology of herpes virus. . Virology 12:, 204–222. [CrossRef] [PubMed]
    [Google Scholar]
  52. Wingfield P. T., Stahl S. J., Thomsen D. R., Homa F. L., Booy F. P., Trus B. L., Steven A. C.. ( 1997; ). Hexon-only binding of VP26 reflects differences between the hexon and penton conformations of VP5, the major capsid protein of herpes simplex virus. . J Virol 71:, 8955–8961.[PubMed]
    [Google Scholar]
  53. Yakushko Y., Hackmann C., Günther T., Rückert J., Henke M., Koste L., Alkharsah K., Bohne J., Grundhoff A.. & other authors ( 2011; ). Kaposi’s sarcoma-associated herpesvirus bacterial artificial chromosome contains a duplication of a long unique-region fragment within the terminal repeat region. . J Virol 85:, 4612–4617. [CrossRef] [PubMed]
    [Google Scholar]
  54. Zhou Z. H., He J., Jakana J., Tatman J. D., Rixon F. J., Chiu W.. ( 1995; ). Assembly of VP26 in herpes simplex virus-1 inferred from structures of wild-type and recombinant capsids. . Nat Struct Biol 2:, 1026–1030. [CrossRef] [PubMed]
    [Google Scholar]
  55. Zhou F. C., Zhang Y. J., Deng J. H., Wang X. P., Pan H. Y., Hettler E., Gao S. J.. ( 2002; ). Efficient infection by a recombinant Kaposi’s sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis. . J Virol 76:, 6185–6196. [CrossRef] [PubMed]
    [Google Scholar]
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