Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1991 Sep;65(9):4991–4998. doi: 10.1128/jvi.65.9.4991-4998.1991

Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1.

L Montross 1, S Watkins 1, R B Moreland 1, H Mamon 1, D L Caspar 1, R L Garcea 1
PMCID: PMC248962  PMID: 1651418

Abstract

Polyomavirus normally assembles in the nucleus of infected mouse cells. Sf9 insect cells expressing the polyomavirus major capsid protein VP1 were examined by electron microscopy. Capsidlike particles of apparently uniform size were found in the nucleus. Immunogold electron microscopy demonstrated abundant VP1 in the cytoplasm which was not assembled into any recognizable higher-order structure. Cytoplasmic VP1 assembled after the cells were treated with the calcium ionophore ionomycin. Purified VP1 aggregates were shown by negative staining and cryoelectron microscopy to consist predominantly of particles similar to the empty T = 7 viral capsid. Thus, polyomavirus VP1 can assemble in vivo into capsids independent of other viral proteins or DNA. Nuclear assembly may result from increased available calcium in this subcellular compartment.

Full text

PDF
4991

Images in this article

4993Image
on p.4993
4993Image
on p.4993
4994Image
on p.4994
4995Image
on p.4995
4996Image
on p.4996
4997Image
on p.4997

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BANFIELD W. G., DAWE C. J., BRINDLEY D. C. Intracellular and extracellular particles in tissue cultures inoculated with parotid-tumor agent (polyoma virus). J Natl Cancer Inst. 1959 Nov;23:1123–1135. [PubMed] [Google Scholar]
  2. Barkan A., Mertz J. E. DNA sequence analysis of simian virus 40 mutants with deletions mapping in the leader region of the late viral mRNA's: mutants with deletions similar in size and position exhibit varied phenotypes. J Virol. 1981 Feb;37(2):730–737. doi: 10.1128/jvi.37.2.730-737.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barkan A., Welch R. C., Mertz J. E. Missense mutations in the VP1 gene of simian virus 40 that compensate for defects caused by deletions in the viral agnogene. J Virol. 1987 Oct;61(10):3190–3198. doi: 10.1128/jvi.61.10.3190-3198.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baumgartner I., Kuhn C., Fanning E. Identification and characterization of fast-sedimenting SV40 nucleoprotein complexes. Virology. 1979 Jul 15;96(1):54–63. doi: 10.1016/0042-6822(79)90172-7. [DOI] [PubMed] [Google Scholar]
  5. Benjamin T. L. Host range mutants of polyoma virus. Proc Natl Acad Sci U S A. 1970 Sep;67(1):394–399. doi: 10.1073/pnas.67.1.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bina M., Blasquez V., Ng S. C., Beecher S. SV40 morphogenesis. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):565–569. doi: 10.1101/sqb.1983.047.01.066. [DOI] [PubMed] [Google Scholar]
  7. Blasquez V., Beecher S., Bina M. Simian virus 40 morphogenetic pathway. An analysis of assembly-defective tsB201 DNA protein complexes. J Biol Chem. 1983 Jul 10;258(13):8477–8484. [PubMed] [Google Scholar]
  8. Brady J. N., Winston V. D., Consigli R. A. Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions. J Virol. 1977 Sep;23(3):717–724. doi: 10.1128/jvi.23.3.717-724.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. CASPAR D. L., KLUG A. Physical principles in the construction of regular viruses. Cold Spring Harb Symp Quant Biol. 1962;27:1–24. doi: 10.1101/sqb.1962.027.001.005. [DOI] [PubMed] [Google Scholar]
  10. Carswell S., Alwine J. C. Simian virus 40 agnoprotein facilitates perinuclear-nuclear localization of VP1, the major capsid protein. J Virol. 1986 Dec;60(3):1055–1061. doi: 10.1128/jvi.60.3.1055-1061.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chandra S., Gross D., Ling Y. C., Morrison G. H. Quantitative imaging of free and total intracellular calcium in cultured cells. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1870–1874. doi: 10.1073/pnas.86.6.1870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Christiansen G., Landers T., Griffith J., Berg P. Characterization of components released by alkali disruption of simian virus 40. J Virol. 1977 Mar;21(3):1079–1084. doi: 10.1128/jvi.21.3.1079-1084.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dubochet J., Adrian M., Schultz P., Oudet P. Cryo-electron microscopy of vitrified SV40 minichromosomes: the liquid drop model. EMBO J. 1986 Mar;5(3):519–528. doi: 10.1002/j.1460-2075.1986.tb04241.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fernandez-Munoz R., Coca-Prados M., Hsu M. T. Intracellular forms of simian virus 40 nucleoprotein complexes. I. Methods of isolation and characterization in CV-1 cells. J Virol. 1979 Feb;29(2):612–623. doi: 10.1128/jvi.29.2.612-623.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. French T. J., Marshall J. J., Roy P. Assembly of double-shelled, viruslike particles of bluetongue virus by the simultaneous expression of four structural proteins. J Virol. 1990 Dec;64(12):5695–5700. doi: 10.1128/jvi.64.12.5695-5700.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. French T. J., Roy P. Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. J Virol. 1990 Apr;64(4):1530–1536. doi: 10.1128/jvi.64.4.1530-1536.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Friedmann T., David D. Structural roles of polyoma virus proteins. J Virol. 1972 Oct;10(4):776–782. doi: 10.1128/jvi.10.4.776-782.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Garber E. A., Seidman M. M., Levine A. J. The detection and characterization of multiple forms of SV40 nucleoprotein complexes. Virology. 1978 Oct 15;90(2):305–316. doi: 10.1016/0042-6822(78)90315-x. [DOI] [PubMed] [Google Scholar]
  19. Garcea R. L., Ballmer-Hofer K., Benjamin T. L. Virion assembly defect of polyomavirus hr-t mutants: underphosphorylation of major capsid protein VP1 before viral DNA encapsidation. J Virol. 1985 May;54(2):311–316. doi: 10.1128/jvi.54.2.311-316.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Garcea R. L., Benjamin T. L. Host range transforming gene of polyoma virus plays a role in virus assembly. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3613–3617. doi: 10.1073/pnas.80.12.3613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Garcea R. L., Benjamin T. L. Isolation and characterization of polyoma nucleoprotein complexes. Virology. 1983 Oct 15;130(1):65–75. doi: 10.1016/0042-6822(83)90118-6. [DOI] [PubMed] [Google Scholar]
  22. Garcea R. L., Salunke D. M., Caspar D. L. Site-directed mutation affecting polyomavirus capsid self-assembly in vitro. Nature. 1987 Sep 3;329(6134):86–87. doi: 10.1038/329086a0. [DOI] [PubMed] [Google Scholar]
  23. Gheysen D., Jacobs E., de Foresta F., Thiriart C., Francotte M., Thines D., De Wilde M. Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells. Cell. 1989 Oct 6;59(1):103–112. doi: 10.1016/0092-8674(89)90873-8. [DOI] [PubMed] [Google Scholar]
  24. Hogle J., Kirchhausen T., Harrison S. C. Divalent cation sites in tomato bushy stunt virus. Difference maps at 2-9 A resolution. J Mol Biol. 1983 Nov 25;171(1):95–100. doi: 10.1016/s0022-2836(83)80315-5. [DOI] [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Leavitt A. D., Roberts T. M., Garcea R. L. Polyoma virus major capsid protein, VP1. Purification after high level expression in Escherichia coli. J Biol Chem. 1985 Oct 15;260(23):12803–12809. [PubMed] [Google Scholar]
  27. Loudon P. T., Roy P. Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: inclusion of the largest protein VP1 in the core and virus-like proteins. Virology. 1991 Feb;180(2):798–802. doi: 10.1016/0042-6822(91)90094-r. [DOI] [PubMed] [Google Scholar]
  28. Luckow V. A., Summers M. D. High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors. Virology. 1989 May;170(1):31–39. doi: 10.1016/0042-6822(89)90348-6. [DOI] [PubMed] [Google Scholar]
  29. Margolskee R. F., Nathans D. Suppression of a VP1 mutant of simian virus 40 by missense mutations in serine codons of the viral agnogene. J Virol. 1983 Nov;48(2):405–409. doi: 10.1128/jvi.48.2.405-409.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mattern C. F., Takemoto K. K., Daniel W. A. Replication of polyoma virus in mouse embryo cells: electron microscopic observations. Virology. 1966 Oct;30(2):242–256. doi: 10.1016/0042-6822(66)90099-7. [DOI] [PubMed] [Google Scholar]
  31. Moreland R. B., Montross L., Garcea R. L. Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1. J Virol. 1991 Mar;65(3):1168–1176. doi: 10.1128/jvi.65.3.1168-1176.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ng S. C., Mertz J. E., Sanden-Will S., Bina M. Simian virus 40 maturation in cells harboring mutants deleted in the agnogene. J Biol Chem. 1985 Jan 25;260(2):1127–1132. [PubMed] [Google Scholar]
  33. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  34. Rasmussen L., Battles J. K., Ennis W. H., Nagashima K., Gonda M. A. Characterization of virus-like particles produced by a recombinant baculovirus containing the gag gene of the bovine immunodeficiency-like virus. Virology. 1990 Oct;178(2):435–451. doi: 10.1016/0042-6822(90)90341-n. [DOI] [PubMed] [Google Scholar]
  35. Rayment I., Baker T. S., Caspar D. L., Murakami W. T. Polyoma virus capsid structure at 22.5 A resolution. Nature. 1982 Jan 14;295(5845):110–115. doi: 10.1038/295110a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Resnick J., Shenk T. Simian virus 40 agnoprotein facilitates normal nuclear location of the major capsid polypeptide and cell-to-cell spread of virus. J Virol. 1986 Dec;60(3):1098–1106. doi: 10.1128/jvi.60.3.1098-1106.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Salunke D. M., Caspar D. L., Garcea R. L. Polymorphism in the assembly of polyomavirus capsid protein VP1. Biophys J. 1989 Nov;56(5):887–900. doi: 10.1016/S0006-3495(89)82735-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Salunke D. M., Caspar D. L., Garcea R. L. Self-assembly of purified polyomavirus capsid protein VP1. Cell. 1986 Sep 12;46(6):895–904. doi: 10.1016/0092-8674(86)90071-1. [DOI] [PubMed] [Google Scholar]
  39. Shenk T. E., Carbon J., Berg P. Construction and analysis of viable deletion mutants of simian virus 40. J Virol. 1976 May;18(2):664–671. doi: 10.1128/jvi.18.2.664-671.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Urakawa T., Ferguson M., Minor P. D., Cooper J., Sullivan M., Almond J. W., Bishop D. H. Synthesis of immunogenic, but non-infectious, poliovirus particles in insect cells by a baculovirus expression vector. J Gen Virol. 1989 Jun;70(Pt 6):1453–1463. doi: 10.1099/0022-1317-70-6-1453. [DOI] [PubMed] [Google Scholar]
  42. Watkins S. C., Samuel J. L., Marotte F., Bertier-Savalle B., Rappaport L. Microtubules and desmin filaments during onset of heart hypertrophy in rat: a double immunoelectron microscope study. Circ Res. 1987 Mar;60(3):327–336. doi: 10.1161/01.res.60.3.327. [DOI] [PubMed] [Google Scholar]
  43. Williams D. A., Becker P. L., Fay F. S. Regional changes in calcium underlying contraction of single smooth muscle cells. Science. 1987 Mar 27;235(4796):1644–1648. doi: 10.1126/science.3103219. [DOI] [PubMed] [Google Scholar]
  44. Williams D. A., Fogarty K. E., Tsien R. Y., Fay F. S. Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2. Nature. 1985 Dec 12;318(6046):558–561. doi: 10.1038/318558a0. [DOI] [PubMed] [Google Scholar]
  45. de Agostini A. I., Watkins S. C., Slayter H. S., Youssoufian H., Rosenberg R. D. Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: antithrombin binding on cultured endothelial cells and perfused rat aorta. J Cell Biol. 1990 Sep;111(3):1293–1304. doi: 10.1083/jcb.111.3.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES