Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Jun 20;92(13):5992–5996. doi: 10.1073/pnas.92.13.5992

Polyomavirus VP1 phosphorylation: coexpression with the VP2 capsid protein modulates VP1 phosphorylation in Sf9 insect cells.

M Li 1, S E Delos 1, L Montross 1, R L Garcea 1
PMCID: PMC41628  PMID: 7597067

Abstract

The polyomavirus virion has an outer capsid comprised of 72 pentamers of the VP1 protein associated with the minor virion proteins, VP2 and VP3, and the viral minichromosome. To investigate the interaction between VP1 and VP2/VP3, we mapped VP1 phosphorylation sites and assayed VP1 recognition by anti-peptide antibodies after coexpression of VP1 with VP2 or VP3 by using recombinant baculovirus vectors. VP1, expressed either alone or with VP3, was phosphorylated on serine residues, which are not modified during polyomavirus infection of mouse cells. When VP1 was coexpressed with VP2, the nonphysiologic serine phosphorylation of VP1 was decreased, and a tryptic peptide containing Thr-63, a site modified during virus infection of mouse cells, was phosphorylated. An anti-peptide antibody directed against the VP1 BC loop domain containing Thr-63 recognized VP1 expressed alone but not VP1 coexpressed with VP2 or VP3. The change in phosphorylation resulting from coexpression of two structural proteins identifies the potential of the baculovirus system for studying protein-protein interactions and defines a functional role for the VP1-VP2 interaction.

Full text

PDF
5992

Images in this article

5994Fig. 1
on p.5994
5995Fig. 3
on p.5995
5995Fig. 4
on p.5995

Selected References

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

  1. Barouch D. H., Harrison S. C. Interactions among the major and minor coat proteins of polyomavirus. J Virol. 1994 Jun;68(6):3982–3989. doi: 10.1128/jvi.68.6.3982-3989.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benjamin T. L. The hr-t gene of polyoma virus. Biochim Biophys Acta. 1982 Dec 21;695(2):69–95. doi: 10.1016/0304-419x(82)90018-x. [DOI] [PubMed] [Google Scholar]
  3. Cole C. N., Landers T., Goff S. P., Manteuil-Brutlag S., Berg P. Physical and genetic characterization of deletion mutants of simian virus 40 constructed in vitro. J Virol. 1977 Oct;24(1):277–294. doi: 10.1128/jvi.24.1.277-294.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Delos S. E., Montross L., Moreland R. B., Garcea R. L. Expression of the polyomavirus VP2 and VP3 proteins in insect cells: coexpression with the major capsid protein VP1 alters VP2/VP3 subcellular localization. Virology. 1993 May;194(1):393–398. doi: 10.1006/viro.1993.1274. [DOI] [PubMed] [Google Scholar]
  5. Emery V. C., Bishop D. H. The development of multiple expression vectors for high level synthesis of eukaryotic proteins: expression of LCMV-N and AcNPV polyhedrin protein by a recombinant baculovirus. Protein Eng. 1987 Aug-Sep;1(4):359–366. doi: 10.1093/protein/1.4.359. [DOI] [PubMed] [Google Scholar]
  6. Forstová J., Krauzewicz N., Wallace S., Street A. J., Dilworth S. M., Beard S., Griffin B. E. Cooperation of structural proteins during late events in the life cycle of polyomavirus. J Virol. 1993 Mar;67(3):1405–1413. doi: 10.1128/jvi.67.3.1405-1413.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fraser M. J. The baculovirus-infected insect cell as a eukaryotic gene expression system. Curr Top Microbiol Immunol. 1992;158:131–172. doi: 10.1007/978-3-642-75608-5_6. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Griffith J. P., Griffith D. L., Rayment I., Murakami W. T., Caspar D. L. Inside polyomavirus at 25-A resolution. Nature. 1992 Feb 13;355(6361):652–654. doi: 10.1038/355652a0. [DOI] [PubMed] [Google Scholar]
  11. Höss A., Moarefi I., Scheidtmann K. H., Cisek L. J., Corden J. L., Dornreiter I., Arthur A. K., Fanning E. Altered phosphorylation pattern of simian virus 40 T antigen expressed in insect cells by using a baculovirus vector. J Virol. 1990 Oct;64(10):4799–4807. doi: 10.1128/jvi.64.10.4799-4807.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Krauzewicz N., Streuli C. H., Stuart-Smith N., Jones M. D., Wallace S., Griffin B. E. Myristylated polyomavirus VP2: role in the life cycle of the virus. J Virol. 1990 Sep;64(9):4414–4420. doi: 10.1128/jvi.64.9.4414-4420.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Li M., Garcea R. L. Identification of the threonine phosphorylation sites on the polyomavirus major capsid protein VP1: relationship to the activity of middle T antigen. J Virol. 1994 Jan;68(1):320–327. doi: 10.1128/jvi.68.1.320-327.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Liddington R. C., Yan Y., Moulai J., Sahli R., Benjamin T. L., Harrison S. C. Structure of simian virus 40 at 3.8-A resolution. Nature. 1991 Nov 28;354(6351):278–284. doi: 10.1038/354278a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Montross L., Watkins S., Moreland R. B., Mamon H., Caspar D. L., Garcea R. L. Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1. J Virol. 1991 Sep;65(9):4991–4998. doi: 10.1128/jvi.65.9.4991-4998.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Piwnica-Worms H., Williams N. G., Cheng S. H., Roberts T. M. Regulation of pp60c-src and its interaction with polyomavirus middle T antigen in insect cells. J Virol. 1990 Jan;64(1):61–68. doi: 10.1128/jvi.64.1.61-68.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Sahli R., Freund R., Dubensky T., Garcea R., Bronson R., Benjamin T. Defect in entry and altered pathogenicity of a polyoma virus mutant blocked in VP2 myristylation. Virology. 1993 Jan;192(1):142–153. doi: 10.1006/viro.1993.1016. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Stehle T., Yan Y., Benjamin T. L., Harrison S. C. Structure of murine polyomavirus complexed with an oligosaccharide receptor fragment. Nature. 1994 May 12;369(6476):160–163. doi: 10.1038/369160a0. [DOI] [PubMed] [Google Scholar]
  24. Streuli C. H., Griffin B. E. Myristic acid is coupled to a structural protein of polyoma virus and SV40. Nature. 1987 Apr 9;326(6113):619–622. doi: 10.1038/326619a0. [DOI] [PubMed] [Google Scholar]
  25. Vik T. A., Sweet L. J., Erikson R. L. Coinfection of insect cells with recombinant baculovirus expressing pp60v-src results in the activation of a serine-specific protein kinase pp90rsk. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2685–2689. doi: 10.1073/pnas.87.7.2685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zullo J., Stiles C. D., Garcea R. L. Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1210–1214. doi: 10.1073/pnas.84.5.1210. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES