1887

Abstract

In rotavirus-infected cells, the non-structural proteins NSP5 and NSP2 localize in complexes called viroplasms, where replication and assembly occur. Recently, we have demonstrated direct interaction of NSP5 with NSP2, and as a consequence of that, up-regulation of NSP5 hyperphosphorylation. To investigate a possible structural role for the NSP2-NSP5 interaction, we analysed the cytoplasmic distribution of the two proteins in transfected cells by immunofluorescence using specific antibodies. Here we report that NSP2 and NSP5 can drive the formation of viroplasm-like structures (VLS) in the absence of other rotaviral proteins and rotavirus replication. Several NSP5 deletion mutants were constructed and expressed in combination with NSP2. Both the N- and C-terminal domains of NSP5 were found to be essential for VLS formation. Only one mutant, with an internal deletion of residues 81–130, was able to interact with NSP2 to form VLS. Analysis of the phosphorylation capacity of the different mutants in vivo indicated that hyperphosphorylation of NSP5 is necessary, but not sufficient, for VLS formation. Our results suggest a role for the non-structural protein NSP5 in the structure of viroplasms mediated by its interaction with NSP2.

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1999-02-01
2024-03-28
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References

  1. Afrikanova I., Fabbretti E., Miozzo M. C., Burrone O. R. 1998; Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2. Journal of General Virology 78:2679–2686
    [Google Scholar]
  2. Afrikanova I., Miozzo M. C., Giambiagi S., Burrone O. R. 1996; Phosphorylation generates different forms of rotavirus NSP5. Journal of General Virology 77:2059–2065
    [Google Scholar]
  3. Aponte C., Poncet D., Cohen J. 1996; Recovery and characterization of a replicase complex in rotavirus-infected cells by using a monoclonal antibody against NSP2. Journal of Virology 70:985–991
    [Google Scholar]
  4. Blackhall J., Fuentes A., Hansen K., Magnusson G. 1997; Serine protein kinase activity associated with rotavirus phosphoprotein NSP5. Journal of Virology 71:138–144
    [Google Scholar]
  5. Chou T. Y., Hart G. W., Dang C. V. 1995; c-Myc is glycosylated at threonine 58, a known phosphorylation site and a mutational hot spot in lymphomas. Journal of Biological Chemistry 270:18961–18965
    [Google Scholar]
  6. Cohen J. 1977; Ribonucleic acid polymerase activity associated with purified calf rotavirus. Journal of General Virology 36:395–402
    [Google Scholar]
  7. Crawford S. E., Labbe M., Cohen J., Burroughs M. H., Zhou Y.-J., Estes M. K. 1994; Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells. Journal of Virology 68:5945–5952
    [Google Scholar]
  8. Estes M. K. 1996; Rotaviruses and their replication. In Fields Virology 3rd edn pp 1625–1655 Edited by Fields B. N., Knipe D. M., Howley P. M. Philadelphia: Lippincott–Raven;
    [Google Scholar]
  9. Estes M. K., Graham D. Y., Gerba C. P., Smith E. M. 1979; Simian rotavirus SA11 replication in cell cultures. Journal of Virology 31:810–815
    [Google Scholar]
  10. Fuerst R. T., Niles G. E., Studier W. F., Moss B. 1986; Eucaryotic transient-expression system based on recombinant vaccinia virus that synthesized bacteriophage T7 RNA polymerase. Proceedings of the National Academy of Sciences, USA 83:8122–8126
    [Google Scholar]
  11. Gonzáles S. A., Affranchino J. L. 1995; Assembly ofdouble-layered virus-like particles in mammalian cells by coexpression ofhuman rotavirus VP2 and VP6. Journal of General Virology 76:2357–2360
    [Google Scholar]
  12. Gonzáles S. A., Burrone O. 1991; Rotavirus NS26 is modified by addition of single O-linked residues of N-acetylglucosamine. Virology 182:8–16
    [Google Scholar]
  13. Kattoura M. D., Clapp L. L., Patton J. T. 1992; The rotavirus non structural protein NS35, possesses RNA-binding activity in vitro and in vivo . Virology 192:698–708
    [Google Scholar]
  14. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  15. Mansell E. A., Ramig R. F., Patton J. T. 1994; Temperature-sensitive lesions in the capsid proteins of the rotavirus mutants ts-F and ts-G that affect virion assembly. Virology 204:69–81
    [Google Scholar]
  16. Patton J. T., Salter-Cid L., Kalbach A., Mansell E. A., Kattoura M. 1993; Nucleotide and amino acid sequence analysis of the rotavirus non structural RNA-binding protein NS35. Virology 192:438–446
    [Google Scholar]
  17. Petrie B. L., Graham D. Y., Hanssen H., Estes M. K. 1982; Localization of rotavirus antigens in infected cells by ultrastructural immunocytochemistry. Journal of General Virology 63:457–467
    [Google Scholar]
  18. Petrie B. L., Greenberg H. B., Graham D. Y., Estes M. K. 1984; Ultrastructural localisation of rotavirus antigens using colloidal gold. Virus Research 1:133–152
    [Google Scholar]
  19. Poncet D., Lindenbaum P., L’Haridon R., Cohen J. 1997; In vivo an in vitro phosphorylation of rotavirus NSP5 correlates with its localization in viroplasms. Journal of Virology 71:34–41
    [Google Scholar]
  20. Snow D. M., Hart G. W. 1998; Nuclear and cytoplasmatic glycosylation. Internal Review of Cytology 181:43–74
    [Google Scholar]
  21. Welch S.-K. W., Crawford S. E., Estes M. K. 1989; Rotavirus SA11 genome segment 11 protein is a nonstructural phosphoprotein. Journal of Virology 63:3974–3982
    [Google Scholar]
  22. Zeng C. Q.-Y., Wentz M. J., Cohen J., Estes M. K., Ramig R. F. 1996; Characterization and replicase activity of double-layered and single-layered particles expressed from baculovirus recombinants. Journal of Virology 70:2736–2742
    [Google Scholar]
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