1887

Abstract

Viroplasms are discrete structures formed in the cytoplasm of rotavirus-infected cells and constitute the replication machinery of the virus. The non-structural proteins NSP2 and NSP5 localize in viroplasms together with other viral proteins, including the polymerase VP1, VP3 and the main inner-core protein, VP2. NSP2 and NSP5 interact with each other, activating NSP5 hyperphosphorylation and the formation of viroplasm-like structures (VLSs). We have used NSP2 and NSP5 fused to the enhanced green fluorescent protein (EGFP) to investigate the localization of both proteins within viroplasms in virus-infected cells, as well as the dynamics of viroplasm formation. The number of viroplasms was shown first to increase and then to decrease with time post-infection, while the area of each one increased, suggesting the occurrence of fusions. The interaction between NSP2 and a series of NSP5 mutants was investigated using two different assays, a yeast two-hybrid system and an binding/immunoprecipitation assay. Both methods gave comparable results, indicating that the N-terminal region (33 aa) as well as the C-terminal part (aa 131–198) of NSP5 are required for binding to NSP2. When fused to the N and C terminus of EGFP, respectively, these two regions were able to confer the ability to localize in the viroplasm and to form VLSs with NSP2.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.19611-0
2004-03-01
2021-10-17
Loading full text...

Full text loading...

/deliver/fulltext/jgv/85/3/vir850625.html?itemId=/content/journal/jgv/10.1099/vir.0.19611-0&mimeType=html&fmt=ahah

References

  1. Afrikanova I., Miozzo M. C., Giambiagi S., Burrone O. 1996; Phosphorylation generates different forms of rotavirus NSP5. J Gen Virol 77:2059–2065
    [Google Scholar]
  2. Afrikanova I., Fabbretti E., Miozzo M. C., Burrone O. R. 1998; Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2. J Gen Virol 79:2679–2686
    [Google Scholar]
  3. Becker M. M., Peters T. R., Dermody T. S. 2003; Reovirus σNS and μNS proteins form cytoplasmic inclusion structures in the absence of viral infection. J Virol 77:5948–5963
    [Google Scholar]
  4. Berois M., Sapin C., Erk I., Poncet D., Cohen J. 2003; Rotavirus nonstructural protein NSP5 interacts with major core protein VP2. J Virol 77:1757–1763
    [Google Scholar]
  5. Blackhall J., Munoz M., Fuentes A., Magnusson G. 1998; Analysis of rotavirus nonstructural protein NSP5 phosphorylation. J Virol 72:6398–6405
    [Google Scholar]
  6. Breeden L., Nasmyth K. 1985; Regulation of the yeast HO gene. Cold Spring Harbor Symp Quant Biol 50:643–650
    [Google Scholar]
  7. Dales S., Gomatos P., Hsu K. 1965; The uptake and development of reovirus in strain L cells followed with labelled viral riboznucleic acid and ferritin-antibody complexes. Virology 25:193–211
    [Google Scholar]
  8. Earl P. L., Moss B. 1993; Generation of recombinant vaccinia viruses. In Current Protocols in Molecular Biology Edited by Ausubel F. M. B., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. New York: Wiley & Sons;
    [Google Scholar]
  9. Eichwald C., Vascotto F., Fabbretti E., Burrone O. R. 2002; Rotavirus NSP5: mapping phosphorylation sites and kinase activation and viroplasm localization domains. J Virol 76:3461–3470
    [Google Scholar]
  10. Estes M. K., Graham D. Y., Gerba C. P., Smith E. M. 1979; Simian rotavirus SA11 replication in cell cultures. J Virol 31:810–815
    [Google Scholar]
  11. Fabbretti E., Afrikanova I., Vascotto F., Burrone O. R. 1999; Two non-structural rotavirus proteins, NSP2 and NSP5, form viroplasm-like structures in vivo . J Gen Virol 80:333–339
    [Google Scholar]
  12. Fillmore G. C., Lin H., Li J. K. 2002; Localization of the single-stranded RNA-binding domains of bluetongue virus nonstructural protein NS2. J Virol 76:499–506
    [Google Scholar]
  13. Gallegos C. O., Patton J. T. 1989; Characterization of rotavirus replication intermediates: a model for the assembly of single-shelled particles. Virology 172:616–627
    [Google Scholar]
  14. Gietz D., St Jean A., Woods R. A., Schiestl R. H. 1992; Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425
    [Google Scholar]
  15. Gillian A. L., Nibert M. L. 1998; Amino terminus of reovirus nonstructural protein sigma NS is important for ssRNA binding and nucleoprotein complex formation. Virology 240:1–11
    [Google Scholar]
  16. Gillian A. L., Schmechel S. C., Livny J., Schiff L. A., Nibert M. L. 2000; Reovirus protein σNS binds in multiple copies to single-stranded RNA and shares properties with single-stranded DNA binding proteins. J Virol 74:5939–5948
    [Google Scholar]
  17. Gonzalez S. A., Burrone O. R. 1991; Rotavirus NS26 is modified by addition of single O -linked residues of N -acetylglucosamine. Virology 182:8–16
    [Google Scholar]
  18. Grimley P. M., Rosenblum E. N., Mims S. J., Moss B. 1970; Interruption by rifampin of an early stage in vaccinia virus morphogenesis: accumulation of membranes which are precursors of virus envelopes. J Virol 6:519–533
    [Google Scholar]
  19. Huismans H., Joklik W. K. 1976; Reovirus-coded polypeptides in infected cells: isolation of two native monomeric polypeptides with affinity for single-stranded and double-stranded RNA, respectively. Virology 70:411–424
    [Google Scholar]
  20. Jayaram H., Taraporewala Z., Patton J. T., Prasad B. V. 2002; Rotavirus protein involved in genome replication and packaging exhibits a HIT-like fold. Nature 417:311–315
    [Google Scholar]
  21. Kattoura M. D., Clapp L. L., Patton J. T. 1992; The rotavirus nonstructural protein, NS35, possesses RNA-binding activity in vitro and in vivo. Virology 191:698–708
    [Google Scholar]
  22. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  23. Mattion N. M., Mitchell D. B., Both G. W., Estes M. K. 1991; Expression of rotavirus proteins encoded by alternative open reading frames of genome segment 11. Virology 181:295–304
    [Google Scholar]
  24. Nagaya A., Pogo B. G., Dales S. 1970; Biogenesis of vaccinia: separation of early stages from maturation by means of rifampicin. Virology 40:1039–1051
    [Google Scholar]
  25. Patton J. T., Gallegos C. O. 1990; Rotavirus RNA replication: single-stranded RNA extends from the replicase particle. J Gen Virol 71:1087–1094
    [Google Scholar]
  26. Patton J. T., Jones M. T., Kalbach A. N., He Y. W., Xiaobo J. 1997; Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome. J Virol 71:9618–9626
    [Google Scholar]
  27. Petrie B. L., Greenberg H. B., Graham D. Y., Estes M. K. 1984; Ultrastructural localization of rotavirus antigens using colloidal gold. Virus Res 1:133–152
    [Google Scholar]
  28. Poncet D., Lindenbaum P., L’Haridon R., Cohen J. 1997; In vivo and in vitro phosphorylation of rotavirus NSP5 correlates with its localization in viroplasms. J Virol 71:34–41
    [Google Scholar]
  29. Rhim J., Jordan L., Mayor H. 1962; Cytochemical, fluorescent-antibody and electron microscopic studies on the growth of reovirus (ECHO 10) in tissue culture. Virology 17:342–355
    [Google Scholar]
  30. Richardson M. A., Furuichi Y. 1985; Synthesis in Escherichia coli of the reovirus nonstructural protein sigma NS. J Virol 56:527–533
    [Google Scholar]
  31. Sambrook J., Fristisch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor;
    [Google Scholar]
  32. Schuck P., Taraporewala Z., McPhie P., Patton J. T. 2001; Rotavirus nonstructural protein NSP2 self-assembles into octamers that undergo ligand-induced conformational changes. J Biol Chem 276:9679–9687
    [Google Scholar]
  33. Silverstein S. C., Schur P. H. 1970; Immunofluorescent localization of double-stranded RNA in reovirus-infected cells. Virology 41:564–566
    [Google Scholar]
  34. Taraporewala Z. F., Patton J. T. 2001; Identification and characterization of the helix-destabilizing activity of rotavirus nonstructural protein NSP2. J Virol 75:4519–4527
    [Google Scholar]
  35. Taraporewala Z. F., Chen D., Patton J. T. 2001; Multimers of the bluetongue virus nonstructural protein, NS2, possess nucleotidyl phosphatase activity: similarities between NS2 and rotavirus NSP2. Virology 280:221–231
    [Google Scholar]
  36. Taraporewala Z. F., Schuck P., Ramig R. F., Silvestri L., Patton J. T. 2002; Analysis of a temperature-sensitive mutant rotavirus indicates that NSP2 octamers are the functional form of the protein. J Virol 76:7082–7093
    [Google Scholar]
  37. Torres-Vega M. A., Gonzalez R. A., Duarte M., Poncet D., Lopez S., Arias C. F. 2000; The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6. J Gen Virol 81:821–830
    [Google Scholar]
  38. Vende P., Taraporewala Z. F., Patton J. T. 2002; RNA-binding activity of the rotavirus phosphoprotein NSP5 includes affinity for double-stranded RNA. J Virol 76:5291–5299
    [Google Scholar]
  39. Visintin M., Tse E., Axelson H., Rabbitts T. H., Cattaneo A. 1999; Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proc Natl Acad Sci U S A 96:11723–11728
    [Google Scholar]
  40. Ward G. A., Stover C. K., Moss B., Fuerst T. R. 1995; Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells. Proc Natl Acad Sci U S A 92:6773–6777
    [Google Scholar]
  41. Welch S. K., Crawford S. E., Estes M. K. 1989; Rotavirus SA11 genome segment 11 protein is a nonstructural phosphoprotein. J Virol 63:3974–3982
    [Google Scholar]
  42. Wentz M. J., Patton J. T., Ramig R. F. 1996; The 3′-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis. J Virol 70:7833–7841
    [Google Scholar]
  43. Zeng C. Q., Estes M. K., Charpilienne A., Cohen J. 1998; The N terminus of rotavirus VP2 is necessary for encapsidation of VP1 and VP3. J Virol 72:201–208
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.19611-0
Loading
/content/journal/jgv/10.1099/vir.0.19611-0
Loading

Data & Media loading...

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