1887

Abstract

Cytoplasmic inclusion bodies, known as viroplasms or viral factories, are assumed to be the sites of replication of members of the family . Immunocytochemical and biochemical analyses were carried out to characterize the poorly understood viroplasms of the phytoreovirus (RDV). Within 6 h of inoculation of cells, viroplasms, namely discrete cytoplasmic inclusions, were formed that contained the non-structural proteins Pns6, Pns11 and Pns12 of RDV, which appeared to be the constituents of the inclusions. Formation of similar inclusions in non-host insect cells upon expression of Pns12 in a baculovirus system and the association of molecules of Pns12 suggested that the inclusions observed in RDV-infected cells were composed basically of Pns12. Core proteins P1, P3, P5 and P7 and core virus particles were identified in the interior region of the inclusions. In contrast, accumulation of the outer capsid proteins P2, P8 and P9 and of intact virus particles was evident in the peripheral regions of the inclusions. These observations suggest that core particles were constructed inside the inclusions, whereas outer capsid proteins were assembled at the periphery of the inclusions. Viral inclusions were shown to be the sites of viral RNA synthesis by labelling infected cells with 5-bromouridine 5′-triphosphate. The number of viroplasms decreased with time post-inoculation as their sizes increased, suggesting that inclusions might fuse with one another during the virus-propagation process. Our results are consistent with a model, proposed for vertebrate reoviruses, in which viroplasms play a pivotal role in virus assembly.

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2006-02-01
2024-12-07
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References

  1. Becker M. M., Peters T. R., Dermody T. S. 2003; Reovirus σ NS and μ NS proteins form cytoplasmic inclusion structures in the absence of viral infection. J Virol 77:5948–5963 [CrossRef]
    [Google Scholar]
  2. 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 [CrossRef]
    [Google Scholar]
  3. Boccardo G., Milne R. G. 1984; Plant reovirus group. In CMI/AAB Descriptions of Plant Viruses no. 294 Edited by Morant A. F., Harrison B. D. Kew, UK: Commonwealth Microbiology Institute/Association of Applied Biology;
    [Google Scholar]
  4. Broering T. J., Parker J. S. L., Joyce P. L., Kim J., Nibert M. L. 2002; Mammalian reovirus nonstructural protein μ NS forms large inclusions and colocalizes with reovirus microtubule-associated protein μ 2 in transfected cells. J Virol 76:8285–8297 [CrossRef]
    [Google Scholar]
  5. Broering T. J., Kim J., Miller C. L., Piggott C. D. S., Dinoso J. B., Nibert M. L., Parker J. S. L. 2004; Reovirus nonstructural protein μ NS recruits viral core surface proteins and entering core particles to factory-like inclusions. J Virol 78:1882–1892 [CrossRef]
    [Google Scholar]
  6. Broering T. J., Arnold M. M., Miller C. L., Hurt J. A., Joyce P. L., Nibert M. L. 2005; Carboxyl-proximal regions of reovirus nonstructural protein μ NS necessary and sufficient for forming factory-like inclusions. J Virol 79:6194–6206 [CrossRef]
    [Google Scholar]
  7. Brookes S. M., Hyatt A. D., Eaton B. T. 1993; Characterization of virus inclusion bodies in bluetongue virus-infected cells. J Gen Virol 74:525–530 [CrossRef]
    [Google Scholar]
  8. Eichwald C., Rodriguez J. F., Burrone O. R. 2004; Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation. J Gen Virol 85:625–634 [CrossRef]
    [Google Scholar]
  9. Estes M. K. 2001; Rotaviruses and their replication. In Fields' Virology . , 4th edn. pp  1747–1785 Edited by Knipe D. M., Howley P. M. Philadelphia, PA: Lippincott Williams & Wilkins;
  10. 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]
  11. Fields B. N., Raine C. S., Baum S. G. 1971; Temperature-sensitive mutants of reovirus type 3: defects in viral maturation as studied by immunofluorescence and electron microscopy. Virology 43:569–578 [CrossRef]
    [Google Scholar]
  12. Fukushi T., Shikata E., Kimura I. 1962; Some morphological characters of rice dwarf virus. Virology 18:192–205 [CrossRef]
    [Google Scholar]
  13. Huismans H., van Dijk A. A., Bauskin A. R. 1987; In vitro phosphorylation and purification of a nonstructural protein of bluetongue virus with affinity for single-stranded RNA. J Virol 61:3589–3595
    [Google Scholar]
  14. Kimura I. 1986; A study of rice dwarf virus in vector cell monolayers by fluorescent antibody focus counting. J Gen Virol 67:2119–2124 [CrossRef]
    [Google Scholar]
  15. Kimura I., Omura T. 1988; Leafhopper cell cultures as a means for phytoreovirus research. Adv Dis Vector Res 5:111–135
    [Google Scholar]
  16. Li Y., Bao Y. M., Wei C. H. & 7 other authors 2004; Rice dwarf phytoreovirus segment S6-encoded nonstructural protein has a cell-to-cell movement function. J Virol 78:5382–5389 [CrossRef]
    [Google Scholar]
  17. Miller C. L., Broering T. J., Parker J. S. L., Arnold M. M., Nibert M. L. 2003; Reovirus σ NS protein localizes to inclusions through an association requiring the μ NS amino terminus. J Virol 77:4566–4576 [CrossRef]
    [Google Scholar]
  18. Miyazaki N., Hagiwara K., Naitow H., Higashi T., Cheng R. H., Tsukihara T., Nakagawa A., Omura T. 2005; Transcapsidation and the conserved interactions of two major structural proteins of a pair of phytoreoviruses confirm the mechanism of assembly of the outer capsid layer. J Mol Biol 345:229–237 [CrossRef]
    [Google Scholar]
  19. Modrof J., Lymperopoulos K., Roy P. 2005; Phosphorylation of bluetongue virus nonstructural protein 2 is essential for formation of viral inclusion bodies. J Virol 79:10023–10031 [CrossRef]
    [Google Scholar]
  20. Mohan K. V. K., Muller J., Som I., Atreya C. D. 2003; The N- and C-terminal regions of rotavirus NSP5 are the critical determinants for the formation of viroplasm-like structures independent of NSP2. J Virol 77:12184–12192 [CrossRef]
    [Google Scholar]
  21. Nakagawa A., Miyazaki N., Taka J. & 9 other authors 2003; The atomic structure of Rice dwarf virus reveals the self-assembly mechanism of component proteins. Structure 11:1227–1238 [CrossRef]
    [Google Scholar]
  22. Nibert M. L., Schiff L. A. 2001; Reoviruses and their replication. In Fields' Virology , 4th edn. pp  1679–1728 Edited by Knipe D. M., Howley P. M. Philadelphia, PA: Lippincott Williams & Wilkins;
    [Google Scholar]
  23. Omura T., Kimura I. 1994; Leafhopper cell culture for virus research. In Arthropod Cell Culture Systems pp  91–107 Edited by Maramorosch K., McIntosh A. H. Philadelphia, PA: CRC Press;
    [Google Scholar]
  24. Omura T., Yan J. 1999; Role of outer capsid proteins in transmission of phytoreovirus by insect vectors. Adv Virus Res 54:15–43
    [Google Scholar]
  25. Parker J. S. L., Broering T. J., Kim J., Higgins D. E., Nibert M. L. 2002; Reovirus core protein μ 2 determines the filamentous morphology of viral inclusion bodies by interacting with and stabilizing microtubules. J Virol 76:4483–4496 [CrossRef]
    [Google Scholar]
  26. 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]
  27. Roy P. 2001; Orbiviruses. In Fields' Virology . , 4th edn. pp  1835–1869 Edited by Knipe D. M., Howley P. M. Philadelphia, PA: Lippincott Williams & Wilkins;
  28. Shikata E. 1969; Electron microscopic studies on rice viruses. In IRRI: The Virus Diseases of the Rice Plant pp  223–240 Baltimore, MD: Johns Hopkins University Press;
    [Google Scholar]
  29. Silvestri L. S., Taraporewala Z. F., Patton J. T. 2004; Rotavirus replication: plus-sense templates for double-stranded RNA synthesis are made in viroplasms. J Virol 78:7763–7774 [CrossRef]
    [Google Scholar]
  30. Suzuki N., Sugawara M., Kusano T. 1992; Rice dwarf phytoreovirus segment S12 transcript is tricistronic in vitro . Virology 191:992–995 [CrossRef]
    [Google Scholar]
  31. Suzuki N., Sugawara M., Kusano T., Mori H., Matsuura Y. 1994; Immunodetection of rice dwarf phytoreoviral proteins in both insect and plant hosts. Virology 202:41–48 [CrossRef]
    [Google Scholar]
  32. Suzuki N., Hosokawa D., Matsuura Y., Kikuchi A., Omura T. 1999; In vivo and in vitro phosphorylation of rice dwarf phytoreovirus Pns12 cytoplasmic nonstructural protein. Arch Virol 144:1371–1380 [CrossRef]
    [Google Scholar]
  33. Taraporewala Z. F., Patton J. T. 2004; Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae . Virus Res 101:57–66 [CrossRef]
    [Google Scholar]
  34. Theron J., Huismans H., Nel L. H. 1996; Identification of a short domain within the non-structural protein NS2 of epizootic haemorrhagic disease virus that is important for single strand RNA-binding activity. J Gen Virol 77:129–137 [CrossRef]
    [Google Scholar]
  35. Thomas C. P., Booth T. F., Roy P. 1990; Synthesis of bluetongue virus-encoded phosphoprotein and formation of inclusion bodies by recombinant baculovirus in insect cells: it binds the single-stranded RNA species. J Gen Virol 71:2073–2083 [CrossRef]
    [Google Scholar]
  36. Ueda S., Masuta C., Uyeda I. 1997; Hypothesis on particle structure and assembly of rice dwarf phytoreovirus: interactions among multiple structural proteins. J Gen Virol 78:3135–3140
    [Google Scholar]
  37. Uyeda I., Kudo H., Yamada N., Matsumura T., Shikata E. 1990; Nucleotide sequence of rice dwarf virus genome segment 4. J Gen Virol 71:2217–2222 [CrossRef]
    [Google Scholar]
  38. Xu H., Li Y., Mao Z. & 8 other authors 1998; Rice dwarf phytoreovirus segment S11 encodes a nucleic acid binding protein. Virology 240:267–272 [CrossRef]
    [Google Scholar]
  39. Zhong B., Kikuchi A., Moriyasu Y., Higashi T., Hagiwara K., Omura T. 2003; A minor outer capsid protein, P9, of Rice dwarf virus . Arch Virol 148:2275–2280 [CrossRef]
    [Google Scholar]
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