Rotavirus genome replication and the first steps of virus morphogenesis take place in cytoplasmic viral factories, called viroplasms, containing four structural (VP1, VP2, VP3 and VP6) and two non-structural (NSP2 and NSP5) proteins. NSP2 and NSP5 have been shown to be essential for viroplasm formation and, when co-expressed in uninfected cells, to form viroplasm-like structures (VLS). In the present work, VLS formation was shown upon co-expression of NSP5 with the core protein VP2 despite the absence of NSP2, indicating a central role for NSP5 in VLS assembly. Since VP2 and NSP2 also induce NSP5 hyperphosphorylation, the possible correlation between VLS formation and the NSP5 phosphorylation status was investigated without evidence of a direct link. In VLS induced by NSP2, the polymerase VP1 was recruited, while the middle layer protein VP6 was not, forming instead tubular structures. On the other hand, VLS induced by VP2 were able to recruit both VP1 and VP6. More importantly, in VLS formed when NSP5 was expressed with both inducers, all viroplasmic proteins were found co-localized, resembling their distribution in viroplasms. Our results suggest a key role for NSP5 in architectural assembly of viroplasms and in recruitment of viroplasmic proteins. A new role for VP2 as an inducer of viroplasms and of NSP5 hyperphosphorylation is also described. These data may contribute to the understanding of rotavirus morphogenesis.


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  1. Abramoff, M. D. & Viergever, M. A.(2002). Computation and visualization of three-dimensional soft tissue motion in the orbit. IEEE Trans Med Imaging 21, 296–304.[CrossRef] [Google Scholar]
  2. Afrikanova, I., Miozzo, M. C., Giambiagi, S. & Burrone, O.(1996). Phosphorylation generates different forms of rotavirus NSP5. J Gen Virol 77, 2059–2065.[CrossRef] [Google Scholar]
  3. 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]
  4. Altenburg, B. C., Graham, D. Y. & Estes, M. K.(1980). Ultrastructural study of rotavirus replication in cultured cells. J Gen Virol 46, 75–85.[CrossRef] [Google Scholar]
  5. Arnoldi, F. & Burrone, O. R.(2009). Role of viral nonstructural proteins in rotavirus replication. Future Virol 4, 185–196.[CrossRef] [Google Scholar]
  6. Arnoldi, F., Campagna, M., Eichwald, C., Desselberger, U. & Burrone, O. R.(2007). Interaction of rotavirus polymerase VP1 with nonstructural protein NSP5 is stronger than that with NSP2. J Virol 81, 2128–2137.[CrossRef] [Google Scholar]
  7. Ayala-Breton, C., Arias, M., Espinosa, R., Romero, P., Arias, C. F. & Lopez, S.(2009). Analysis of the kinetics of transcription and replication of the rotavirus genome by RNA interference. J Virol 83, 8819–8831.[CrossRef] [Google Scholar]
  8. 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]
  9. Boyce, M. & Roy, P.(2007). Recovery of infectious bluetongue virus from RNA. J Virol 81, 2179–2186.[CrossRef] [Google Scholar]
  10. Boyce, M., Celma, C. C. & Roy, P.(2008). Development of reverse genetics systems for bluetongue virus: recovery of infectious virus from synthetic RNA transcripts. J Virol 82, 8339–8348.[CrossRef] [Google Scholar]
  11. Cabral-Romero, C. & Padilla-Noriega, L.(2006). Association of rotavirus viroplasms with microtubules through NSP2 and NSP5. Mem Inst Oswaldo Cruz 101, 603–611.[CrossRef] [Google Scholar]
  12. Campagna, M., Eichwald, C., Vascotto, F. & Burrone, O. R.(2005). RNA interference of rotavirus segment 11 mRNA reveals the essential role of NSP5 in the virus replicative cycle. J Gen Virol 86, 1481–1487.[CrossRef] [Google Scholar]
  13. Campagna, M., Budini, M., Arnoldi, F., Desselberger, U., Allende, J. E. & Burrone, O. R.(2007). Impaired hyperphosphorylation of rotavirus NSP5 in cells depleted of casein kinase 1α is associated with the formation of viroplasms with altered morphology and a moderate decrease in virus replication. J Gen Virol 88, 2800–2810.[CrossRef] [Google Scholar]
  14. Charpilienne, A., Lepault, J., Rey, F. & Cohen, J.(2002). Identification of rotavirus VP6 residues located at the interface with VP2 that are essential for capsid assembly and transcriptase activity. J Virol 76, 7822–7831.[CrossRef] [Google Scholar]
  15. 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.[CrossRef] [Google Scholar]
  16. Eichwald, C., Jacob, G., Muszynski, B., Allende, J. E. & Burrone, O. R.(2004a). Uncoupling substrate and activation functions of rotavirus NSP5: phosphorylation of Ser-67 by casein kinase 1 is essential for hyperphosphorylation. Proc Natl Acad Sci U S A 101, 16304–16309.[CrossRef] [Google Scholar]
  17. Eichwald, C., Rodriguez, J. F. & Burrone, O. R.(2004b). Characterisation of rotavirus NSP2/NSP5 interaction and dynamics of viroplasms formation. J Gen Virol 85, 625–634.[CrossRef] [Google Scholar]
  18. Esparza, J., Gorziglia, M., Gil, F. & Romer, H.(1980). Multiplication of human rotavirus in cultured cells: an electron microscopic study. J Gen Virol 47, 461–472.[CrossRef] [Google Scholar]
  19. Estes, M. & Kapikian, A.(2007). Rotaviruses. In Fields Virology, 5th edn, pp. 1917–1974. Edited by D. M. Knipe & P. M. Howley Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins.
  20. 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]
  21. 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]
  22. Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B.(1986). Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83, 8122–8126.[CrossRef] [Google Scholar]
  23. 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.[CrossRef] [Google Scholar]
  24. Graham, A., Kudesia, G., Allen, A. M. & Desselberger, U.(1987). Reassortment of human rotavirus possessing genome rearrangements with bovine rotavirus: evidence for host cell selection. J Gen Virol 68, 115–122.[CrossRef] [Google Scholar]
  25. Kar, A. K., Bhattacharya, B. & Roy, P.(2007). Bluetongue virus RNA binding protein NS2 is a modulator of viral replication and assembly. BMC Mol Biol 8, 4[CrossRef] [Google Scholar]
  26. Kattoura, M. D., Chen, X. & Patton, J. T.(1994). The rotavirus RNA-binding protein NS35 (NSP2) forms 10S multimers and interacts with the viral RNA polymerase. Virology 202, 803–813.[CrossRef] [Google Scholar]
  27. Kobayashi, T., Antar, A. A., Boehme, K. W., Danthi, P., Eby, E. A., Guglielmi, K. M., Holm, G. H., Johnson, E. M., Maginnis, M. S. & other authors(2007). A plasmid-based reverse genetics system for animal double-stranded RNA viruses. Cell Host Microbe 1, 147–157.[CrossRef] [Google Scholar]
  28. Komoto, S., Sasaki, J. & Taniguchi, K.(2006). Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus. Proc Natl Acad Sci U S A 103, 4646–4651.[CrossRef] [Google Scholar]
  29. Lopez, T., Rojas, M., Ayala-Breton, C., Lopez, S. & Arias, C. F.(2005). Reduced expression of the rotavirus NSP5 gene has a pleiotropic effect on virus replication. J Gen Virol 86, 1609–1617.[CrossRef] [Google Scholar]
  30. Martin, D., Duarte, M., Lepault, J. & Poncet, D.(2009). Sequestration of free tubulin molecules by the viral protein NSP2 induces microtubule depolymerization during rotavirus infection. J Virol 84, 2522–2532. [Google Scholar]
  31. Miller, C. L., Arnold, M. M., Broering, T. J., Hastings, C. E. & Nibert, M. L.(2010). Localization of mammalian orthoreovirus proteins to cytoplasmic factory-like structures via nonoverlapping regions of μNS. J Virol 84, 867–882.[CrossRef] [Google Scholar]
  32. Mohan, K. V., Muller, J. & 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]
  33. Montero, H., Rojas, M., Arias, C. F. & Lopez, S.(2008). Rotavirus infection induces the phosphorylation of eIF2α but prevents the formation of stress granules. J Virol 82, 1496–1504.[CrossRef] [Google Scholar]
  34. Parker, J. S., Broering, T. J., Kim, J., Higgins, D. E. & Nibert, M. L.(2002). Reovirus core protein μ2 determines the filamentous morphology of viral inclusion bodies by interacting with and stabilizing microtubules. J Virol 76, 4483–4496.[CrossRef] [Google Scholar]
  35. Patton, J. T., Vasquez-Del Carpio, R., Tortorici, M. A. & Taraporewala, Z. F.(2007). Coupling of rotavirus genome replication and capsid assembly. Adv Virus Res 69, 167–201. [Google Scholar]
  36. 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.[CrossRef] [Google Scholar]
  37. Pim, D., Massimi, P., Dilworth, S. M. & Banks, L.(2005). Activation of the protein kinase B pathway by the HPV-16 E7 oncoprotein occurs through a mechanism involving interaction with PP2A. Oncogene 24, 7830–7838.[CrossRef] [Google Scholar]
  38. 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]
  39. Vascotto, F., Campagna, M., Visintin, M., Cattaneo, A. & Burrone, O. R.(2004). Effects of intrabodies specific for rotavirus NSP5 during the virus replicative cycle. J Gen Virol 85, 3285–3290.[CrossRef] [Google Scholar]
  40. Wei, T., Uehara-Ichiki, T., Miyazaki, N., Hibino, H., Iwasaki, K. & Omura, T.(2009). Association of rice gall dwarf virus with microtubules is necessary for viral release from cultured insect vector cells. J Virol 83, 10830–10835.[CrossRef] [Google Scholar]
  41. Zeng, C. Q., Wentz, M. J., Cohen, J., Estes, M. K. & Ramig, R. F.(1996). Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants. J Virol 70, 2736–2742. [Google Scholar]
  42. 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]

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vol. , part 7, pp. 1782 - 1793

Confocal immunofluorescence of viroplasms and VLS-NSP2i

VLS-VP2i in NSP5–EGFP/MA104 cells

3D reconstruction of VLS

VLS formation upon transient co-transfection of NSP5–EGFP

Cellular distribution of VP6 [Single PDF file](1003 KB)


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