Advertisement

Structure and function of rotavirus nonstructural protein NSP3

  • D. Poncet
  • C. Aponte
  • J. Cohen
Conference paper
Part of the Archives of Virology book series (ARCHIVES SUPPL, volume 12)

Summary

The genomes of viruses in the family Reoviridae consist of segmented double-stranded RNA. There are 10 to 12 segments depending on the genus. The 5′ ends and the 3′ ends of the RNAs present conserved motifs for each virus genus. These conserved motifs have been hypothesized to play a role in genomic segment assortment during virus morphogenesis. Using a set of monoclonal antibodies we have tried to identify rotaviral proteins that bind to RNA during infection in cell culture. This methodology takes advantage of being able to label RNA in vitro to high specific activity and also of solid phase processing of RNA-protein complexes. After cross-linking the RNA to protein in infected cells, protein-RNA complexes are precipitated with a specific MAb; then, the RNA in the complex is labeled in vitro and the protein or nucleic acid moieties are analyzed by usual protocols. This paper describes results using an anti NSP3 MAb.

In infected cells, we have shown that NSP3 binds to the eleven messenger RNAs, and that a sequence from nucleotides 8 to 15 is protected from digestion with RNAse T1 by NSP3 in the RNA-protein complex. The availability of recombinant protein NSP3 expressed in the baculovirus-insect cell system has allowed the sequence specificity of NSP3 to be studied in vitro. The minimal sequence recognized by NSP3 is GACC. The role of NSP3 in rotavirus replication is discussed based on these results and by comparison with other RNA-binding proteins of members of the Reoviridae family.

Keywords

Viral mRNAs NSP3 Gene Protein NSP3 Solid Phase Processing Blue Tongue Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Aponte C, Mattion NM, Estes MK, Charpilienne A, Cohen J (1993) Expression of two bovine rotavirus non-structural proteins (NSP-2, NSP-3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins. Arch Virol 133: 85–95PubMedCrossRefGoogle Scholar
  2. 2.
    Boyle J and Holmes K (1986) RNA-binding proteins of bovine rotavirus. J Virol 58: 561–568PubMedGoogle Scholar
  3. 3.
    Cohen J, Charpilienne A, Chilmonczyk S, Estes MK (1989) Nucleotide sequence of bovine rotavirus gene 1 and expression of the gene product in baculovirus. Virology 171: 131–140PubMedCrossRefGoogle Scholar
  4. 4.
    Eiden JJ (1993) Gene-5 of the IDIR agent (Group-B rotavirus) encodes a protein equivalent to NS34 of Group-A rotavirus. Virology 196: 298–302PubMedCrossRefGoogle Scholar
  5. 5.
    Estes MK, Cohen J (1989) Rotavirus gene structure and function. Microbiol Rev 53: 410–449PubMedGoogle Scholar
  6. 6.
    Gallegos CO, Patton JT (1989) Characterization of rotavirus replication intermediates a model for the assembly of single-shelled particles. Virology 172: 616–627PubMedCrossRefGoogle Scholar
  7. 7.
    Hua J, Chen X, Patton JT (1994) Deletion mapping of the rotavirus metalloprotein NS53 (NSP1): the conserved cysteine-rich region is essential for virus-specific RNA binding. J Virol 68: 3990–4000PubMedGoogle Scholar
  8. 8.
    Kattoura MD, Chen X, Patton JT (1994) The rotavirus RNA-binding protein NS35 (NSP2) forms 105 multimers and interacts with the viral RNA polymerase. Virology 202: 803–813PubMedCrossRefGoogle Scholar
  9. 9.
    Kattoura MD, Clapp LL, Patton JT (1992) The rotavirus nonstructural protein, NS35, possesses RNA-binding activity in vitro and in vivo. Virology 191: 698–708PubMedCrossRefGoogle Scholar
  10. 10.
    L’Haridon R, Scherrer R (1976) In vitro culture of a rotavirus associated with neonatal calf scours. Annales de Recherches Vétérinaires 7: 373–381PubMedGoogle Scholar
  11. 11.
    Langland JO, Pettiford S, Jiang BM, Jacobs BL (1994) Products of the porcine group C rotavirus NSP3 gene bind specifically to double-stranded RNA and inhibit activation of the interferon-induced protein kinase PKR. J Virol 68: 3821–3829PubMedGoogle Scholar
  12. 12.
    Mattion NM, Cohen J, Estes MK (1993) The rotavirus proteins. In: Kapikian A (ed) Virus infection of the gastrointestinal tract. Marcel Dekker, New York, pp 169–249Google Scholar
  13. 13.
    Mattion NM, Cohen J, Aponte C, Estes MK (1992) Characterization of an oligomerization domain and RNA binding properties on rotavirus nonstructural protein NS34. Virology 190: 68–83PubMedCrossRefGoogle Scholar
  14. 14.
    Poncet D, Aponte C, Cohen J (1993) Rotavirus protein NSP3 (NS34) is bound to the 3′ end consensus sequence of viral mRNAs in infected cells. J Virol 67: 3159–3165PubMedGoogle Scholar
  15. 15.
    Poncet D, Laurent S, Cohen J (1994) Four nucleotides are the minimal requirement for RNA recognition by rotavirus by non-structural protein NSP3. EMBO J 13: 4165–4173PubMedGoogle Scholar
  16. 16.
    Rao CD, Das M, Ilango P, Lalwani R, Rao BS, Gowda K (1995) Comparative nucleotide and amino acid sequence analysis of the sequence-specific RNA-binding rotavirus nonstructural protein NSP3. Virology 207: 327–333PubMedCrossRefGoogle Scholar
  17. 17.
    Stamatos NM, Gomatos PJ (1992) Binding to selected regions of reovirus mRNAs by a nonstructural reovirus protein. Proc Natl Acad Sci USA 79: 3457–3461CrossRefGoogle Scholar
  18. 18.
    Van-Staden-V, Huismans-H (1991) A comparison of the genes which encode nonstructural protein NS3 of different orbiviruses. J Gen Virol 72: 1073–1079PubMedCrossRefGoogle Scholar
  19. 19.
    Wentz M, Zeng CQ-Y, Patton JT, Estes MK, Ramig RF (1995) Defining the cis-acting elements for replication of rotavirus RNA. Am Soc Virol meeting Austin TX.Google Scholar

Copyright information

© Springer-Verlag Wien 1996

Authors and Affiliations

  • D. Poncet
    • 1
  • C. Aponte
    • 1
  • J. Cohen
    • 1
  1. 1.Laboratoire de Virologie et Immunologie Moleculaires INRAC.R.J., Domaine de VilvertJouy-en-JosasFrance

Personalised recommendations