1887

Abstract

The avian reovirus non-structural protein NS has previously been shown to bind single-stranded (ss) RNA in a sequence-independent manner. The results of the present study further reveal that NS binds poly(A), poly(U) and ssDNA, but not poly(C), poly(G) or duplex nucleic acids, suggesting that NS has some nucleotide-sequence specificity for ssRNA binding. The current findings also show that NS is present in large ribonucleoprotein complexes in the cytoplasm of avian reovirus-infected cells, indicating that it exists in intimate association with ssRNAs . Removal of RNA from the complexes generates a NS protein form that sediments between 4·5 and 7 S, suggesting that RNA-free NS associates into small oligomers. Expression and purification of recombinant NS in insect cells allowed us to generate specific antibodies and to perform a variety of assays. The results of these assays revealed that: (i) RNA-free NS exists as homodimers and homotrimers; (ii) the minimum RNA size for NS binding is between 10 and 20 nt; (iii) NS does not have a preference for viral mRNA sequences; and (iv) its RNA-binding activity is conformation-dependent. Baculovirus expression of point and deletion NS mutants in insect cells showed that the five conserved basic amino acids that are important for RNA binding and ribonucleoprotein-complex formation are dispersed throughout the entire NS sequence, suggesting that this protein binds ssRNA through conformational domains. Finally, the properties of the avian reovirus protein NS are compared with those of its mammalian reovirus counterpart.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.80491-0
2005-04-01
2019-11-22
Loading full text...

Full text loading...

/deliver/fulltext/jgv/86/4/vir861159.html?itemId=/content/journal/jgv/10.1099/vir.0.80491-0&mimeType=html&fmt=ahah

References

  1. Bodelón, G., Labrada, L., Martínez-Costas, J. & Benavente, J. ( 2001; ). The avian reovirus genome segment S1 is a functionally tricistronic gene that expresses one structural and two nonstructural proteins in infected cells. Virology 290, 181–191.[CrossRef]
    [Google Scholar]
  2. Boyle, J. F. & Holmes, K. V. ( 1986; ). RNA-binding proteins of bovine rotavirus. J Virol 58, 561–568.
    [Google Scholar]
  3. Burd, C. G. & Dreyfuss, G. ( 1994; ). Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615–621.[CrossRef]
    [Google Scholar]
  4. Chiu, C. J. & Lee, L. H. ( 1997; ). Cloning and nucleotide sequencing of the S4 genome segment of avian reovirus S1133. Arch Virol 142, 2515–2520.[CrossRef]
    [Google Scholar]
  5. Cordingley, M. G., LaFemina, R. L., Callahan, P. L. & 8 other authors ( 1990; ). Sequence-specific interaction of Tat protein and Tat peptides with the transactivation-responsive sequence element of human immunodeficiency virus type 1 in vitro. Proc Natl Acad Sci U S A 87, 8985–8989.[CrossRef]
    [Google Scholar]
  6. Fillmore, G. C., Lin, H. & Li, J. K.-K. ( 2002; ). Localization of the single-stranded RNA-binding domains of bluetongue virus nonstructural protein NS2. J Virol 76, 499–506.[CrossRef]
    [Google Scholar]
  7. Gillian, A. L. & Nibert, M. L. ( 1998; ). Amino terminus of reovirus nonstructural protein σNS is important for ssRNA binding and nucleoprotein complex formation. Virology 240, 1–11.[CrossRef]
    [Google Scholar]
  8. 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.[CrossRef]
    [Google Scholar]
  9. Gomatos, P. J., Prakash, O. & Stamatos, N. M. ( 1981; ). Small reovirus particles composed solely of sigma NS with specificity for binding different nucleic acids. J Virol 39, 115–124.
    [Google Scholar]
  10. González, S. & Ortín, J. ( 1999; ). Characterization of influenza virus PB1 protein binding to viral RNA: two separate regions of the protein contribute to the interaction domain. J Virol 73, 631–637.
    [Google Scholar]
  11. Grande, A. & Benavente, J. ( 2000; ). Optimal conditions for the growth, purification and storage of the avian reovirus S1133. J Virol Methods 85, 43–54.[CrossRef]
    [Google Scholar]
  12. Hsiao, J., Martínez-Costas, J., Benavente, J. & Vakharia, V. N. ( 2002; ). Cloning, expression, and characterization of avian reovirus guanylyltransferase. Virology 296, 288–299.[CrossRef]
    [Google Scholar]
  13. 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.[CrossRef]
    [Google Scholar]
  14. Jones, R. C. ( 2000; ). Avian reovirus infections. Rev Sci Tech 19, 614–625.
    [Google Scholar]
  15. Kenan, D. J., Query, C. C. & Keene, J. D. ( 1991; ). RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci 16, 214–220.[CrossRef]
    [Google Scholar]
  16. Kochan, G., Gonzalez, D. & Rodriguez, J. F. ( 2003; ). Characterization of the RNA-binding activity of VP3, a major structural protein of Infectious bursal disease virus. Arch Virol 148, 723–744.[CrossRef]
    [Google Scholar]
  17. Lazinski, D., Grzadzielska, E. & Das, A. ( 1989; ). Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif. Cell 59, 207–218.[CrossRef]
    [Google Scholar]
  18. Martínez-Costas, J., Varela, R. & Benavente, J. ( 1995; ). Endogenous enzymatic activities of the avian reovirus S1133: identification of the viral capping enzyme. Virology 206, 1017–1026.[CrossRef]
    [Google Scholar]
  19. Martínez-Costas, J., Gónzalez-López, C., Vakharia, V. N. & Benavente, J. ( 2000; ). Possible involvement of the double-stranded RNA-binding core protein σA in the resistance of avian reovirus to interferon. J Virol 74, 1124–1131.[CrossRef]
    [Google Scholar]
  20. Merrill, B. M., Stone, K. L., Cobianchi, F., Wilson, S. H. & Williams, K. R. ( 1988; ). Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotein. J Biol Chem 263, 3307–3313.
    [Google Scholar]
  21. Miller, C. L., Broering, T. J., Parker, J. S. L., Arnold, M. M. & Nibert, M. L. ( 2003; ). Reovirus σNS protein localizes to inclusions through an association requiring the μNS amino terminus. J Virol 77, 4566–4576.[CrossRef]
    [Google Scholar]
  22. Query, C. C., Bentley, R. C. & Keene, J. D. ( 1989; ). A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell 57, 89–101.[CrossRef]
    [Google Scholar]
  23. Richardson, M. A. & Furuichi, Y. ( 1985; ). Synthesis in Escherichia coli of the reovirus nonstructural protein σ NS. J Virol 56, 527–533.
    [Google Scholar]
  24. Robertson, M. D. & Wilcox, G. E. ( 1986; ). Avian reovirus. Vet Bull 56, 155–174.
    [Google Scholar]
  25. Rould, M. A., Perona, J. J., Söll, D. & Steitz, T. A. ( 1989; ). Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2·8 Å resolution. Science 246, 1135–1142.[CrossRef]
    [Google Scholar]
  26. Schnitzer, T. J. ( 1985; ). Protein coding assignment of the S genes of the avian reovirus S1133. Virology 141, 167–170.[CrossRef]
    [Google Scholar]
  27. Shelton, I. H., Kasupski, G. J., Jr, Oblin, C. & Hand, R. ( 1981; ). DNA binding of a nonstructural reovirus protein. Can J Biochem 59, 122–130.[CrossRef]
    [Google Scholar]
  28. Spandidos, D. A. & Graham, A. F. ( 1976; ). Physical and chemical characterization of an avian reovirus. J Virol 19, 968–976.
    [Google Scholar]
  29. Taraporewala, Z. F., Chen, D. & Patton, J. T. ( 1999; ). Multimers formed by the rotavirus nonstructural protein NSP2 bind to RNA and have nucleoside triphosphatase activity. J Virol 73, 9934–9943.
    [Google Scholar]
  30. Tourís-Otero, F., Cortez-San Martín, M., Martínez-Costas, J. & Benavente, J. ( 2004a; ). Avian reovirus morphogenesis occurs within viral factories and begins with the selective recruitment of σNS and λA to μNS inclusions. J Mol Biol 341, 361–374.[CrossRef]
    [Google Scholar]
  31. Tourís-Otero, F., Martínez-Costas, J., Vakharia, V. N. & Benavente, J. ( 2004b; ). Avian reovirus nonstructural protein μNS forms viroplasm-like inclusions and recruits protein σNS to these structures. Virology 319, 94–106.[CrossRef]
    [Google Scholar]
  32. van der Heide, L. ( 2000; ). The history of avian reovirus. Avian Dis 44, 638–641.[CrossRef]
    [Google Scholar]
  33. Varela, R. & Benavente, J. ( 1994; ). Protein coding assignment of avian reovirus strain S1133. J Virol 68, 6775–6777.
    [Google Scholar]
  34. Yin, H. S. & Lee, L. H. ( 1998; ). Identification and characterization of RNA-binding activities of avian reovirus non-structural protein σNS. J Gen Virol 79, 1411–1413.
    [Google Scholar]
  35. Yin, H. S. & Lee, L. H. ( 2000; ). Characterization of avian reovirus non structural protein σNS synthesized in Escherichia coli. Virus Res 67, 1–9.[CrossRef]
    [Google Scholar]
  36. Yin, H. S., Shien, J.-H. & Lee, L. H. ( 2000; ). Synthesis in Escherichia coli of avian reovirus core protein σA and its dsRNA-binding activity. Virology 266, 33–41.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.80491-0
Loading
/content/journal/jgv/10.1099/vir.0.80491-0
Loading

Data & Media loading...

Most Cited This Month

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