1887

Abstract

Analysis of the amino acid sequence of core protein μA of avian reovirus has indicated that it may share similar functions to protein μ2 of mammalian reovirus. Since μ2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant μA ( μA) was designed and used to test these activities. μA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that  μA possessed NTPase activity that enabled the protein to hydrolyse the phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of  μA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein  μA. The activity of  μA is dependent on the divalent cations Mg or Mn, but not Ca or Zn. Optimal NTPase activity of  μA was achieved between pH 5.5 and 6.0. In addition,  μA enzymic activity increased with temperature up to 40 °C and was almost totally inhibited at temperatures higher than 55 °C. Tests of phosphate release from RNA substrates with  μA or K408A/K412A  μA indicated that  μA, but not K408A/K412A  μA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of  μA might be carried out at the same active site, and that protein μA could play important roles during viral RNA synthesis.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.82592-0
2007-06-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/88/6/1797.html?itemId=/content/journal/jgv/10.1099/vir.0.82592-0&mimeType=html&fmt=ahah

References

  1. Attoui H., Fang Q., Mohd Jaafar F., Cantaloube J. F., Biagini P., de Micco P., de Lamballerie X. 2002; Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus (genus Aquareovirus , family Reoviridae ). J Gen Virol 83:1941–1951
    [Google Scholar]
  2. Bisaillon M., Lemay G. 1997; Characterization of the reovirus λ 1 protein RNA 5′-triphosphatase activity. J Biol Chem 272:29954–29957 [CrossRef]
    [Google Scholar]
  3. Bisaillon M., Bergeron J., Lemay G. 1997; Characterization of the nucleotide triphosphate phosphohydrolase and helicase activities of the reovirus λ 1 protein. J Biol Chem 272:18298–18303 [CrossRef]
    [Google Scholar]
  4. Bodelon G., Labrada L., Martinez-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]
  5. Diaz-Ruiz J. R., Kaper J. M. 1978; Isolation of viral double stranded RNAs using a LiCl fractionation procedures. Prep Biochem 8:1–17
    [Google Scholar]
  6. Duncan R. 1996; The low pH-dependent entry of avian reovirus is accompanied by two specific cleavages of the major outer capsid protein μ2C. Virology 219:179–189 [CrossRef]
    [Google Scholar]
  7. Gross C. H., Shuman S. 1996; The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding. J Virol 70:1706–1713
    [Google Scholar]
  8. Kim J., Parker J. S., Murray K. E., Nibert M. L. 2004; Nucleoside and RNA triphosphatase activities of orthoreovirus transcriptase cofactor μ2. J Biol Chem 279:4394–4403
    [Google Scholar]
  9. Kuo M. D., Chin C., Hsu S. L., Shiao J. Y., Wang T. M., Lin J. H. 1996; Characterization of the NTPase activity of Japanese encephalitis virus NS3 protein. J Gen Virol 77:2077–2084 [CrossRef]
    [Google Scholar]
  10. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  11. Martinez-Costas J., Gonzalez-Lopez C., Vakharia V. N., Benavente J. 2000; Possible involvement of the double-stranded RNA-binding core protein σ A in the resistance of avian reovirus to interferon. J Virol 74:1124–1131 [CrossRef]
    [Google Scholar]
  12. Myette J. R., Niles E. G. 1996; Characterization of the vaccinia virus RNA 5′-triphosphatase and nucleotide triphosphate phosphohydrolase activities. Demonstration that both activities are carried out at the same active site. J Biol Chem 271:11945–11952 [CrossRef]
    [Google Scholar]
  13. Noad L., Shou J., Coombs K. M., Duncan R. 2006; Sequences of avian reovirus M1, M2 and M3 genes and predicted structure/function of the encoded μproteins. Virus Res 116:45–57 [CrossRef]
    [Google Scholar]
  14. Noble S., Nibert M. L. 1997a; Characterization of an ATPase activity in reovirus cores and its genetic association with core-shell protein λ 1. J Virol 71:2182–2191
    [Google Scholar]
  15. Noble S., Nibert M. L. 1997b; Core protein μ2 is a second determinant of nucleoside triphosphatase activities by reovirus cores. J Virol 71:7728–7735
    [Google Scholar]
  16. Paoletti E., Moss B. 1974; Two nucleic acid-dependent nucleoside triphosphate phosphohydrolases from vaccinia virus. Nucleoside substrate and polynucleotide cofactor specificities. J Biol Chem 249:3281–3286
    [Google Scholar]
  17. Perkins D. N., Pappin D. J., Creasy D. M., Cottrell J. S. 1999; Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567 [CrossRef]
    [Google Scholar]
  18. Ramadevi N., Roy P. 1998; Bluetongue virus core protein VP4 has nucleoside triphosphate phosphohydrolase activity. J Gen Virol 79:2475–2480
    [Google Scholar]
  19. Rikkonen M., Peranen J., Kaariainen L. 1994; ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2. J Virol 68:5804–5810
    [Google Scholar]
  20. Schnitzer T. J., Ramos T., Gouvea V. 1982; Avian reovirus polypeptides: analysis of intracellular virus-specified products, virions, top component, and cores. J Virol 43:1006–1014
    [Google Scholar]
  21. Shmulevitz M., Yameen Z., Dawe S., Shou J., O'Hara D., Holmes I., Duncan R. 2002; Sequential partially overlapping gene arrangement in the tricistronic S1 genome segments of avian reovirus and Nelson Bay reovirus: implications for translation initiation. J Virol 76:609–618 [CrossRef]
    [Google Scholar]
  22. Spandidos D. A., Graham A. F. 1976; Physical and chemical characterization of an avian reovirus. J Virol 19:968–976
    [Google Scholar]
  23. Su Y. P., Su B. S., Shien J. H., Liu H. J., Lee L. H. 2006; The sequence and phylogenetic analysis of avian reovirus genome segments M1, M2, and M3 encoding the minor core protein μA, the major outer capsid protein μB, and the nonstructural protein μNS. J Virol Methods 133:146–157 [CrossRef]
    [Google Scholar]
  24. Taraporewala Z., 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]
  25. Tomazic-Allen S. J. 1991; Recombinant bacterial alkaline phosphatase as an immunodiagnostic enzyme. Ann Biol Clin (Paris) 49:287–290
    [Google Scholar]
  26. Varela R., Benavente J. 1994; Protein coding assignment of avian reovirus strain S1133. J Virol 68:6775–6777
    [Google Scholar]
  27. Vasiljeva L., Merits A., Auvinen P., Kaariainen L. 2000; Identification of a novel function of the alphavirus capping apparatus. RNA 5′-triphosphatase activity of Nsp2. J Biol Chem 275:17281–17287 [CrossRef]
    [Google Scholar]
  28. Vasquez-Del Carpio R., Gonzaleg-Nilo F. D., Riadi G., Taraporewala Z. F., Patton J. T. 2006; Histidine triad-like motif of the rotavirus NSP2 octamer mediates both RTPase and NTPase activities. J Mol Biol 362:539–554 [CrossRef]
    [Google Scholar]
  29. Warrener P., Tamura J. K., Collett M. S. 1993; RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria. J Virol 67:989–996
    [Google Scholar]
  30. Wiener J. R., Bartlett J. A., Joklik W. K. 1989; The sequences of reovirus serotype 3 genome segments M1 and M3 encoding the minor protein μ2 and the major nonstructural protein μNS, respectively. Virology 169:293–304 [CrossRef]
    [Google Scholar]
  31. Wu W. Y., Shien J. H., Lee L. H., Shieh H. K. 1994; Analysis of the double-stranded RNA genome segments among avian reovirus field isolates. J Virol Methods 48:119–122 [CrossRef]
    [Google Scholar]
  32. Yin H. S., Lee L. H. 2000; Characterization of avian reovirus nonstructural protein σ NS synthesized in Escherichia coli . Virus Res 67:1–9 [CrossRef]
    [Google Scholar]
  33. Yin H. S., Shieh H. K., Lee L. H. 1997; Characterization of the double-stranded RNA genome segment S3 of avian reovirus. J Virol Methods 67:93–101 [CrossRef]
    [Google Scholar]
  34. 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]
  35. Yin H. S., Su Y. P., Lee L. H. 2002; Evidence of nucleotidyl phosphatase activity associated with core protein σ A of avian reovirus S1133. Virology 293:379–385 [CrossRef]
    [Google Scholar]
  36. Yin P., Keirstead N. D., Broering T. J., Arnold M. M., Parker J. S., Nibert M. L., Coombs K. M. 2004; Comparisons of the M1 genome segments and encoded μ2 proteins of different reovirus isolates. Virol J 1:6 [CrossRef]
    [Google Scholar]
  37. Yu L., Shuman S. 1996; Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme. J Virol 70:6162–6168
    [Google Scholar]
  38. Zou S., Brown E. G. 1992; Nucleotide sequence comparison of the M1 genome segment of reovirus type 1 Lang and type 3 Dearing. Virus Res 22:159–164 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.82592-0
Loading
/content/journal/jgv/10.1099/vir.0.82592-0
Loading

Data & Media loading...

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