1887

Abstract

Alanine substitution mutations in the Sendai virus nucleocapsid (NP) protein have defined highly conserved hydrophobic and charged residues from amino acids (aa) 362 to 371 that are essential for function of the protein in RNA replication. Mutant NP362, which had the change F362A, was incapable of supporting in vitro RNA replication. NP362 expressed alone formed extended oligomers which exhibited an abnormal morphology and density suggesting that these particles were not associated with any RNA. Mutant NP364, which had changes L362A and G365A, was also inactive in RNA replication; however, this was because the protein was unstable and did not form NP-NP complexes. Mutant NP370 mutant, which had changes K370A and D371A, was inactive in in vitro replication, although it could form the required NP0-P and NP-NP protein complexes. The self-assembled nucleocapsid-like particles formed by NP370 alone had a morphology like that of wild-type NP and banded in CsCl as ribonucleoprotein particles, suggesting that they contained cellular RNA. These data suggest that the replication defect of NP370 may be in the ability to specifically encapsidate Sendai virus genome RNA. Mutant NP373, where nonconserved charged residues at aa 373 and 375 were substituted with alanine, gave a wild-type phenotype. Thus these amino acids are not required for either protein-protein interactions or in vitro Sendai virus RNA replication.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-80-6-1383
1999-06-01
2022-05-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/80/6/0801383a.html?itemId=/content/journal/jgv/10.1099/0022-1317-80-6-1383&mimeType=html&fmt=ahah

References

  1. Amrute S. B., Abdul-Manan Z., Pandey V., Williams K. R., Modak M. J. 1994; Purification and nucleic acid binding properties of a fragment of type C1 /C2 heterogeneous nuclear ribonucleoprotein from thymic nuclear extracts. Biochemistry 33:8282–8291
    [Google Scholar]
  2. Bass S. H., Mulkerrin M. G., Wells J. A. 1991; A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor. Proceedings of the National Academy of Sciences, USA 88:4498–4502
    [Google Scholar]
  3. Blumberg B. M., Giorgi C., Kolakofsky D. 1983; N protein of vesicular stomatitis virus selectively encapsidates leader RNA in vitro. Cell 32:559–567
    [Google Scholar]
  4. Brennan C. A., Platt T. 1991; Mutations in an RNP1 consensus sequence of rho protein reduce RNA binding affinity but facilitate helicase turnover. Journal of Biological Chemistry 266:17296–17305
    [Google Scholar]
  5. Buchholz C. J., Spehner D., Drillien R., Neubert W. J., Homann H.E. 1993; The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly. Journal of Virology 67:5803–5812
    [Google Scholar]
  6. Buchholz C. J., Retzler C., Homann H. E., Neubert W. J. 1994; The carboxyl-terminal domain of Sendai virus nucleocapsid protein is involved in complex formation between phosphoprotein and nucleocapsid-like particles. Virology 204:770–776
    [Google Scholar]
  7. Burd C. G., Dreyfuss G. 1994; Conserved structures and diversity of functions of RNA-binding proteins. Science 265:615–621
    [Google Scholar]
  8. Calain P., Roux L. 1993; The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. Journal of Virology 67:4822–4830
    [Google Scholar]
  9. Carlsen S. R., Peluso R. W., Moyer S. A. 1985; In vitro replication of Sendai virus wild-type and defective interfering particle genome RNAs. Journal of Virology 54:493–500
    [Google Scholar]
  10. Chandrika R., Horikami S. M., Smallwood S., Moyer S. A. 1995a; Mutations in conserved domain I of the Sendai virus L polymerase protein uncouple transcription and replication. Virology 213:352–363
    [Google Scholar]
  11. Chandrika R., Myers T., Moyer S. A. 1995b; Measles virus nucleocapsid protein can function in Sendai virus defective interfering particle genome synthesis in vitro. Virology 206:777–782
    [Google Scholar]
  12. Chenik M., Chebli K., Gaudin Y., Blondel D. 1994; In vivo interaction of rabies virus phosphoprotein (P) and nucleoprotein (N): existence of two N-binding sites on P protein. Journal of General Virology 75:2889–2896
    [Google Scholar]
  13. Cunningham B. C., Wells J. A. 1989; High -resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244:1081–1085
    [Google Scholar]
  14. Curran J., Boeck R., Kolakofsky D. 1991; The Sendai virus P gene expresses both an essential protein and an inhibitor of RNA synthesis by shuffling modules via mRNA editing. EMBO Journal 10:3079–3085
    [Google Scholar]
  15. Curran J., Homann H., Buchholz C., Rochat S., Neubert W., Kolakofsky D. 1993; The hypervariable C-terminal tail of the Sendai paramyxovirus nucleocapsid protein is required for template function but not for RNA encapsidation. Journal of Virology 67:4358–4364
    [Google Scholar]
  16. Curran J., Marq J. -B., Kolakofsky D. 1995; An N-terminal domain of the Sendai paramyxovirus P protein acts as a chaperone for the NP protein during the nascent chain assembly step of genome replication. Journal of Virology 69:849–855
    [Google Scholar]
  17. Davis N. L., Arnheiter H., Wertz G. W. 1986; Vesicular stomatitis virus N and NS proteins form multiple complexes. Journal of Virology 59:751–754
    [Google Scholar]
  18. Diamond S. E., Kirkegaard K. 1994; Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperature-sensitive mutants defective in RNA synthesis. Journal of Virology 68:863–876
    [Google Scholar]
  19. Fooks A. R., Stephenson J. R., Warnes A., Dowsett A. B., Rima B. K., Wilkinson G.W.G. 1993; Measles virus nucleocapsid protein expressed in insect cells assembles into nucleocapsid-like structures. Journal of General Virology 74:1439–1444
    [Google Scholar]
  20. Fu Z. F., Zheng Y., Wunner W. H., Koprowski H., Deitzschold B. 1994; Both the N- and the C-terminal domains of the nominal phosphoprotein of rabies virus are involved in binding to the nucleoprotein. Virology 200:590–597
    [Google Scholar]
  21. 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. Proceedings of the National Academy of Sciences, USA 83:8122–8126
    [Google Scholar]
  22. Garcia J., Garcia-Barreno B., Vivo A., Melero J. A. 1993; Cytoplasmic inclusions of respiratory syncytial virus-infected cells–formation of inclusion-bodies in transfected cells that co-express the nucleoprotein, the phosphoprotein and the 22K protein. Virology 195:243–247
    [Google Scholar]
  23. Higuchi R., Krummel B., Saiki R. K. 1988; A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Research 16:7351–7367
    [Google Scholar]
  24. Homann H. E., Willenbrink W., Buchholz C. J., Neubert W. J. 1991; Sendai virus protein–protein interactions studied by a protein-blotting protein-overlay technique: mapping of domains on NP protein required for binding to P protein. Journal of Virology 65:1304–1309
    [Google Scholar]
  25. Horikami S. M., Curran J., Kolakofsky D., Moyer S. A. 1992; Complexes of Sendai virus NP–P and P–L proteins are required for defective interfering particle genome replication in vitro. Journal of Virology 66:4901–4908
    [Google Scholar]
  26. Howard M., Wertz G. 1989; Vesicular stomatitis virus RNA replication: a role for the NS protein. Journal of General Virology 70:2683–2694
    [Google Scholar]
  27. Huber M., Cattaneo R., Spielhofer P., Orvell C., Norrby E., Messerli M., Perriard J.-C., Billeter M. A. 1991; Measles virus phospho-protein retains the nucleocapsid protein in the cytoplasm. Virology 185:299–308
    [Google Scholar]
  28. Kingsbury D. 1991 (editor) The Paramyxoviruses New York: Plenum Press;
    [Google Scholar]
  29. Lamb R. A., Kolakofsky D. 1996; Paramyxoviridae: the viruses and their replication. In Fields Virology pp 1177–1204 Edited by Fields N. B., Knipe D. M., Howley P. M. Philadelphia: Lippincott–Raven;
    [Google Scholar]
  30. Miyahara K., Kitada S., Yoshimoto M., Matsumura H., Kawano M., Komada H., Tsurudome M., Kusugawa S., Nishio M., Ito Y. 1992; Molecular evolution of paramyxoviruses: nucleotide sequence analyses of the human parainfluenza type I virus NP and M protein genes and construction of phylogenetic trees for all the human paramyxoviruses. Archives of Virology 124:255–268
    [Google Scholar]
  31. Morgan E. M., Re G. G., Kingsbury D. 1984; Complete sequence of the Sendai virus NP gene from a cloned insert. Virology 135:279–287
    [Google Scholar]
  32. Moyer S. A., Smallwood-Kentro S., Haddad A., Prevec L. 1991; Assembly and transcription of synthetic vesicular stomatitis virus nucleocapsids. Journal of Virology 65:2170–2178
    [Google Scholar]
  33. Myers T. M., Moyer S. A. 1997; An amino-terminal domain of the Sendai virus nucleocapsid protein is required for template function in viral RNA synthesis. Journal of Virology 71:918–924
    [Google Scholar]
  34. Myers T. M., Pieters A., Moyer S. A. 1997; A highly conserved region of the Sendai virus nucleocapsid protein contributes to the NP–NP binding domain. Virology 229:322–335
    [Google Scholar]
  35. Nishio M., Tsurudome M., Kawano M., Watanabe N., Ohgimoto S., Ito M., Komada H., Ito Y. 1996; Interaction between nucleocapsid protein (NP) and phosphoprotein (P) of human parainfluenza virus type 2: one of the two NP binding sites on P is essential for granule formation. Journal of General Virology 77:2457–2463
    [Google Scholar]
  36. Peluso R. W., Moyer S. A. 1988; Viral proteins required for the in vitro replication of vesicular stomatitis virus defective interfering particle genome RNA. Virology 162:369–376
    [Google Scholar]
  37. Precious B., Young D. F., Bermingham A., Fearns R., Ryan M., Randall R. E. 1995; Inducible expression of the P, V and NP genes of the paramyxovirus SV5 in cell-lines and an examination of NP: P and NP: V interactions. Journal of Virology 69:8001–8010
    [Google Scholar]
  38. Predki P. F., Nayak L. M., Gottlieb M. B. C., Regan L. 1995; Dissecting RNA–protein interactions: RNA–RNA recognition by Rop. Cell 80:41–50
    [Google Scholar]
  39. Smallwood S., Moyer S. A. 1993; Promoter analysis of the vesicular stomatitis virus RNA polymerase. Virology 192:254–263
    [Google Scholar]
  40. Spehner D., Kirn A., Drillien R. 1991; Assembly of nucleocapsid-like structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein. Journal of Virology 65:6296–6300
    [Google Scholar]
  41. Spehner D., Drillien R., Howley P. M. 1997; The assembly of the measles virus nucleoprotein into nucleocapsid-like particles is modulated by the phosphoprotein. Virology 232:260–268
    [Google Scholar]
  42. Sprague J., Condra J. H., Arnheiter H., Lazzarini R. A. 1983; Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein. Journal of Virology 45:773–781
    [Google Scholar]
  43. Zhao H., Banerjee A. K. 1995; Interaction between the nucleocapsid protein and the phosphoprotein of human parainfluenza virus 3. Journal of Biological Chemistry 270:12485–12490
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-80-6-1383
Loading
/content/journal/jgv/10.1099/0022-1317-80-6-1383
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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