1887

Abstract

Nucleocapsid (N) proteins from representative viruses of three genera within the were expressed in insect cells using recombinant baculoviruses. RNA-containing structures, which appear morphologically identical to viral nucleocapsids, were isolated and subsequently imaged under a transmission electron microscope. Analysis of these images revealed marked differences in nucleocapsid morphology among the genera investigated, most notably between viruses of the and the subfamilies. Helical pitch measurements were made, revealing that measles virus (MV, a within the subfamily ) N protein produces helices that adopt multiple conformations with varying degrees of flexibility, while that of the simian virus type 5 (SV5, subfamily ) produces more rigid structures with a less heterogeneous pitch distribution. Nucleocapsids produced by respiratory syncytial virus (RSV, subfamily ) appear significantly narrower than those of MV and SV5 and have a longer pitch than the most extended form of MV. In addition to helical nucleocapsids, ring structures were also produced, image analysis of which has demonstrated that rings assembled from MV N protein consist of 13 subunits. This is consistent with previous reports that Sendai virus nucleocapsids have 13·07 subunits per turn. It was determined, however, that SV5 subnucleocapsid rings have 14 subunits, while rings derived from the radically different RSV nucleocapsid have been found to contain predominantly 10 subunits.

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2002-08-01
2024-04-25
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References

  1. Bermingham A., Collins P. L. 1999; The M2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription. Proceedings of the National Academy of Sciences, USA 96:11259–11264
    [Google Scholar]
  2. 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]
  3. Collins P. L., Hill M. G., Cristina J., Grosfeld H. 1996; Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus. Proceedings of the National Academy of Sciences, USA 93:81–85
    [Google Scholar]
  4. Collins P. L., Chanock R. M., Murphy B. R. 2001; Respiratory syncytial virus. In Fields Virology pp 1443–1485 Edited by Knipe D. M., Howley P. M. Philadelphia: Lippincott Williams & Wilkins;
    [Google Scholar]
  5. Coronel E. C., Takimoto T., Murti K. G., Varich N., Portner A. 2001; Nucleocapsid incorporation into parainfluenza virus is regulated by specific interaction with matrix protein. Journal of Virology 75:1117–1123
    [Google Scholar]
  6. Curran J. 1996; Reexamination of the Sendai virus P protein domains required for RNA synthesis: a possible supplemental role for the P protein. Virology 221:130–140
    [Google Scholar]
  7. 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]
  8. Egelman E. H., Wu S. S., Amrein M., Portner A., Murti G. 1989; The Sendai virus nucleocapsid exists in at least four different helical states. Journal of Virology 63:2233–2243
    [Google Scholar]
  9. Fearns R., Peeples M. E., Collins P. L. 1997; Increased expression of the N protein of respiratory syncytial virus stimulates minigenome replication but does not alter the balance between the synthesis of mRNA and antigenome. Virology 236:188–201
    [Google Scholar]
  10. Fearns R., Collins P. L., Peeples M. E. 2000; Functional analysis of the genomic and antigenomic promoters of human respiratory syncytial virus. Journal of Virology 74:6006–6014
    [Google Scholar]
  11. Fooks A. R., Stephenson J. R., Warnes A., Dowsett A. B., Rima B. K., Wilkinson G. W. 1993; Measles virus nucleocapsid protein expressed in insect cells assembles into nucleocapsid-like structures. Journal of General Virology 74:1439–1444
    [Google Scholar]
  12. Frank J., Radermacher M., Penczek P., Zhu J., Li Y., Ladjadj M., Leith A. 1996; spider and web: processing and visualization of images in 3D electron microscopy and related fields. Journal of Structural Biology 116:190–199
    [Google Scholar]
  13. Galinski M. S. 1991; Paramyxoviridae : transcription and replication. Advances in Virus Research 39:129–162
    [Google Scholar]
  14. Gubbay O., Curran J., Kolakofsky D. 2001; Sendai virus genome synthesis and assembly are coupled: a possible mechanism to promote viral RNA polymerase processivity. Journal of General Virology 82:2895–2903
    [Google Scholar]
  15. Heggeness M. H., Scheid A., Choppin P. W. 1980; Conformation of the helical nucleocapsids of paramyxoviruses and vesicular stomatitis virus: reversible coiling and uncoiling induced by changes in salt concentration. Proceedings of the National Academy of Sciences, USA 77:2631–2635
    [Google Scholar]
  16. 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]
  17. Iseni F., Barge A., Baudin F., Blondel D., Ruigrok R. W. 1998; Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures. Journal of General Virology 79:2909–2919
    [Google Scholar]
  18. Kato A., Ohnishi Y., Kohase M., Saito S., Tashiro M., Nagai Y. 2001; Y2, the smallest of the Sendai virus C proteins, is fully capable of both counteracting the antiviral action of interferons and inhibiting viral RNA synthesis. Journal of Virology 75:3802–3810
    [Google Scholar]
  19. Lamb R. A., Kolakofsky D. 2001; Paramyxoviridae : The viruses and their replication. In Fields Virology pp 1305–1340 Edited by Knipe D. M., Howley P. M. Philadelphia: Lippincott Williams & Wilkins;
    [Google Scholar]
  20. Meric C., Spehner D., Mazarin V. 1994; Respiratory syncytial virus nucleocapsid protein (N) expressed in insect cells forms nucleocapsid-like structures. Virus Research 31:187–201
    [Google Scholar]
  21. Mountcastle W. E., Compans R. W., Caliguiri L. A., Choppin P. W. 1970; Nucleocapsid protein subunits of simian virus 5, Newcastle disease virus, and Sendai virus. Journal of Virology 6:677–684
    [Google Scholar]
  22. Mountcastle W. E., Compans R. W., Lackland H., Choppin P. W. 1974; Proteolytic cleavage of subunits of the nucleocapsid of the paramyxovirus simian virus 5. Journal of Virology 14:1253–1261
    [Google Scholar]
  23. Murphy S. K., Parks G. D. 1997; Genome nucleotide lengths that are divisible by six are not essential but enhance replication of defective interfering RNAs of the paramyxovirus simian virus 5. Virology 232:145–157
    [Google Scholar]
  24. Murray J., Loney C., Murphy L. B., Graham S., Yeo R. P. 2001; Characterization of monoclonal antibodies raised against recombinant respiratory syncytial virus nucleocapsid (N) protein: identification of a region in the carboxy terminus of N involved in the interaction with P protein. Virology 289:252–261
    [Google Scholar]
  25. Myers T. M., Smallwood S., Moyer S. A. 1999; Identification of nucleocapsid protein residues required for Sendai virus nucleocapsid formation and genome replication. Journal of General Virology 80:1383–1391
    [Google Scholar]
  26. Penczek P., Radermacher M., Frank J. 1992; Three-dimensional reconstruction of single particles embedded in ice. Ultramicroscopy 40:33–53
    [Google Scholar]
  27. Pringle C. R. 1997; The order Mononegavirales : current status. Archives of Virology 142:2321–2326
    [Google Scholar]
  28. Pringle C. R. 1999; Virus taxonomy at the XIth International Congress of Virology, Sydney, Australia, 1999. Archives of Virology 144:2065–2070
    [Google Scholar]
  29. Radecke F., Spielhofer P., Schneider H., Kaelin K., Huber M., Dotsch C., Christiansen G., Billeter M. A. 1995; Rescue of measles viruses from cloned DNA. EMBO Journal 14:5773–5784
    [Google Scholar]
  30. Samal S. K., Collins P. L. 1996; RNA replication by a respiratory syncytial virus RNA analog does not obey the rule of six and retains a nonviral trinucleotide extension at the leader end. Journal of Virology 70:5075–5082
    [Google Scholar]
  31. Schoehn G., Iseni F., Mavrakis M., Blondel D., Ruigrok R. W. 2001; Structure of recombinant rabies virus nucleoprotein–RNA complex and identification of the phosphoprotein binding site. Journal of Virology 75:490–498
    [Google Scholar]
  32. Schroeter J. P., Bretaudiere J. P. 1996; suprim: easily modified image processing software. Journal of Structural Biology 116:131–137
    [Google Scholar]
  33. Spehner D., Kirn A., Drillien R. 1991; Assembly of nucleocapsidlike structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein. Journal of Virology 65:6296–6300
    [Google Scholar]
  34. 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]
  35. Tapparel C., Maurice D., Roux L. 1998; The activity of Sendai virus genomic and antigenomic promoters requires a second element past the leader template regions: a motif (GNNNNN)3 is essential for replication. Journal of Virology 72:3117–3128
    [Google Scholar]
  36. van Heel M., Frank J. 1981; Use of multivariate statistics in analysing the images of biological macromolecules. Ultramicroscopy 6:187–194
    [Google Scholar]
  37. Vulliemoz D., Roux L. 2001; ‘Rule of six’: how does the Sendai virus RNA polymerase keep count?. Journal of Virology 75:4506–4518
    [Google Scholar]
  38. Whittaker M., Carragher B. O., Milligan R. A. 1995; phoelix: a package for semi-automated helical reconstruction. Ultramicroscopy 58:245–259
    [Google Scholar]
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