This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tellinghuisen, T. L.
Right arrow Articles by Kuhn, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tellinghuisen, T. L.
Right arrow Articles by Kuhn, R. J.

 Previous Article  |  Next Article 

Journal of Virology, May 2000, p. 4302-4309, Vol. 74, No. 9
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Nucleic Acid-Dependent Cross-Linking of the Nucleocapsid Protein of Sindbis Virus

Timothy L. Tellinghuisen and Richard J. Kuhn*

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

Received 17 December 1999/Accepted 2 February 2000

The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway.

* Corresponding author. Mailing address: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907. Phone: (765) 494-1164. Fax: (765) 496-1189. E-mail: rjkuhn{at}bragg.bio.purdue.edu.

Journal of Virology, May 2000, p. 4302-4309, Vol. 74, No. 9
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Warrier, R., Linger, B. R., Golden, B. L., Kuhn, R. J. (2008). Role of Sindbis Virus Capsid Protein Region II in Nucleocapsid Core Assembly and Encapsidation of Genomic RNA. J. Virol. 82: 4461-4470 [Abstract] [Full Text]  
  • Hong, E. M., Perera, R., Kuhn, R. J. (2006). Alphavirus capsid protein helix I controls a checkpoint in nucleocapsid core assembly.. J. Virol. 80: 8848-8855 [Abstract] [Full Text]  
  • Kiermayr, S., Kofler, R. M., Mandl, C. W., Messner, P., Heinz, F. X. (2004). Isolation of Capsid Protein Dimers from the Tick-Borne Encephalitis Flavivirus and In Vitro Assembly of Capsid-Like Particles. J. Virol. 78: 8078-8084 [Abstract] [Full Text]  
  • Ma, Y. M., Vogt, V. M. (2004). Nucleic Acid Binding-Induced Gag Dimerization in the Assembly of Rous Sarcoma Virus Particles In Vitro. J. Virol. 78: 52-60 [Abstract] [Full Text]  
  • LINGER, B. R., KUNOVSKA, L., KUHN, R. J., GOLDEN, B. L. (2004). Sindbis virus nucleocapsid assembly: RNA folding promotes capsid protein dimerization. RNA 10: 128-138 [Abstract] [Full Text]  
  • Perera, R., Navaratnarajah, C., Kuhn, R. J. (2003). A Heterologous Coiled Coil Can Substitute for Helix I of the Sindbis Virus Capsid Protein. J. Virol. 77: 8345-8353 [Abstract] [Full Text]  
  • Jones, C. T., Ma, L., Burgner, J. W., Groesch, T. D., Post, C. B., Kuhn, R. J. (2003). Flavivirus Capsid Is a Dimeric Alpha-Helical Protein. J. Virol. 77: 7143-7149 [Abstract] [Full Text]  
  • Wootton, S. K., Yoo, D. (2003). Homo-Oligomerization of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein and the Role of Disulfide Linkages. J. Virol. 77: 4546-4557 [Abstract] [Full Text]  
  • Zhang, W., Mukhopadhyay, S., Pletnev, S. V., Baker, T. S., Kuhn, R. J., Rossmann, M. G. (2002). Placement of the Structural Proteins in Sindbis Virus. J. Virol. 76: 11645-11658 [Abstract] [Full Text]  
  • Mukhopadhyay, S., Chipman, P. R., Hong, E. M., Kuhn, R. J., Rossmann, M. G. (2002). In Vitro-Assembled Alphavirus Core-Like Particles Maintain a Structure Similar to That of Nucleocapsid Cores in Mature Virus. J. Virol. 76: 11128-11132 [Abstract] [Full Text]  
  • Ma, Y. M., Vogt, V. M. (2002). Rous Sarcoma Virus Gag Protein-Oligonucleotide Interaction Suggests a Critical Role for Protein Dimer Formation in Assembly. J. Virol. 76: 5452-5462 [Abstract] [Full Text]  
  • Skoging-Nyberg, U., Liljeström, P. (2001). M-X-I Motif of Semliki Forest Virus Capsid Protein Affects Nucleocapsid Assembly. J. Virol. 75: 4625-4632 [Abstract] [Full Text]  
  • Tellinghuisen, T. L., Perera, R., Kuhn, R. J. (2001). In Vitro Assembly of Sindbis Virus Core-Like Particles from Cross-Linked Dimers of Truncated and Mutant Capsid Proteins. J. Virol. 75: 2810-2817 [Abstract] [Full Text]  
  • Perera, R., Owen, K. E., Tellinghuisen, T. L., Gorbalenya, A. E., Kuhn, R. J. (2001). Alphavirus Nucleocapsid Protein Contains a Putative Coiled Coil {alpha}-Helix Important for Core Assembly. J. Virol. 75: 1-10 [Abstract] [Full Text]  
  • Gaspar, L. P., Terezan, A. F., Pinheiro, A. S., Foguel, D., Rebello, M. A., Silva, J. L. (2001). The Metastable State of Nucleocapsids of Enveloped Viruses as Probed by High Hydrostatic Pressure. J. Biol. Chem. 276: 7415-7421 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»