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Journal of Virology, July 1999, p. 5309-5319, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

In Vitro Assembly of Alphavirus Cores by Using Nucleocapsid Protein Expressed in Escherichia coli

Timothy L. Tellinghuisen, Agnes E. Hamburger,dagger Bonnie R. Fisher,Dagger Ralf Ostendorp,§ and Richard J. Kuhn*

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

Received 25 January 1999/Accepted 25 March 1999

The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19-264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19-264) or could inhibit assembly if its molar ratio relative to CP(19-264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly.

* Corresponding author. Mailing address: Department of Biological Sciences, Purdue University, Lilly Hall of Life Sciences, West Lafayette, IN 47907-1392. Phone: (765) 494-1164. Fax: (765) 496-1189. E-mail: rjkuhn{at}bragg.bio.purdue.edu.

dagger Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

Dagger Present address: Department of Chemistry, University of California, Davis, CA 95616.

§ Present address: MorphoSys AG, D-82152 Martinsried/Munich, Germany.

Journal of Virology, July 1999, p. 5309-5319, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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