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Journal of Virology, July 1999, p. 5309-5319, Vol. 73, No. 7
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.
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


*
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.
Present address: Department of Biology, Massachusetts Institute of
Technology, Cambridge, MA 02139.
Present address: Department of Chemistry, University of
California, Davis, CA 95616.
§
Present address: MorphoSys AG, D-82152 Martinsried/Munich, Germany.
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