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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.
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