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Journal of Virology, January 2001, p. 490-498, Vol. 75, No. 1
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.490-498.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Structure of Recombinant Rabies Virus
Nucleoprotein-RNA Complex and Identification of the Phosphoprotein
Binding site
Guy
Schoehn,1
Frédéric
Iseni,1,
Manos
Mavrakis,1
Danielle
Blondel,2 and
Rob
W. H.
Ruigrok1,*
European Molecular Biology Laboratory
Grenoble Outstation, 38042 Grenoble cedex 9,1
and Laboratoire de Génétique des Virus, Centre
National de la Recherche Scientifique, 91198 Gif-sur-Yvette
cedex,2 France
Received 18 July 2000/Accepted 30 September 2000
Rabies virus nucleoprotein (N) was produced in insect cells, in
which it forms nucleoprotein-RNA (N-RNA) complexes that are biochemically and biophysically indistinguishable from rabies virus
N-RNA. We selected recombinant N-RNA complexes that were bound to short
insect cellular RNAs which formed small rings containing 9 to 11 N
monomers. We also produced recombinant N-RNA rings and viral N-RNA that
were treated with trypsin and that had lost the C-terminal quarter of
the nucleoprotein. Trypsin-treated N-RNA no longer bound to recombinant
rabies virus phosphoprotein (the viral polymerase cofactor), so the
presence of the C-terminal part of N is needed for binding of the
phosphoprotein. Both intact and trypsin-treated recombinant N-RNA rings
were analyzed with cryoelectron microscopy, and three-dimensional
models were calculated from single-particle image analysis combined
with back projection. Nucleoprotein has a bilobed shape, and each
monomer has two sites of interaction with each neighbor. Trypsin
treatment cuts off part of one of the lobes without shortening the
protein or changing other structural parameters. Using negative-stain
electron microscopy, we visualized phosphoprotein bound to the tips of
the N-RNA rings, most likely at the site that can be removed by
trypsin. Based on the shape of N determined here and on structural
parameters derived from electron microscopy on free rabies virus N-RNA
and from nucleocapsid in virus, we propose a low-resolution model for
rabies virus N-RNA in the virus.
*
Corresponding author. Mailing address: EMBL Grenoble
Outstation, c/o ILL, Polygone Scientifique, 6 rue Jules Horowitz, 38000 Grenoble, France. Phone: 33-4-76-20-72-73. Fax: 33-4-76-20-71-99. E-mail: ruigrok{at}embl-grenoble.fr.

Present address: Department of Microbiology, University of Geneva
Medical School, CH-1211 Geneva 4,
Switzerland.
Journal of Virology, January 2001, p. 490-498, Vol. 75, No. 1
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.490-498.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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