Isolation and crystallization of a unique size category of recombinant Rabies virus Nucleoprotein-RNA rings.
In order to study the packaging of rabies virus RNA inside the viral nucleocapsid, rabies nucleoprotein was expressed in insect cells. In the cells, it binds to cellular RNA to form long, helical or short circular complexes, depending on the length of the bound RNA. The circular complexes contained from 9 up to 13 N-protomers per ring. Separation of the rings into defined size classes was impossible through regular column chromatographies or gradient centrifugation. The size classes could be separated by native polyacrylamide gel electrophoresis. A large-scale separation was achieved with a 4% native gel using a preparative electrophoresis apparatus. Crystallization trials were set up with N-RNA rings from three size classes and crystals were obtained in all cases. The best diffracting crystals, diffracting up to 6A, contained rings with 11 N-protomers plus an RNA molecule of 99 nucleotides. The diffraction limit was improved to 3.5A by air dehydration prior to flash freezing.
Albertini
Aurélie A V
AA
Institut de Virologie Moléculaire et Structurale, FRE 2854 Université Joseph Fourier-CNRS, IVMS, c/o EMBL, BP 181, 38042 Grenoble cedex 9, France.
Clapier
Cedric R
CR
Wernimont
Amy K
AK
Schoehn
Guy
G
Weissenhorn
Winfried
W
Ruigrok
Rob W H
RW
D000818:Animals; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D009698:Nucleoproteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D012367:RNA, Viral / Q000737:chemistry / Q000648:ultrastructure*; D011820:Rabies virus / Q000737:chemistry / Q000648:ultrastructure*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D014721:Vesicular stomatitis Indiana virus / Q000737:chemistry / Q000648:ultrastructure; D019065:Virus Assembly
D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D012367:RNA, Viral; D011994:Recombinant Proteins