Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures

Rabies virus nucleoprotein (N) was produced in insect cells using the baculovirus expression system described by Prehaud et al, (Virology 178, 486-497, 1990), The protein was either purified on a CsCl gradient, resulting in a mixture of nucleocapsid-like structures and beaded rings, as observed by electron microscopy, or on a glycerol gradient that resulted in a preparation of the rings only, The rings and nucleocapsid-like structures had the same morphological characteristics as viral nucleocapsids, N in these structures is an 84 Angstrom long and thin molecule that is spaced at around 34 Angstrom along the length of the nucleocapsid, identical in shape and spacing as the nucleoprotein in nucleocapsids of rabies virus and very similar to those of vesicular stomatitis virus. The recombinant nucleocapsids contained RNA with a stoichiometry similar to that found in viral nucleocapsids. The RNA bound in the beaded rings was a subset of the insect cellular RNA. One of the RNA species was partially sequenced and, although a positive identification could not be made, could correspond to a tRNA, With respect to sensitivity to trypsin and RNase digestion, the recombinant and viral nucleocapsids behaved similar, Trypsin cleaved a 17 kDa fragment from the carboxy terminus of N with only a very small effect on the morphology of the nucleocapsids. RNase A completely digested the resident RNA in both viral and recombinant nucleocapsids into fragments of 4-5 nt long, again with no effect on the morphology of the nucleocapsids, Thus, when the RNA is cleaved, the structure must be maintained by protein-protein contacts, Experiments to remove the resident RNA from viral and recombinant rabies virus nucleocapsids failed, whereas the same methods used to eliminate the RNA from vesicular stomatitis virus nucleocapsids was successful.