Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures

Rabies virus nucleoprotein (N) was produced in insect cells using the bacul ovirus 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 e lectron microscopy, or on a glycerol gradient that resulted in a preparatio n of the rings only, The rings and nucleocapsid-like structures had the sam e morphological characteristics as viral nucleocapsids, N in these structur es is an 84 Angstrom long and thin molecule that is spaced at around 34 Ang strom along the length of the nucleocapsid, identical in shape and spacing as the nucleoprotein in nucleocapsids of rabies virus and very similar to t hose 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 identi fication could not be made, could correspond to a tRNA, With respect to sen sitivity to trypsin and RNase digestion, the recombinant and viral nucleoca psids behaved similar, Trypsin cleaved a 17 kDa fragment from the carboxy t erminus of N with only a very small effect on the morphology of the nucleoc apsids. RNase A completely digested the resident RNA in both viral and reco mbinant nucleocapsids into fragments of 4-5 nt long, again with no effect o n the morphology of the nucleocapsids, Thus, when the RNA is cleaved, the s tructure must be maintained by protein-protein contacts, Experiments to rem ove the resident RNA from viral and recombinant rabies virus nucleocapsids failed, whereas the same methods used to eliminate the RNA from vesicular s tomatitis virus nucleocapsids was successful.