Structure of recombinant rabies virus nucleoprotein-RNA complex and identification of the phosphoprotein binding site

Rabies virus nucleoprotein (N) was produced in insect cells, in which it fo rms nucleoprotein-RNA (N-RNA) complexes that are biochemically and biophysi cally indistinguishable from rabies virus N-RNA. We selected recombinant N- RNA complexes that were bound to short insect cellular RNAs which formed sm all 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 b ound to recombinant rabies virus phosphoprotein (the viral polymerase cofac tor), 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 w ere calculated from single-particle image analysis combined with back proje ction. 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 param eters. Using negative-stain electron microscopy, we visualized phosphoprote in bound to the tips of the N-RNA rings, most likely at the site that can b e removed by trypsin. Based on the shape of N determined here and on struct ural parameters derived from electron microscopy on free rabies virus N-RNA and from nucleocapsid in virus, we propose a low-resolution model for rabi es virus N-RNA in the virus.