Rabies virus chaperone: Identification of the phosphoprotein peptide that keeps nucleoprotein soluble and free from non-specific RNA

The genomic RNA of rabies virus is always complexed with the viral nucleoprotein (N). This N–RNA complex is the template for viral transcription and replication. The viral phosphoprotein (P) has two functions during the infection process: it binds through its carboxy-terminus to N in the N–RNA complex and at the same time with an amino-terminal domain to the polymerase and in this way fixes the polymerase to its template. The second function of P is to bind to newly produced N in the infected cell in order to prevent that N binds non-specifically and irreversibly to cellular RNA. In order to identify the part of the phosphoprotein that binds to N and keeps the latter soluble, we isolated the N–P complex, performed sequential protease digestions, and determined the identity of the remaining N and P peptides in the purified digested complex. Although the digestion steps removed short sequences of N, most of N remained intact and soluble, indicating that the overall structure was not affected. Most of P, including the carboxy-terminal N–RNA-binding domain, was removed during the first digestion step. N-terminal sequencing and mass spectrometry analysis identified a P peptide containing residues 4–40 that remained associated with N. Coexpression and coimmunoprecipitation experiments and yeast two-hybrid experiments showed that this peptide alone could bind to N in vivo.