Binding of Rabies Virus Polymerase Cofactor to Recombinant Circular Nucleoprotein–RNA Complexes

In rabies virus, the attachment of the L polymerase (L) to the viral nucleocapsids (NCs)—a nucleoprotein (N)–RNA complex that serves as template for RNA transcription and replication—is mediated by the polymerase cofactor, the phosphoprotein (P). P forms dimers (P2) that bind through their C-terminal domains (PCTD) to the C-terminal region of the N. Recombinant circular Nm–RNA complexes containing 9 to 12 protomers of N (hereafter, the subscript m denotes the number of N protomers) served here as model systems for studying the binding of P to NC-like Nm–RNA complexes. Titration experiments show that there are only two equivalent and independent binding sites for P dimers on the Nm–RNA rings and that each P dimer binds through a single PCTD. A dissociation constant in the nanomolar range (160 ± 20 nM) was measured by surface plasmon resonance, indicating a strong interaction between the two partners. Small-angle X-ray scattering (SAXS) data and small-angle neutron scattering data showed that binding of two PCTD had almost no effect on the size and shape of the Nm–RNA rings, whereas binding of two P2 significantly increased the size of the complexes. SAXS data and molecular modeling were used to add flexible loops (NNTD loop, amino acids 105–118; NCTD loop, amino acids 376–397) missing in the recently solved crystal structure of the circular N11–RNA complex and to build a model for the N10–RNA complex. Structural models for the Nm–RNA–(PCTD)2 complexes were then built by docking the known PCTD structure onto the completed structures of the circular N10–RNA and N11–RNA complexes. A multiple-stage flexible docking procedure was used to generate decoys, and SAXS and biochemical data were used for filtering the models. In the refined model, the PCTD is bound to the C-terminal top of one N protomer (Ni), with the C-terminal helix (α6) of PCTD lying on helix α14 of Ni. By an induced-fit mechanism, the NCTD loop of the same protomer (Ni) and that of the adjacent one (Ni − 1) mold around the PCTD, making extensive protein–protein contacts that could explain the strong affinity of P for its template. The structural model is in agreement with available biochemical data and provides new insights on the mechanism of attachment of the polymerase complex to the NC template.