Rabies virus is a non-segmented negative sense RNA virus that causes disease in humans with a 100% case fatality rate, resulting in c. 60,000 deaths/year world-wide. There are currently no treatments for rabies disease, but a number of critical interactions of viral proteins provide potential targets to develop new antiviral compounds. Of particular interest are the interactions of viral nucleo (N), phospho (P), and large/polymerase (L) wherein the N-protein encapsidates genomic RNA to form the helical nucleocapsid (N-RNA) that serves as the template for viral transcription and replication by the RNA-dependent polymerase complex, composed of the enzymatic L-protein and non-catalytic polymerase cofactor P-protein. P-protein is critical in attaching L to the N-RNA template via an interaction between the P-protein C-terminal domain (PCTD) and the C-terminal trypsin-sensitive peptide of N-protein (N-pep). This interdomain interface between N-pep and PCTD provides a potentially valuable target, but the complex structure remains only partially resolved.
We have commenced a project to characterize the precise molecular interactions of P- and N-protein. The PCTD and N-pep have been expressed in Escherichia coli, generating protein at high yield and purity. NMR titrations of PCTD with N-pep suggest a single binding mode with a micromolar affinity at the positive patch of PCTD and the flexible loop region of N-pep. The mutagenesis study indicates that the interaction between N-pep and PCTD is mainly mediated through electrostatic and hydrophobic interactions. By introducing mutations and cyclizing N-pep, the binding affinity has been improved about 200-fold, which enables elucidation of the complex structure by X-ray crystallography and NMR. The ongoing research seeks to investigate the structural features of the PCTD-N-pep complex, which will reveal the replication machinery of rabies virus as well as the strategies by which P-protein mediates diverse functions in viral replication and immune evasion.