Rabies virus is a non-segmented negative sense RNA virus that causes encephalitis in humans with a 100% case fatality rate, resulting in > 61,000 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). As a multifunctional protein, P also forms multiple interactions with host factors that underlie diverse roles in the virus-host interface, particularly in immune evasion. The PCTD alone contains binding sites for host factors including STAT1, PML protein, microtubules, and two phosphorylation sites. The strategies by which P mediates diverse functions in viral replication and immune evasion by coordinating its multiple interactions with viral proteins and host cellular proteins remain unknown.
We have commenced a project to investigate the structure and dynamics of PCTD and characterise its precise molecular interactions with N-pep. 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 N-pep/PCTD interaction is governed by electrostatic and hydrophobic forces. By introducing mutations and cyclizing N-pep, the binding affinity has been improved 200-fold. The structural and dynamic study of PCTD and its phosphomimetics using X-ray crystallography and NMR suggests that there is no conformational change of PCTD upon phosphorylation. NMR titration experiments further confirm that the single binding site model of PCTD/N-pep is not affected by the phosphorylation state of PCTD.