Structural analysis of critical interactions in the replication machinery of rabies virus

Abstract

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 (P-CTD) and the C-terminal trypsin-sensitive peptide of N protein (N-pep), which provides a potentially valuable target for anti-viral drug design. As a multifunctional protein, P also forms multiple interactions with host factors that underlie diverse roles in the virus-host interface, particular in immune evasion. Its C-terminal domain (P-CTD) alone bears binding sites for host factors including STAT1, microtubule and PML protein and two phosphorylation sites, apart from N-binding site. The strategies by which P mediates diverse functions in viral replication and immune evasion by coordinating its multiple interactions with viral protein and host cellular proteins remain unknown.

We have commenced a project to investigate the structure and dynamics of P-CTD and characterize its precise molecular interactions with N-pep. The P-CTD and N-pep have been expressed in Escherichia coli, generating protein at high yield and purity. NMR titrations of P-CTD with N-pep suggest a single binding mode with a micromolar affinity at the positive patch of P-CTD and the flexible loop region of N-pep. A mutagenesis study indicates that the interaction between N-pep and P-CTD is mainly mediated through electrostatic and hydrophobic interactions. By introducing mutations and cyclizing N-pep, the binding affinity has been improved 200-fold. The structural and dynamic study of P-CTD and its phosphomimetics using X-ray crystallography and NMR suggests that there is no conformational change of P-CTD upon phosphorylation. NMR titration experiments further confirmed that the single binding site model of P-CTD/N-pep is not affected by the phosphorylation state of P-CTD.