Rabies is a currently incurable lethal disease, which has a 100% case-fatality rate, the highest of known infectious diseases. Rabies is caused by lyssaviruses, including rabies virus and Australian bat lyssavirus; although lyssaviruses make only five proteins, they can both mediate viral replication and arrest potent control over the biology of the infected host cell and the host immune system. Central to this is the multifunctional ‘P protein’ that is positioned at the core of the virus-host interface where it forms a myriad of interactions with viral and host proteins critical to replication and disease progression. We have shown that preventing such interactions, we can prevent an otherwise invariable lethal disease in vivo, identifying the P protein axis as an important target for antiviral approaches. However, the mechanisms by which the P protein is able to coordinate and regulate its interactions, and the structural basis of these interactions remain unresolved.
Biophysical and structural-biology approaches including NMR are been used to define the molecular basis of P-protein C-terminal domain (CTD) interactions with host immune (STAT1) and viral replication (N) proteins. These analyses aim to determine the residues that mediate the CTD-STAT1 interaction, and thereby gain insight into the mechanism by which P-protein inhibits IFN/STAT1 responses. We expressed and purified the CTD and STAT1 at high yields, overcoming key hurdles to studies of STAT1 interactions. We have used cross-saturation transfer NMR on 2H,15N CTD and unlabelled STAT1 to identify key interacting residues on the CTD. We will present novel insights from these studies concerning the extent and composition of the molecular interface, which represent the first direct structural data for a viral IFN-antagonist-STAT1 interaction, and discuss ongoing studies of the structural and functional aspects of CTD-STAT1 interaction as well as our approaches to design broad-spectrum antiviral drugs targeting this interface.