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The genus Lyssavirus comprises fifteen viral species including the archetype rabies virus (RABV). Lyssaviruses cause rabies disease that has a 100% case:fatality rate in humans, resulting in over 61,000 deaths/year and an annual financial burden of US$ 6 billion worldwide. A major aspect of viral infection is the capacity to evade host interferon (IFN)-mediated innate antiviral immunity, and it has been reported that RABV uses a unique mechanism to achieve this through selective targeting of IFN-activated STAT (signal transducer and activator of transcription) proteins by the viral phosphoprotein (P). The interaction of P-protein with STAT1 and STAT2 has been assumed to be critical to disease and to thereby represent a potential target for antiviral drugs and virus attenuation strategies for vaccine development. However, at commencement of the project described in this thesis, the precise binding site and specific residues of P-protein involved in the interaction with STATs had not been identified, so that no virus defective for this interaction had been generated. As a result, the genuine importance of P-protein:STAT interaction in infection and disease and its value as a therapeutic target was unknown. To address this, a comparative study of IFN/STAT antagonist functions of P-proteins from a panel of lyssaviruses representative of the evolutionary diversity in the genus, and of differing pathogenicity was performed. This revealed that the selective targeting of IFN-activated STATs by P-proteins is broadly conserved among lyssaviruses, indicative of importance to infection. However, subtle differences were observed in the interaction and inhibition of STATs by specific P-proteins. This enabled targeted sequence/structure analysis and site-directed mutagenesis to characterise the STAT-binding site for the first time, by identifying specific residues within a hydrophobic pocket of the P-protein C-terminal domain (CTD), the ‘W-hole’, that are critical for P-protein:STAT interaction and, thereby, antagonism of IFN-dependent antiviral responses. Importantly, although RABV P-protein is a multifunctional protein with essential roles in viral genome replication and transcription, we showed that mutation of the W-hole does not affect these functions, indicating that this region represents a highly specific, distinct site for STAT interaction. These findings enabled the generation of the first mutant lyssavirus specifically deficient for STAT interaction (STAT-blind virus). Growth of STAT-blind virus was indistinguishable from parental wild-type (WT) virus in cells that cannot produce IFN, but was strongly impaired following exposure to IFN, indicative of greatly increased IFN sensitivity. This showed that immune evasion functions of RABV P-protein are separable from its other key functions in infection, and provided the first structural insights into how P-protein can coordinate its diverse functions, a fundamentally important aspect of viral biology. Importantly, infection of mice indicated that growth of the STAT-blind virus is strongly attenuated in the brain, producing no major neurological symptoms or fatalities compared with the invariably lethal parental WT virus. This provides the first direct evidence that P-protein interaction with STATs in the central nervous system (CNS) is critical to the development of lethal rabies in vivo, and that STAT antagonism per se represents a potent target for viral attenuation. The characterisation of the P-protein STAT-binding interface, and of specific attenuating mutations therein, is significant to efforts to develop new vaccines/therapeutics for currently incurable lyssavirus infection. |
