133: Mechanisms of lyssavirus evasion of innate immunity and roles in lethal rabies disease

Abstract

Lyssaviruses, including rabies virus (RABV) and Australian bat lyssavirus (ABL) are a globally distributed genus of zoonotic pathogens that cause rabies disease with a case-fatality rate in humans of 100%, resulting in over 61,000 deaths/year. To understand the mechanisms underlying lyssavirus evasion of interferon (IFN)-mediated antiviral immune responses, we have used a multidisciplinary approach combining techniques including viral reverse genetics and in vivo infection, next-generation sequencing/bioinformatics, single live-cell imaging (including novel live-cell microtubule-association assays), super-resolution d STORM microscopy, immune signalling assays, and protein–protein interaction analysis. This work has determined that the lyssavirus IFN-antagonist P-protein forms a complex array of molecular interactions in host cells, including with STATs 1 and 2, microtubules, nuclear import and export receptors, and intranuclear structures [1] , [2] , [3] , [4] . Recently, we have identified novel interactions with STAT3, which enable inhibition of signalling by several cytokines [3] , and with nucleoli, and have found that P-protein specifically alters microtubule structure and dynamics. Using mutagenesis to modify specific association sequences in P-protein, we have determined that many of these interactions are critical to infection in vivo [1] , [4] . In particular, we found that interactions of lyssavirus P-proteins with STATs are conserved across the lyssavirus genus [2] , and that mutagenic inhibition of these interactions results in strongly reduced growth of virus in the CNS of infected mice to attenuate pathogenicity, such that the mutated virus causes no neurological symptoms or death compared with the invariably lethal wild-type strain [1] . Similarly, our recent studies using mutation of virus to prevent P-protein subcellular trafficking and modulation of microtubule structure indicate that this strongly impairs IFN-antagonist function, resulting in attenuation in vivo . Together, these studies demonstrate critical roles in disease of diverse interactions that underlie viral antagonism of cytokine signalling; this identifies novel targets for the generation of attenuated vaccines and antiviral drugs. Our current studies, which include structural analysis using NMR and crystallography, seek to exploit this knowledge to generate new approaches to combat rabies disease.