Elucidation of the roles of the lyssavirus P3 protein-microtubule interface in viral immune evasion and pathogenesis

© 2018 Dr. Aaron Mitchell BriceCentral to the capacity of many viruses to cause disease are their ability to subvert the host-cell’s innate antiviral immune defence, the interferon (IFN) response, through expression of IFN antagonist proteins. For rabies virus (RABV) and other members of the genus Lyssavirus, antagonism of IFN signalling is mediated by the products of their P gene, the phosphoproteins (P1 – P5). These isoforms target the vital IFN-activated signalling transcription factor signal transducers and activators of transcription 1 (STAT1) through diverse mechanisms related to their distinct intracellular localisations and association with different cellular proteins/structures. In particular, the P3 isoform interacts with the cellular microtubule (MT) cytoskeleton and induces STAT1-MT colocalisation as part of its strategy to inhibit STAT1 signalling. The contributions of this mechanism to viral pathogenesis in vivo, and the molecular details of the P3-MT interaction, however, remain unresolved. To address this, we used confocal imaging, single-molecule localisation microscopy, immune signalling assays and proteomics to perform quantitative comparative analyses of the intracellular phenotypes and IFN antagonist function of P3 proteins from a pathogenic RABV strain, Nishigahara (Ni), and a non-pathogenic, Ni-derivative strain, Ni-CE. We found that the MT-association of Ni-CE-P3 was significantly impaired compared to Ni-P3, and this correlated with a defective capacity to antagonise STAT1 signalling. Furthermore, we identified a single Ni-CE-P mutation, N226-H, that alone was sufficient to partially inhibit these processes and attenuate the capacity for RABV to cause lethal disease in mice. These data indicate that the P3-MT interface contributes significantly to RABV pathogenesis. Further analysis of the molecular mechanisms underlying P3-MT association identified several residues of P3 protein that may contribute MT-binding, including a possible regulatory mechanism involving phosphorylation of a previously identified protein kinase C target site, S210. We also present evidence that P3 protein induces bundling of MT filaments, which was also inhibited by the Ni-CE-P mutations. As this was similarly observed in cells infected with RABV in a P gene-dependent manner, this may indicate that MT-bundling may not only provide an indicator of P3-MT interaction in transfected cells, but might also be an important element of P3 protein activity in infected cells. Finally, examination of the P3 proteins expressed by diverse lyssaviruses revealed that they exhibit highly heterologous intracellular localisations, but retain a conserved targeting of STAT1. Despite this diversity, our data does suggest roles for MT-association in the STAT1 antagonist function of many lyssavirus P3 proteins. Together, the data presented in this thesis has advanced our understanding of the contributions of the P3-MT interface to lyssavirus immune evasion