Abstract
Although microtubules (MTs) are known to have important roles in intracellular transport of many viruses, a number of reports suggest that specific viral MT-associated proteins (MAPs) target MTs to subvert distinct MT-dependent cellular processes. The precise functional importance of these interactions and their roles in pathogenesis, however, remain largely unresolved. To assess the association with disease of the rabies virus (RABV) MAP, P3, we quantitatively compared the phenotypes of P3 from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derivative strain, Ni-CE. Using confocal/live-cell imaging and dSTORM super-resolution microscopy to quantify protein interactions with the MT network and with individual MT filaments, we found that the interaction by Ni-CE-P3 is significantly impaired compared with Ni-P3. This correlated with an impaired capacity to effect association of the transcription factor STAT1 with MTs and to antagonize interferon (IFN)/STAT1-dependent antiviral signaling. Importantly, we identified a single mutation in Ni-CE-P3 that is sufficient to inhibit MT-association and IFN-antagonist function of Ni-P3, and showed that this mutation alone attenuates the pathogenicity of RABV. These data provide evidence that the viral protein-MT interface has important roles in pathogenesis, suggesting that this interface could provide targets for vaccine/antiviral drug development.
Highlights
P protein isoforms have been shown to have antagonistic function toward antiviral innate immune signaling by type-I interferon (IFN) cytokines, by inhibiting both IFN induction and IFN-dependent signaling[11,12,13,14,15,16]
To assess P3-MT interactions, we used confocal laser scanning microscopy (CLSM) for a newly developed quantitative live-cell assay of protein-MT association, and super-resolution direct stochastic optical reconstruction microscopy. The latter is a single-molecule localization technique that detects photon emissions from single, spatially and temporally distinct, fluorescent molecules to precisely localize individual fluorophores; this can achieve c. 20 nm spatial resolution to enable analysis of structural changes to single MT filaments (c. 25 nm diameter). Using these approaches together with quantitative colocalization analysis, yeast2-hybrid assays, IFN-dependent reporter gene assays, and viral reverse genetics/animal infection, we show that MT-interaction and IFN/STAT1 antagonism differs significantly between P3 protein from the pathogenic and non-pathogenic viruses
Ni-CE rabies virus (RABV) strains fused to green fluorescent protein (GFP; Fig. 1) in a panel of mammalian cell lines from neuronal and extraneural tissues of several RABV susceptible/host species[23,24,25,26], including COS-7 (African green monkey kidney), NSC-34, SK-N-SH, HeLa and NA
Summary
Aaron Brice[1], Donna R. Using confocal/live-cell imaging and dSTORM super-resolution microscopy to quantify protein interactions with the MT network and with individual MT filaments, we found that the interaction by Ni-CE-P3 is significantly impaired compared with Ni-P3 This correlated with an impaired capacity to effect association of the transcription factor STAT1 with MTs and to antagonize interferon (IFN)/STAT1-dependent antiviral signaling. To assess P3-MT interactions, we used confocal laser scanning microscopy (CLSM) for a newly developed quantitative live-cell assay of protein-MT association, and super-resolution direct stochastic optical reconstruction microscopy (dSTORM) The latter is a single-molecule localization technique that detects photon emissions from single, spatially and temporally distinct, fluorescent molecules to precisely localize individual fluorophores; this can achieve c. We further identify a single residue mutation (N226-H) that significantly inhibits both processes, enabling the first direct analysis of the role of this interaction in viral pathogenicity in mice
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