Super-resolution microscopy reveals important roles for microtubule bundling by rabies virus P3 protein in immune evasion and disease — ASN Events
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Super-resolution microscopy reveals important roles for microtubule bundling by rabies virus P3 protein in immune evasion and disease (#228)

Aaron M Brice 1 , Donna R Whelan 2 , Kim Lieu 3 , Naoto Ito 4 , Linda Wiltzer 3 , Camden Y Lo 5 , Danielle Blondel 6 , Toby DM Bell 2 , David A Jans 3 , Gregory W Moseley 1
  1. Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia
  2. School of Chemistry, Monash University, Melbourne, Victoria, Australia
  3. Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia
  4. Faculty of Applied Biological Sciences and the United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan
  5. Monash Micro Imaging, Melbourne, Victoria, Australia
  6. Unité de Virologie Moleculaire et Structurale, CNRS, Gif sur Yvette Cedex, France

Rabies virus (RABV) is the causative agent of rabies, a severe neurological disease with a c.100% case-fatality rate that results in >55,000 human deaths per year.  The high lethality of RABV is thought to be dependent on its ability to inhibit host interferon (IFN)-mediated antiviral immune signalling via the association of its IFN-antagonist P3 protein and STAT immune signalling proteins.  In vitro studies have indicated that P3 association with the microtubule (MT) cytoskeleton is important to STAT signalling inhibition but the molecular details of the P3-STAT-MT interaction and its roles in disease in vivo are currently unresolved.

Using a unique RABV pathogenicity model, viral reverse genetics, immune signalling assays and a novel, quantitative in vivo MT tracing assay using live 3D confocal laser scanning microscopy (CLSM), we identified a specific mutation (N226-H) that can impair the MT-association, as detected by CLSM, and IFN-antagonistic function of P3.  Introduction of the N226-H mutation into an invariably lethal strain of RABV strongly impaired disease progression in vivo in mice.  Importantly, analysis utilising super-resolution dSTORM (direct STochastic Optical Reconstruction Microscopy) revealed that P3 induces significant MT bundling, and that N226-H is defective in this respect.  However, N226-H does not affect the physical interaction of P3 with MT fractions or tubulin, indicating that the mutation specifically impacts P3’s capacity to modify MT structure.  This indicates a vital link between MT bundling and inhibition of immune signalling by P3 and this mechanism is crucial to RABV pathogenicity, a novel discovery among viruses.

This data indicates that RABV and potentially other pathogenic viruses exploit/modulate dynamic MT functions to manipulate host cell biology.  This study has clear implications for our understanding of viral disease mechanisms and their potential targeting for vaccine/therapeutic development and highlights the power of super-resolution microscopic approaches to probe molecular events at the host-pathogen interface.  Ongoing research aims to characterise in detail the molecular events at the MT interface of RABV, Ebola and HIV-1.