Super-resolution imaging of subcellular remodelling by viral proteins

Rozario AM1, Zwettler F2, Rawlinson S3, Brice A3, Sauer M2, Whelan D1, Moseley G3 and Bell TDM1

  1. School of Chemistry, Monash University, Clayton, Victoria.
  2. Department of Biotechnology and Biophysics, University of Wuerzburg, Bavaria, Germany.
  3. School of Biomedical Sciences, Monash University, Clayton, Victoria.

Viruses are microscopic infectious agents capable of evading immune responses and causing fatal human disease. Investigating viral mechanisms and viral-host interactions using light microscopy is limited due to diffraction of light (~250 nm). Super-resolution fluorescence microscopy (SRFM) can achieve resolutions as good as 20 nm, making possible observation of nanoscale changes in virally altered cell structures. Lyssavirus phosphoproteins (P1-P5) interact with STAT1 to antagonize interferon-mediated antiviral responses. Previously, we observed rabies lyssavirus (RABV) P3 bind onto microtubules (MTs) and induce bundling. Mutations to P3 diminish MT bundling, correlating with improved interferon response and reduced lethality in mice models (Brice et al., Sci Rep. 2016, doi: 10.1038/srep33493). However, the precise role of MT bundling in lyssavirus disease progression is still unclear. Here we report on using SRFM to analyse bundling effects of P3 from other lyssaviruses, finding significant divergence in MT interactions between the related pathogens. To elucidate bundle structure, we have developed assays for expansion microscopy (ExM), a method that physically enlarges samples ~4-fold for improved imaging resolution, potentially down to <10 nm. Additionally, we have investigated pathogenic henipavirus matrix protein (HeV M) which localises in subnucleolar puncta and binds Treacle, a protein involved in DNA-damage response (DDR) machinery. We have imaged for the first time using SRFM subnucleolar Treacle puncta (100-200 nm) and effects thereon of HeV M, and show that M protein subverts Treacle and supresses rRNA synthesis to a similar extent as during a DDR (Rawlinson et al., doi: