Poster Presentation 12th Australasian Virology Society Meeting 2024

Nucleolar Hendra Virus Interactions Visualised By Expansion Microscopy (#251)

Nathan Sos 1 , Patrick Veugelers 1 2 , Ilya Glinin 1 , Gregory Moseley 2 , Stephen Rawlinson 2 , Toby Bell 1
  1. School of Chemistry, Monash University, Melbourne, Victoria, Australia
  2. Department of Microbiology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia

Hendra Virus (HeV) is a bat-borne virus associated with fatal human and equine infection, for which no available treatment currently exists [1]. Among its six viral proteins, the Matrix (M) protein is crucial in viral assembly and budding, and, intriguingly, also traffics to the nucleus and nucleolus during infection. Previously, we demonstrated that HeV M hijacks the nucleolar DNA damage response by binding to Treacle protein within a sub-nucleolar compartment corresponding to fibrillar centre/dense fibrillar component (FC-DFC), leading to suppression of ribosomal RNA biogenesis [2]. While confocal microscopy suggests that Treacle and M colocalise in these discreet nucleolar sub-compartments [2], the diffraction limit of light (~200 nm) and the small size of these sub-nucleolar compartments have thus far limited efforts to elucidate their relative spatial organisation in greater detail.

The previous two decades have seen tremendous strides in biological imaging with the inception of super-resolution microscopy – a suite of techniques capable of circumventing the diffraction limit. Alongside the many optical avenues to improve imaging fidelity, novel sample preparation strategies by way of Expansion Microscopy (ExM) have provided alternative routes to resolution gain. ExM samples are embedded in polyelectrolyte hydrogels that swell in water, such that acquisition of sub-diffraction detail with diffraction-limited hardware is possible.

This work presents the application of Protein-Retention Expansion Microscopy (proExM) [3] in visualising HeV M-Treacle interactions within nucleoli. ProExM conferred an approximate four-fold sample enlargement; allowing us to observe HeV M and Treacle in the FC-DFC. We have also studied two mutants of HeV M protein (K258A and R57D) which show strikingly different sub-nuclear distributions (impaired and heightened FC-DFC accumulation, respectively). Preliminary R57D M data suggest there is a localisation offset between R57D M and Treacle, whereby Treacle appears to form shells around R57D M-occupied compartments.

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