Following infection, viral proteins form extensive interactions with host proteins and organelles, which underlie mechanisms to modulate critical cellular processes, including those involved in antiviral immune responses. Understanding of these mechanisms is limited, however, by the poor resolving power of standard light microscopy. Super-resolution fluorescence microscopy using single molecule localizations (SML) captures images of biological samples with resolution in the order of tens of nanometers, well below the diffraction limit of light (~200 – 300 nm) [1]. This enables resolution of individual subcellular nanostructures like microtubules (~30 nm wide) and sub-nucleolar puncta (~150 nm diameter). Sample preparation for SML imaging requires only mild fixative and immunolabelling reagents, preserving cells without significant structural perturbation. Optical manipulation of single molecule fluorescence using astigmatism [2] enables 3D super-resolution imaging of these subcellular nanostructures.
We have applied SLM to analyse key aspects of the intracellular virus-host interface. Analysis of the interactions of rabies virus (RABV) P3 protein with microtubules (MT) have revealed that P3 induces large bundles (up to 300 nm wide) that correlate with pathogenesis, such that P3 mutations prevent MT bundling and inhibits disease lethality in vivo [3]. Here, we report recent analyses of MT alterations caused by P3 from different rabies strains and related lyssaviruses, which indicate significant divergence in MT interactions between these dangerous pathogens. We also report the first use of SML, including 3D modelling, to measure sub-nucleolar puncta, and effects thereon by nucleolus-targeting proteins of pathogenic henipaviruses. Finally, we will report recent progress in developing dual-colour super-resolution imaging that will enable direct observation of interactions between viral proteins and host cell structures at unprecedented resolution.