Mapping RNA virus replication factories in molecular detail

Technology advances in electron imaging and sample preparation mean that is now possible not only to generate high resolution structures of isolated virus particles (for example Zhu et al, 2016) but also to begin to map the key events in the viral life cycle within the cell at molecular resolution.

The virus systems selected for this are representatives from two families of RNA virus that we have studied for some years, the Picornaviridae and the Reoviridae. From these families which include a number of important human and animal pathogens we will work with model viruses that do not require containment, namely bovine enterovirus and bovine rotavirus. Both viruses can be grown to high titre in tissue culture.

Ultimately we wish to understand the interactions between cell and virus through the processes of cell entry and uncoating through to virus replication and assembly. Reverse genetics is available for enteroviruses providing an opportunity for fluorescently labelling virus components. As yet reverse genetics is not available for rotaviruses. There is the opportunity to use advancing microscopy, including live cell imaging and cryo-fluorescent microscopy to perform correlative light-electron imaging.

To provide a tractable system this project will address the replication factories that are established by picornaviruses (Belov & Sztul, 2014 ) [these virus replication systems still need a proper name!] and the, potentially very large, viroplasm structures that are formed, apparently via interactions with lipid drops in rotavirus infected cells (Crawford & Desselberger, 2016). Both of these are complex systems and are large enough to be easily recognised in infected cells. The direction of attack will be directly structural – cells will be grown on electron microscope grids, infected and analysed by electron tomography. To obtain sufficiently thin sections the frozen grid will be milled using a focussed ion beam in a scanning electron microscope. The cryo-electron tomography will be performed using a phase plate, to enhance contrast. It is unknown how far the results will be interpretable in terms of known structures and it is likely that considerable further analysis will be required to unravel the details. This will be facilitated by labelling of components, where appropriate with endogenous fluorescent tags, otherwise via specific antibodies. Such labels will then provide a handle for, eg proximity analysis using FLIM-FRET or correlative microscopy with light and electrons. Where appropriate crystallography or electron microscopy will be enrolled to provide higher resolution views of components or sub-complexes. 

Contact Supervisor

Professor David Stuart FRS

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