Correlation of cryo-X-ray tomogram of viral infected cells with cryo-SIM data, left panel shows the ...
A virus life cycle can be simplified into four fundamental steps: entry, replication, assembly, and release of progeny viruses. For enveloped viruses, the entry step is well characterized while for non-enveloped viruses there is a critical lack of understanding of the mechanisms driving host-membrane penetration by the viral particles. Following entry, viruses establish complex strategies to initiate replication. The newly synthesised viral proteins and genomes are selectively recruited and packaged into assembling virus particles, in areas of the cell known as viral factories (VFs) or viral inclusion bodies. In this project, we will use the non-enveloped virus, mammalian reovirus, as a model to study the mechanisms leading to host-membrane penetration by a non-enveloped virus as well as to understand how the ultrastructural organisation of VFs drive viral particle assembly and genome packaging.
Cryo soft X-ray microscopy enables the visualisation of membranes in intact living cells in remarkable detail. In this proposal, we aim to investigate by cryo-X-ray tomography the entry step of the non-enveloped virus reovirus. We will use fluorescently labelled viral particles to visualize in unaltered infected cells the mechanism developed by reovirus to penetrate the host-membrane. In addition, we are interested in gaining novel ultrastructural insights of VFs using cryo-X-ray-microscopy and correlate this information with our previous EM tomograms of fixed stained VFs. This will allow us to better understand how the ultrastructural organization of VFs participate in the coordination of virus assembly and genome packaging.
The aims of this project are to use B24, the Diamond Life Sciences soft X-ray microscope beamline, to: 1) characterize the mechanisms by which non-enveloped reoviruses bypass the host cellular membrane by visualizing single viral particles (circa 60nm in diameter; preliminary data on particle imaging from B24; Figure 1) being released from endosomes using CLEM and cryo-X-ray tomography approaches; 2) characterize how the liquid-like phase VFs coordinate virus replication and assembly using X-ray-tomography and cryo-FIB-SEM approaches; (3) document virus-induced membrane remodelling (multivesicular body formation) within the context of cellular ultrastructure?
We have established a fluorescent reporter system allowing us to visualize single viral particles breaching the host membrane barrier. In brief, viral particles are fluorescently labelled and used to infect cells expressing galectin3 (Gal3-cherry). Gal3 is a lectin protein that binds carbohydrate moieties. We exploit this function to detect penetrating particles as during this process, carbohydrates normally localized outside the cell will be exposed to the cytosol and then sensed by Gal3. By performing live single viral particle microscopy combined with cryo-CLEM and cryo-X ray tomography approaches, we aim at visualizing at high resolution the “ruptured” endosomes to determine how the non-enveloped virus reovirus penetrates the host membrane.
Project reference number: 1020
|Professor Jonathan M Grimes||Structural Biology||Oxford University, Henry Wellcome Building of Genomic Medicine||GBRfirstname.lastname@example.org|
|Professor David Stuart FRS||Structural Biology||Oxford University, Henry Wellcome Building of Genomic Medicine||GBRemail@example.com|
Mammalian orthoreoviruses are believed to replicate in distinctive, cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. The viral nonstructural protein muNS has been implicated in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly. In this study, we sought to identify the regions of muNS that are involved in forming factory-like inclusions in transfected cells in the absence of infection or other viral proteins. Sequences in the carboxyl-terminal one-third of the 721-residue muNS protein were linked to this activity. Deletion of as few as eight residues from the carboxyl terminus of muNS resulted in loss of inclusion formation, suggesting that some portion of these residues is required for the phenotype. A region spanning residues 471 to 721 of muNS was the smallest one shown to be sufficient for forming factory-like inclusions. The region from positions 471 to 721 (471-721 region) includes both of two previously predicted coiled-coil segments in muNS, suggesting that one or both of these segments may also be required for inclusion formation. Deletion of the more amino-terminal one of the two predicted coiled-coil segments from the 471-721 region resulted in loss of the phenotype, although replacement of this segment with Aequorea victoria green fluorescent protein, which is known to weakly dimerize, largely restored inclusion formation. Sequences between the two predicted coiled-coil segments were also required for forming factory-like inclusions, and mutation of either one His residue (His570) or one Cys residue (Cys572) within these sequences disrupted the phenotype. The His and Cys residues are part of a small consensus motif that is conserved across muNS homologs from avian orthoreoviruses and aquareoviruses, suggesting this motif may have a common function in these related viruses. The inclusion-forming 471-721 region of muNS was shown to provide a useful platform for the presentation of peptides for studies of protein-protein association through colocalization to factory-like inclusions in transfected cells. Hide abstract
Reovirus replication occurs in the cytoplasm of the host cell, in virally induced mini-organelles called virus factories. On the basis of the serotype of the virus, the virus factories can manifest as filamentous (type 1 Lang strain) or globular structures (type 3 Dearing strain). The filamentous factories morphology is dependent on the microtubule cytoskeleton; however, the exact function of the microtubule network in virus replication remains unknown. Using a combination of fluorescent microscopy, electron microscopy, and tomography of high-pressure frozen and freeze-substituted cells, we determined the ultrastructural organisation of reovirus factories. Cells infected with the reovirus microtubule-dependent strain display paracrystalline arrays of progeny virions resulting from their tiered organisation around microtubule filaments. On the contrary, in cells infected with the microtubule-independent strain, progeny virions lacked organisation. Conversely to the microtubule-dependent strain, around half of the viral particles present in these viral factories did not contain genomes (genome-less particles). Complementarily, interference with the microtubule filaments in cells infected with the microtubule-dependent strain resulted in a significant increase of genome-less particle number. This decrease of genome packaging efficiency could be rescued by rerouting viral factories on the actin cytoskeleton. These findings demonstrate that the scaffolding properties of the microtubule, and not biochemical nature of tubulin, are critical determinants for reovirus efficient genome packaging. This work establishes, for the first time, a functional correlation between ultrastructural organisation of reovirus factories with genome packaging efficiency and provides novel information on how viruses coordinate assembly of progeny particles. Hide abstract