Assembly intermediates of orthoreovirus captured in the cell

Abstract

Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected ‘single shelled’ intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is ‘collapsed’ compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.

CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy

“…Rapid vitrification of biological specimens by plunge-freezing ( Dubochet et al, 1988 ) helps minimize preparative artifacts typically associated with conventional EM procedures of chemical fixation, dehydration, resin-embedding, and pre or post heavy-metal staining ( Baker et al, 2017 ; Dillard et al, 2018 ; Lučić et al, 2013 ; Romero-Brey and Bartenschlager, 2015 ). Sample vitrification followed by cryo-EM imaging allows for the direct visualization of macromolecular structures on the nanometer- to sub-nanometer-resolution levels in a native frozen-hydrated state ( Ertel et al, 2017 ; Ke et al, 2020a, 2018b, 2018c; Koning et al, 2018 ; Li et al, 2016 ; Liu et al, 2020 ; Luque & Castón, 2020 ; Peukes et al, 2020 ; Riedel et al, 2017 ; Strauss et al, 2016 ; Sutton et al, 2020 ; Unchwaniwala et al, 2020 ; Wan et al, 2017 ; Zeev-Ben-Mordehai et al, 2016 ). Cryo-CLEM has proven to be a powerful method for in situ structural studies, including time-dependent events, especially those associated with viral entry, replication, and egress ( Briegel et al, 2010 ; Carter et al, 2020 ; Hampton et al, 2017 ; Jun et al, 2011 , 2019 ; Zhang, 2013 ).…”

Reovirus Low-Density Particles Package Cellular RNA

“…Therefore, some other mechanism must prevent complete viral RNA packaging into TC particles. Recent detection of collapsed, single-shelled particles in reovirus-infected cells suggests that the inner capsid may be assembled prior to being filled with RNA and RdRps [55]. However, TC particles appear to encapsidate the RdRp but not a complete viral genome.…”

In situ Alphavirus Assembly and Budding Mechanism Revealed by Cellular CryoET

“…While cryoEM is now commonly used to produce 3D reconstructions, the single-molecule nature of imaging also allows for analysis of a macromolecule's entire conformational landscape at atomic detail through static images of its many individual states. Analysis of CHIKV assembly states through the entire progression of virion budding differs significantly from past in situ cryoET and subvolume averaging studies of non-enveloped viruses with apparently distinct assembly intermediate populations in the cells (Dai et al, 2013;Sutton et al, 2020;Vijayakrishnan et al, 2020). Our discrete-state method of classifying and averaging those individual budding states with compositional and conformational heterogeneity into ensemble 3D classes is an important step in studying progression of a transient assembly process on the cell membrane.…”