Assembly intermediates of orthoreovirus captured in the cell

Abstract

Traditionally molecular assembly pathways for viruses have been inferred from high resolution structures of stable intermediates purified in vitro, and from low resolution images of cell sections as well as from genetic approaches including conditionally lethal mutants. Here, we directly visualise a previously unsuspected ‘single shelled’ icosahedral intermediate for a mammalian orthoreovirus, in addition to the expected virions, in cryo-preserved infected cells by cryo-electron tomography of cellular lamellae1,2. Particle classification and averaging yielded structures at resolutions as high as 5.6 Å, sufficient to identify secondary structural elements and place known molecular structures, allowing us to produce an atomic model of the intermediate, comprising 120 copies of protein λ1 and 120 copies of σ2. This λ1 shell is in a ‘collapsed’ form compared to the mature virions, with the molecules pushed inwards at the icosahedral 5-folds by ~100 Å. This grossly indented shell, although produced by a mammalian reovirus, is reminiscent of the first assembly intermediate of prokaryotic dsRNA viruses belonging to a different virus family3, adding weight to the supposition that these diverse viruses share a common ancestor, and suggesting mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds enormous promise for the dissection of the replication cycle of many viruses.