Structures of Reoviridae virions and subviral particles examined by cryo-electron microscopy and image analysis

The Reoviridae family consists of unenveloped icosahedral viruses which have a segmented dsRNA genome encapsidated by a multi-layered protein shell. These large viruses ($\sim$70-80 nm in diameter) contain all the enzymes required for dsRNA transcription. To better understand the structural basis of viral infectivity and morphogenesis, I computed reconstructions of two Reoviridae members, rotavirus and reovirus, as well as several subviral particles, at $\sim$25-35A resolution using cryo-electron microscopy and image analysis. All the particles studied are well characterized biochemically. Because both internal and external features are revealed in the density maps, the combination of structural and biochemical data provides a powerful means to localize the constituent protein subunits and thereby establish structure-function relationships. Rotavirus is formed by six structural proteins arranged in three capsid shells. The reconstructions of rhesus rotavirus and of self-assembled particles of two bovine rotavirus proteins demonstrate that the two outermost capsids are organized on a T = 13(laevo) icosahedral lattice. A total of 780 copies of VP7 form the thin outer capsid, and the viral hemagglutinin, VP4, extends from the particle surface as bi-lobed spikes. The middle shell consists of 260 columnar VP6 trimers, and VP2 constitutes the major core capsid protein. The structures of three nested particles of reovirus were also studied. Proteolysis of virions converts them into infectious subviral particles (ISVPs), which lack $\sigma$3 and exhibit a conformational change in $\sigma$1, and further proteolysis removes $\mu$1 and $\sigma$1 leaving core particles. Difference maps between the three reconstructions permit $\sigma$3 to be unequivocally assigned to densities on the outer surface of virions; $\mu$1 to trimers in ISVPs; and $\sigma$1 to portions of density which adopt different conformations in virions and ISVPs. $\lambda$2 constitutes the major portion of the twelve vertex-associated spikes in the core. Additional density, which extends into the interior of empty ($-$dsRNA) particles, is attributed to $\lambda$3. Conformational changes in specific features distinguish the separate particles and suggest the presence of distinct functional states. For example, pentamers of $\lambda$2 enclose a cavity in virions and ISVPs but adopt an open conformation which exposes a channel in the transcriptionally-active core