Reovirus is an icosahedral, double-stranded (ds) RNA virus that uses viral polymerases packaged within the viral core to transcribe its ten distinct plus-strand RNAs. To localize these polymerases, the structure of the reovirion was refined to a resolution of 7.6 Å by cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image reconstruction. X-ray crystal models of reovirus proteins, including polymerase λ3, were then fitted into the density map. Each copy of λ3 was found anchored to the inner surface of the icosahedral core shell, making major contacts with three molecules of shell protein λ1 and overlapping, but not centering on, a five-fold axis. The overlap explains why only one copy of λ3 is bound per vertex. λ3 is furthermore oriented with its transcript exit channel facing a small channel through the λ1 shell, suggesting how the nascent RNA is passed into the large external cavity of the pentameric capping enzyme complex formed by protein λ2.All viruses with either a single-stranded, minus-sense RNA genome (such as influenza and measles viruses) or a dsRNA genome (such as reoviruses and rotaviruses) must package virally encoded RNA-dependent RNA polymerase (RdRp) molecules within virions to transcribe the genome into single-stranded, plus-sense RNA for translation and replication during infection. The dsRNA viruses are distinct, however, in having icosahedral particles within which the RdRp molecules are regularly bound and the genome remains enclosed throughout transcription. dsRNA virus particles thus provide useful models in which to study structural aspects of RNA-dependent transcription.Reovirus, a member of the Reoviridae family of animal and plant viruses, has a genome comprising ten dsRNA segments, which are surrounded in virions by a multilayered, icosahedral protein capsid (diameter ~850 Å; mass ~110 MDa 1 ). The outer capsid layer is arranged with quasi T = 13(laevo) symmetry and includes 200 heterohexamers of the μ1 and σ3 proteins (μ1 3 σ3 3 ) (refs. 2,3) and 12 homotrimers of the σ1 protein [4][5][6][7] . These proteins are © 2003 Nature Publishing Group Correspondence should be addressed to T.S.B. (tsb@bilbo.bio.purdue.edu). COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.Note: Supplementary information is available on the Nature Structural Biology website. NIH Public Access Author ManuscriptNat Struct Biol. Author manuscript; available in PMC 2014 September 03. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript released during cell entry after fulfilling their roles in stability (σ3), attachment (σ1) and membrane penetration (μ1) (refs. 8-10). The 15-MDa, 23.5-kilobase pair genome is encased by an inner capsid layer having T = 1 symmetry and composed of 60 dimers of the λ1 protein 11 . This inner layer remains surrounding the genome after cell entry and may protect it from eliciting dsRNA-dependent host responses 12,13 . Also in the subviral 'core' that enters the cytoplasm are two proteins external ...

Mammalian reoviruses, prototype members of the Reoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins-1, 1, and 3-to enter host cells. 1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of 1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by "recoating" genome-containing core particles that lacked 1, 1, and 3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to 1. The recoated particles bound to and infected cultured cells in a 1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant 1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the 1 protein. Additional experiments showed that recoated particles containing 1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound 1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of 1 with respect to its structure, assembly into particles, and roles in entry.

Reovirus outer-capsid proteins μ1, ς3, and ς1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed μ1 and ς3 onto purified cores that lack μ1, ς3, and ς1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein ς1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of μ1 and ς3 onto cores had induced rearrangement of the pentameric λ2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH4Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the ς1 protein. To examine the utility of r-cores for genetic dissections of μ1 functions in reovirus entry, we generated r-cores containing a mutant form of μ1 that had been engineered to resist cleavage at the δ:φ junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in δ:φ cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH4Cl, providing new evidence that this cleavage is dispensable for productive infection.

Among members of the genus Orthoreovirus, family Reoviridae, a group of non-enveloped viruses with genomes comprising ten segments of double-stranded RNA, only the "non-fusogenic" mammalian orthoreoviruses (MRVs) have been studied to date by electron cryomicroscopy and three-dimensional image reconstruction. In addition to MRVs, this genus comprises other species that induce syncytium formation in cultured cells, a property shared with members of the related genus Aquareovirus. To augment studies of these "fusogenic" orthoreoviruses, we used electron cryomicroscopy and image reconstruction to analyze the virions of a fusogenic avian orthoreovirus (ARV). The structure of the ARV virion, determined from data at an effective resolution of 14.6 A, showed strong similarities to that of MRVs. Of particular note, the ARV virion has its pentameric lambda-class core turret protein in a closed conformation as in MRVs, not in a more open conformation as reported for aquareovirus. Similarly, the ARV virion contains 150 copies of its monomeric sigma-class core-nodule protein as in MRVs, not 120 copies as reported for aquareovirus. On the other hand, unlike that of MRVs, the ARV virion lacks "hub-and-spokes" complexes within the solvent channels at sites of local sixfold symmetry in the incomplete T=13l outer capsid. In MRVs, these complexes are formed by C-terminal sequences in the trimeric mu-class outer-capsid protein, sequences that are genetically missing from the homologous protein of ARVs. The channel structures and C-terminal sequences of the homologous outer-capsid protein are also genetically missing from aquareoviruses. Overall, the results place ARVs between MRVs and aquareoviruses with respect to the highlighted features.

Structure-function studies with mammalian reoviruses have been limited by the lack of a reverse-genetic system for engineering mutations into the viral genome. To circumvent this limitation in a partial way for the major outer-capsid protein ς3, we obtained in vitro assembly of large numbers of virion-like particles by binding baculovirus-expressed ς3 protein to infectious subvirion particles (ISVPs) that lack ς3. A level of ς3 binding approaching 100% of that in native virions was routinely achieved. The ς3 coat in these recoated ISVPs (rcISVPs) appeared very similar to that in virions by electron microscopy and three-dimensional image reconstruction. rcISVPs retained full infectivity in murine L cells, allowing their use to study ς3 functions in virus entry. Upon infection, rcISVPs behaved identically to virions in showing an extended lag phase prior to exponential growth and in being inhibited from entering cells by either the weak base NH4Cl or the cysteine proteinase inhibitor E-64. rcISVPs also mimicked virions in being incapable of in vitro activation to mediate lysis of erythrocytes and transcription of the viral mRNAs. Last, rcISVPs behaved like virions in showing minor loss of infectivity at 52°C. Since rcISVPs contain virion-like levels of ς3 but contain outer-capsid protein μ1/μ1C mostly cleaved at the δ-φ junction as in ISVPs, the fact that rcISVPs behaved like virions (and not ISVPs) in all of the assays that we performed suggests that ς3, and not the δ-φ cleavage of μ1/μ1C, determines the observed differences in behavior between virions and ISVPs. To demonstrate the applicability of rcISVPs for genetic studies of protein functions in reovirus entry (an approach that we call recoating genetics), we used chimeric ς3 proteins to localize the primary determinants of a strain-dependent difference in ς3 cleavage rate to a carboxy-terminal region of the ISVP-bound protein.