IRIS Explorer Software for Radial-Depth Cueing Reovirus Particles and Other Macromolecular Structures Determined by Cryoelectron Microscopy and Image Reconstruction

Spencer, Stephan M.; Sgro, Jean-Yves; Dryden, Kelly A.; Baker, Timothy S.; Nibert, Max L.

doi:10.1006/jsbi.1997.3902

Cited by 23 publications

(11 citation statements)

References 49 publications

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“…10), became visible when the contour level was raised by only a small amount for T3D cores (Fig. 6F), as previously described for type 1 Lang (T1L) cores (Spencer et al, 1997). These pores were not visible in the T3D spikeless cores displayed at even higher contour levels (Figs.…”

Section: Figsupporting

confidence: 77%

Loss of Activities for mRNA Synthesis Accompanies Loss of λ2 Spikes from Reovirus Cores: An Effect of λ2 on λ1 Shell Structure

et al. 2002

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The 144-kDa lambda2 protein, a component of the transcriptionally active reovirus core particle, catalyzes the last three enzymatic activities for formation of the 5' cap 1 structure on the viral plus-strand transcripts. Limited evidence suggests it may also play a role in transcription per se. Particle-associated lambda2 forms pentameric turrets ("spikes") around the fivefold axes of the icosahedral core. To address the requirements for lambda2 in core functions other than the known functions in RNA capping, particles depleted of lambda2 were generated from cores in vitro by a series of treatments involving heat, protease, and ionic detergent. The resulting particles contained less than 5% of pretreatment levels of lambda2 but showed negligible loss of the other four core proteins or the 10 double-stranded RNA genome segments. Transmission cryo-electron microscopy (cryo-TEM) and scanning cryo-electron microscopy demonstrated loss of the lambda2 spikes from these otherwise intact particles. In functional analyses, the "spikeless cores" showed greatly reduced activities not only for RNA capping but also for transcription and nucleoside triphosphate hydrolysis, suggesting enzymatic or structural roles for lambda2 in all these activities. Comparison of the core and spikeless core structures obtained by cryo-TEM and three-dimensional image reconstruction revealed changes in the lambda1 core shell that accompany lambda2 loss, most notably the elimination of small pores that span the shell near the icosahedral fivefold axes. Changes in the shell may explain the reductions in transcriptase-related activities by spikeless cores.

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Section: Figsupporting

confidence: 77%

Loss of Activities for mRNA Synthesis Accompanies Loss of λ2 Spikes from Reovirus Cores: An Effect of λ2 on λ1 Shell Structure

et al. 2002

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“…4a-c). This region of λ1 has been identified as an opening to a small channel through the shell 47,48 (Figs. 4a,c and 6).…”

Section: Orientation Of λ3 and Route Of Transcript Exitmentioning

confidence: 99%

Reovirus polymerase λ3 localized by cryo-electron microscopy of virions at a resolution of 7.6 Å

Zhang

Walker

Chipman

et al. 2003

Nat Struct Mol Biol

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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 ...

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“…The arrow in (B) identifies the heart-shaped cavity within the outer capsid that is surrounded by the 2 protein. Color in (C), (F), and (I) was added as a function of radial depth in the particles (Spencer et al, 1997) as indicated in the associated color legend. Shifts in hue were incorporated to designate the major layers in orthoreovirus particles: blue, outer capsid; lavender, core shell; yellow, internal to the core shell.…”

Section: Figmentioning

confidence: 99%

Internal/Structures Containing Transcriptase-Related Proteins in Top Component Particles of Mammalian Orthoreovirus

et al. 1998

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The structure of mammalian orthoreovirus top component particles, which are profoundly deficient in the content of double-stranded RNA genome, was determined at 30 A resolution by transmission cryoelectron microscopy and three-dimensional image reconstruction. Previously undetected, ordered densities, appearing primarily as pentameric flowers in the reconstruction, were seen to extend 65 A inwardly from the inner capsid at the icosahedral fivefold axes. Identically positioned but lower density elements were observed in two types of partially uncoated top component particles obtained by limited proteolysis. The levels of three inner-capsid proteins-lamda 1, lamda 3, and mu 2-were reduced in concert with the internal densities during proteolytic uncoating. Since lamda 3 contains the catalytic regions of the viral RNA polymerase and since both lamda 1 and mu 2 appear to play roles in transcription or mRNA capping, the internal structures are concluded to be complexes of the viral transcriptase-related enzymes. The findings have implications for the mechanisms of transcription and mRNA capping by orthoreovirus particles.

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IRIS Explorer Software for Radial-Depth Cueing Reovirus Particles and Other Macromolecular Structures Determined by Cryoelectron Microscopy and Image Reconstruction

Cited by 23 publications

References 49 publications

Loss of Activities for mRNA Synthesis Accompanies Loss of λ2 Spikes from Reovirus Cores: An Effect of λ2 on λ1 Shell Structure

Loss of Activities for mRNA Synthesis Accompanies Loss of λ2 Spikes from Reovirus Cores: An Effect of λ2 on λ1 Shell Structure

Reovirus polymerase λ3 localized by cryo-electron microscopy of virions at a resolution of 7.6 Å

Internal/Structures Containing Transcriptase-Related Proteins in Top Component Particles of Mammalian Orthoreovirus

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