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

Luongo, Cindy; Xing, Zhang; Walker, Stephen B.; Ya, Chen; Broering, Teresa J.; Farsetta, Diane L.; Bowman, Valorie D.; Baker, Timothy S.; Nibert, Max L.

doi:10.1006/viro.2001.1258

Cited by 15 publications

(11 citation statements)

References 47 publications

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“…Reovirus strains Type 1 Lang (T1L) and T3D were our laboratory stocks, originally obtained from Dr. B. N. Fields. Rabbit polyclonal antiserum and purified polyclonal antibodies specific for NS or 3 have been described previously (18,19). Monoclonal antibody (mAb) HA.11 specific for influenza virus hemagglutinin (HA) epitope was obtained from Covance Research Products, and rabbit polyclonal antibody specific for the HA epitope was obtained from Zymed Laboratories Inc. mAb DO-1 specific for p53 (Santa Cruz Biotechnology) was a generous gift from Dr. D. M. Knipe (Harvard Medical School).…”

Section: Methodsmentioning

confidence: 99%

Virus-derived Platforms for Visualizing Protein Associations inside Cells

Miller

Arnold

Broering³

et al. 2007

Molecular & Cellular Proteomics

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Protein-protein associations are vital to cellular functions. Here we describe a helpful new method to demonstrate protein-protein associations inside cells based on the capacity of orthoreovirus protein NS to form large cytoplasmic inclusions, easily visualized by light microscopy, and to recruit other proteins to these structures in a specific manner. We introduce this technology by the identification of a sixth orthoreovirus protein, RNA-dependent RNA polymerase 3, that was recruited to the structures through an association with NS. We then established the broader utility of this technology by using a truncated, fluorescently tagged form of NS as a fusion platform to present the mammalian tumor suppressor p53, which strongly recruited its known interactor simian virus 40 large T antigen to the NS-derived structures. In both examples, we further localized a region of the recruited protein that is key to its recruitment. Using either endogenous p53 or a second fluorescently tagged fusion of p53 with the rotavirus NSP5 protein, we demonstrated p53 oligomerization as well as p53 association with another of its cellular interaction partners, the CREB-binding proteins, within the inclusions. Furthermore using the p53-fused fluorescent NS platform in conjunction with threecolor microscopy, we identified a ternary complex comprising p53, simian virus 40 large T antigen, and retinoblastoma protein. The new method is technically simple, uses commonly available resources, and is adaptable to high throughput formats. Molecular & Cellular Proteomics 6:1027-1038, 2007.Protein-protein associations are vital to cellular functions. Numerous methods are available for addressing whether proteins associate either directly ("interaction") or indirectly (through bridging molecules), but many of these methods can be technically demanding, can be limited by the strength of the association, or cannot resolve whether the association indeed occurs inside cells. In addition, many current objectives and strategies demand methods that are simple and adaptable to high throughput formats. In this report, we describe a new such method that is based on an unusual property of the orthoreovirus-encoded NS protein.Double-stranded RNA viruses of the taxonomic family Reoviridae (e.g. orbiviruses, orthoreoviruses, and rotaviruses) form characteristically shaped, non-membrane-bound inclusions in the cytoplasm of infected cells. These structures are variously called viral inclusion bodies (orbiviruses) (1), viral factories (orthoreoviruses) (2, 3), or viroplasms (rotaviruses) (4) and are thought to be the sites of viral RNA replication and packaging into assembly intermediates that can later mature into infectious virions. The viral factories of some orthoreoviruses (such as certain isolates of strain Type 3 Dearing (T3D) 1 ), for example, have a globular morphology (5, 6) and can attain large diameters (10 m or more) that make them easily identifiable by light microscopy (see Fig. 1a, left panel).Previous studies have identified the medium-sized (-c...

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Section: Methodsmentioning

confidence: 99%

Virus-derived Platforms for Visualizing Protein Associations inside Cells

Miller

Arnold

Broering³

et al. 2007

Molecular & Cellular Proteomics

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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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“…13 In addition, research undertaken on mammalian reovirus, another member of the Reoviridae family, has demonstrated that removal of the pentonal l2 protein results in complete loss of RNA transcriptase activity. 27 Taken together, these observations suggest that the reduced occupancy of VP6 trimers at the 5-fold vertices may lead to the diminished yield of RNA from transcribing Wa DLPs.…”

Section: Absence Of Pentonal Vp6 Trimers In Human Wa Dlp and The Funcmentioning

confidence: 94%

Structural Determinants of Rotavirus Subgroup Specificity Mapped by Cryo-electron Microscopy

Greig

Berriman

O’Brien

et al. 2006

Journal of Molecular Biology

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

Cited by 15 publications

References 47 publications

Virus-derived Platforms for Visualizing Protein Associations inside Cells

Virus-derived Platforms for Visualizing Protein Associations inside Cells

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

Structural Determinants of Rotavirus Subgroup Specificity Mapped by Cryo-electron Microscopy

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