Ultrastructural localization of rotavirus antigens using colloidal gold

Petrie, Betty L.; Greenberg, Harry B.; Graham, David Y.; Estes, Mary K.

doi:10.1016/0168-1702(84)90069-8

Cited by 145 publications

(133 citation statements)

References 26 publications

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“…The mAb's 7D9 and 8D3 (VP6-specific, IgA), 1E11 and SGI-255/60 (VP6-specific, IgG), B4-2 (NSP4-specific, IgG), and 159 (VP7-specific, IgG) have been described previously (4,17,27,28 Concentrations of the mAb preparations were calculated based on the standard curves generated from purified mouse IgG or IgA standards and adjusted to 1 mg/ml for purified mAb's and 3 mg/ml for ascites.…”

Section: Cells and Virusesmentioning

confidence: 99%

Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6–specific IgA mAb

Feng

Lawton²,

Gilbert³

et al. 2002

J. Clin. Invest.

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Rotaviruses are the leading cause of severe diarrheal disease in young children. Intestinal mucosal IgA responses play a critical role in protective immunity against rotavirus reinfection. Rotaviruses consist of three concentric capsid layers surrounding a genome of 11 segments of double-stranded RNA. The outer layer proteins, VP4 and VP7, which are responsible for viral attachment and entry, are targets for protective neutralizing antibodies. However, IgA mAb's directed against the intermediate capsid protein VP6, which do not neutralize the virus, have also been shown to protect mice from rotavirus infection and clear chronic infection in SCID mice. We investigated whether the anti-VP6 IgA (7D9) mAb could inhibit rotavirus replication inside epithelial cells and found that 7D9 acted at an early stage of infection to neutralize rotavirus following antibody lipofection. Using electron cryomicroscopy, we determined the three-dimensional structure of the virus-antibody complex. The attachment of 7D9 IgA to VP6 introduces a conformational change in the VP6 trimer, rendering the particle transcriptionally incompetent and preventing the elongation of initiated transcripts. Based on these observations, we suggest that anti-VP6 IgA antibodies confers protection in vivo by inhibiting viral transcription at the start of the intracellular phase of the viral replication cycle.

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Section: Cells and Virusesmentioning

confidence: 99%

Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6–specific IgA mAb

Feng

Lawton²,

Gilbert³

et al. 2002

J. Clin. Invest.

Self Cite

154

124

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“…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.…”

mentioning

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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“…The viral nonstructural membrane protein NSP4 executes a key role in this step. As a tetramer on the ER membrane (Bowman et al 2000), NSP4 recruits DLP and VP4 to the ER membrane's cytosolic surface (Petrie et al 1984). The energy source required to deform the ER membrane to bud DLP into the ER lumen is not clear, although multivalent interactions between NSP4 and DLP may trigger this reaction (Taylor et al 1993).…”

Section: Viruses Co-opt the Er For Infectionmentioning

confidence: 99%