Protein-RNA Interactions in an Icosahedral Virus at 3.0 Å Resolution

Chen, Zhongguo; Stauffacher, Cynthia V.; Li, Yunge; Schmidt, Timothy R.; Bomu, Wu; Kamer, Greg; Shanks, Michael; Lomonossoff, George P.; Johnson, John E.

doi:10.1126/science.2749253

Cited by 199 publications

(150 citation statements)

References 25 publications

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“…A similar arrangement of stem-loop RNA elements was earlier proposed for southern bean mosaic virus [10] and for bean pod mottle virus [11]. The distribution of stem-loops, based on the stem sizes deduced from crystallography, and the loop size, based on modeling [12], which requires at least nine nucleotides to create a stereochemically acceptable model, would consume a minimum of 80% of the 1058 nucleotides.…”

Section: Introductionmentioning

confidence: 88%

Satellite tobacco mosaic virus RNA: structure and implications for assembly

Larson

McPherson

2001

Current Opinion in Structural Biology

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Section: Introductionmentioning

confidence: 88%

Satellite tobacco mosaic virus RNA: structure and implications for assembly

Larson

McPherson

2001

Current Opinion in Structural Biology

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“…The basic protein environment and characteristic electron density strongly suggested this to be nucleic acid. As in bean pod mottle virus 48 and CPV 41 , some of the nucleic acid is bound to inside cavities of the icosahedral shell and, therefore, has itself attained some icosahedral order. The most extensive ordered DNA structure occurs in three regions of the cavities between the F protein α barrel and the rest of the capsid, including a region close to the ordered part of the J protein.…”

Section: Dna Structure and G Protein Ion Channelmentioning

confidence: 99%

Atomic structure of single-stranded DNA bacteriophage ΦX174 and its functional implications

McKenna

Xia

Willingmann

et al. 1992

Nature

215

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The mechanism of DNA ejection, viral assembly and evolution are related to the structure of bacteriophage ΦX174. The F protein forms a T = 1 capsid whose major folding motif is the eightstranded antiparallel β barrel found in many other icosahedral viruses. Groups of 5 G proteins form 12 dominating spikes that enclose a hydrophilic channel containing some diffuse electron density. Each G protein is a tight β barrel with its strands running radially outwards and with a topology similar to that of the F protein. The 12 'pilot' H proteins per virion may be partially located in the putative ion channel. The small, basic J protein is associated with the DNA and is situated in an interior cleft of the F protein. Tentatively, there are three regions of partially ordered DNA structure, accounting for about 12% of the total genome.Bacteriophage ΦX174 is a small icosahedral virus that contains a single-stranded, closed circular DNA molecule with 5,386 nucleotide bases (for a recent review, see ref. 1). Of the 11 gene products, four (J, F, G and H) participate in the structure of the virion. There are 60 copies each of the J, F and G proteins and 12 copies of the H protein per virion [2][3][4] (Table 1). Electron microscopy of shadowed or negatively stained particles [5][6][7][8] , as well as a threedimensional reconstruction of frozen-hydrated virions (Fig. 1a), clearly show large spikes at the vertices of the icosahedral particles (N.H.O., T.S.B., P.W. and N.L.I., manuscript in preparation). These spikes are about 32 Å long and have a mean diameter of 70 Å. Edgell et al. 3 have shown that these spikes, which can be removed from the capsid with 4 M urea treatment, are composed of G and H proteins. Assuming all 12 spikes are identical, then each spike will contain five G and one H protein 2 . The capsid itself has an external diameter of roughly 260 Å, with a protein shell that is about 30 Å thick, and contains the F and J proteins as well as the single-stranded (ss) DNA. The phage recognizes a lipopolysaccharide (LPS) receptor on the outer cell membrane of Escherichia coli [8][9][10][11] . Host range mutants, which determine which strains of E. coli are infected by ΦX174, map into the F, G and H genes [12][13][14][15] The infectious virion has a relative molecular mass of 6.2 × 10 6 (M r 6,200K) (26% is DNA) 32 . Lysates of infected E. coli contain two components which can be separated by sedimentation through a sucrose gradient 32,33 . The fast band contains the infectious 114S particles whereas the slow band contains noninfectious, 70S particles 32,34,35 . The latter contains about 20% of the normal DNA content with all of the genome represented in the population of particles. Both types of particles can be crystallized into isomorphous monoclinic crystals with space group P2 1 , and cell dimensions a = 305.6, b = 360.8, c = 299.5 Å, β = 92.89° that diffract at least to 2.6-Å resolution 33 . The asymmetric unit of the crystal cell contains one complete particle. We report here the three-dimensional structure...

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“…Comoviruses are plant viruses whose structural and biological properties are strikingly similar to the animal picornaviruses 6,7 . For example, the protein capsids of CPMV, the type member of the comovirus group, and poliovirus, the most extensively studied picornavirus, are comparable (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…Three-dimensional reconstructions of native cowpea mosaic virus (CMPV) and a complex of CPMV saturated with a Fab fragment of a monoclonal antibody against the virus have been determined at 23 Å resolution from low-irradiation images of unstained, frozen-hydrated samples. Despite the nominal resolution of the complex, the physical footprint of the Fab on the capsid surface and the orientation and position of the Fab have been determined to within a few angstroms by fitting atomic models of CPMV 4 and Fab (Kol) 5 to reconstructed density maps.Comoviruses are plant viruses whose structural and biological properties are strikingly similar to the animal picornaviruses 6,7 . For example, the protein capsids of CPMV, the type member of the comovirus group, and poliovirus, the most extensively studied picornavirus, are comparable (Fig.…”

mentioning

confidence: 99%

Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography

Wang

Porta

Chen

et al. 1992

Nature

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Biological processes frequently require the formation of multi-protein or nucleoprotein complexes. Some of these complexes have been produced in homogeneous form, crystallized, and analysed at high resolution by X-ray crystallography (for example, see refs 1-3). Most, however, are too large or too unstable to crystallize. Individual components of such complexes can often be purified and analysed by crystallography. Here we report how the coordinated application of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography provides a powerful approach to study large, unstable macromolecular complexes. Three-dimensional reconstructions of native cowpea mosaic virus (CMPV) and a complex of CPMV saturated with a Fab fragment of a monoclonal antibody against the virus have been determined at 23 Å resolution from low-irradiation images of unstained, frozen-hydrated samples. Despite the nominal resolution of the complex, the physical footprint of the Fab on the capsid surface and the orientation and position of the Fab have been determined to within a few angstroms by fitting atomic models of CPMV 4 and Fab (Kol) 5 to reconstructed density maps.Comoviruses are plant viruses whose structural and biological properties are strikingly similar to the animal picornaviruses 6,7 . For example, the protein capsids of CPMV, the type member of the comovirus group, and poliovirus, the most extensively studied picornavirus, are comparable (Fig. 1a). The CPMV coat contains 60 copies each of large (relative molecular mass 42,000 (M r 42K) and small (24K) protein subunits arranged with icosahedral symmetry. Each large subunit (L) is composed of two antiparallel β-barrel domains which are positioned close to the icosahedral 3-fold axes. The β-barrel domain at the amino-terminal end of L corresponds to the VP2 subunit in poliovirus and the β-barrel at the carboxy-terminal end of L corresponds to VP3 in poliovirus. The small subunits (S), packed near the icosahedral 5-fold axes, correspond to VP1 in poliovirus.CPMV is strongly immunogenic when injected into rabbits or mice. We prepared nine monoclonal antibodies against CPMV using standard methods 8 . The binding properties of these antibodies were analysed by enzyme-linked immunosorbent assay (ELISA) and all nine would only bind to virus particles presented in a double antibody sandwich-type ELISA © 1992 Nature Publishing Group Correspondence to: John E. Johnson. G.W. and C.P. are joint first authors. in which the virus is bound to a polyclonal antibody attached to the plastic plate. Denaturation of the virus by its direct adsorption onto a plastic surface destroys the antigenic sites for all nine monoclonal antibodies. Only monoclonal antibodies 5B2 and 10B7 reacted strongly with CPMV that was free in solution (antibody binding was assayed by electron microscopy analysis of negatively stained complexes marked with gold-labelled anti-mouse immunoglobulin). Fab fragments from monoclonal antibody 5B2 were prepared by digestion of IgGs with insoluble...

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Protein-RNA Interactions in an Icosahedral Virus at 3.0 Å Resolution

Cited by 199 publications

References 25 publications

Satellite tobacco mosaic virus RNA: structure and implications for assembly

Satellite tobacco mosaic virus RNA: structure and implications for assembly

Atomic structure of single-stranded DNA bacteriophage ΦX174 and its functional implications

Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography

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