Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions

Brady, John; Winston, Vern; Consigli, Richard A.

doi:10.1128/jvi.23.3.717-724.1977

Cited by 129 publications

(81 citation statements)

References 24 publications

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“…Divalent cations preserved TGEV core structure. This requirement has also been observed for polyomavirus, rotavirus, and plant viruses (turnip crinkle virus) among others (6,19,29,30).…”

Section: Discussionmentioning

confidence: 64%

The Membrane M Protein Carboxy Terminus Binds to Transmissible Gastroenteritis Coronavirus Core and Contributes to Core Stability

Escors

Ortego

Laude

et al. 2001

J Virol

170

172

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The architecture of transmissible gastroenteritis coronavirus includes three different structural levels, the envelope, an internal core, and the nucleocapsid that is released when the core is disrupted. Starting from purified virions, core structures have been reproducibly isolated as independent entities. The cores were stabilized at basic pH and by the presence of divalent cations, with Mg 2؉ ions more effectively contributing to core stability. Core structures showed high resistance to different concentrations of detergents, reducing agents, and urea and low concentrations of monovalent ions (<200 mM). Cores were composed of the nucleoprotein, RNA, and the C domain of the membrane (M) protein. At high salt concentrations (200 to 300 mM), the M protein was no longer associated with the nucleocapsid, which resulted in destruction of the core structure. A specific ionic interaction between the M protein carboxy terminus and the nucleocapsid was demonstrated using three complementary approaches: (i) a binding assay performed between a collection of M protein amino acid substitution or deletion mutants and purified nucleocapsids that led to the identification of a 16-amino-acid (aa) domain (aa 237 to 252) as being responsible for binding the M protein to the nucleocapsid; (ii) the specific inhibition of this binding by monoclonal antibodies (MAbs) binding to a carboxy-terminal M protein domain close to the indicated peptide but not by MAbs specific for the M protein amino terminus; and (iii) a 26-residue peptide, including the predicted sequence (aa 237 to 252), which specifically inhibited the binding. Direct binding of the M protein to the nucleoprotein was predicted, since degradation of the exposed RNA by RNase treatment did not affect the binding. It is proposed that the M protein is embedded within the virus membrane and that the C region, exposed to the interior face of the virion in a population of these molecules, interacts with the nucleocapsid to which it is anchored, forming the core. Only the C region of the M protein is part of the core.Transmissible gastroenteritis virus (TGEV) is a member of the Coronaviridae family and affects animals, causing severe illness (17,28). TGEV is an enveloped virus with a singlestrand positive-sense RNA genome of 28.5 kb (16; Z. Penzes and L. Enjuanes, submitted for publication), for which an infectious cDNA has been engineered (1). The RNA is bound to the nucleoprotein (N protein), forming a helical nucleocapsid (40). The viral membrane contains three proteins, the spike (S) protein, the membrane (M) protein, and the envelope (E) protein (11,21,24,25). Other coronaviruses also contain an additional membrane glycoprotein, the hemagglutinin esterase (8). The S protein binds to the cellular receptor, the aminopeptidase N (15), and is a determinant of the virus tropism (3, 44). Both the M and E proteins are essential for coronavirus morphogenesis (5,13,18,47). Interactions between these two proteins seem to drive coronavirus envelope assembly, producing virus-like partic...

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“…Divalent cations preserved TGEV core structure. This requirement has also been observed for polyomavirus, rotavirus, and plant viruses (turnip crinkle virus) among others (6,19,29,30).…”

Section: Discussionmentioning

confidence: 64%

The Membrane M Protein Carboxy Terminus Binds to Transmissible Gastroenteritis Coronavirus Core and Contributes to Core Stability

Escors

Ortego

Laude

et al. 2001

J Virol

170

172

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“…Virus was purified from the infected-cell lysate either as described previously for small volumes (17) or as described by Friedmann and Haas (10) by polyethyleneglycol precipitation for large volumes. The CsCl gradients used to purify the virus were prepared as described by Brunck and Leick (2) and described in greater detail previously (1).…”

Section: Materlals and Methodsmentioning

confidence: 99%

“…Dehydrated grids were stained with freshly prepared 1% uranyl acetate. The preparation of capsomeres for electron microscopy has been described previously (1). Observations were made with a Phillips 201 electron microscope operated at 60 kV.…”

Section: Materlals and Methodsmentioning

confidence: 99%

“…The involvement of proteins VP1, VP2, and VP3 in the capsid structure and their relationship to the hexons and pentons is unknown. In addition, it is not clear whether the virus-specific structural proteins are in any way associated with the viral DNA. To investigate these questions, we have examined the dissociation products of a new in vitro dissociation method which we have recently developed for polyoma virions (1). This t Contribution no.…”

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confidence: 99%

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Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N'-tetraacetic acid and dithiothreitol

Brady¹,

Winston²,

Consigli³

1978

J Virol

Self Cite

106

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Treatment of polyoma virions with ethyleneglycol-bis-N,N'-tetraacetic acid (EGTA) and dithiothreitol (DTT) at pH 8.5 resulted in the dissociation of the virions into a DNA-protein complex and individual structural capsomere subunits. The sedimentation value of the DNA-protein complex in sucrose gradients was approximately 48S, and it had a density of 1.45 g/cm3 in equilibrium CsCl gradients. Alkaline sucrose analysis of the DNA within this DNA-protein complex demonstrated that approximately 75% of the DNA is component 1. The proteins associated with the DNA were dissociated by treatment with either NaCl or the anionic detergent Sarkosyl. VP1 and the histone proteins VP4-7 were the major proteins associated with the DNA. Treatment of the DNA-protein complex with alkaline pH resulted in the specific removal of VP1. Electron microscopy of the 48S DNA-protein complex demonstrated that it is a very tightly coiled structure that is slightly larger than the intact virion. Treatment of the complex with either NaCl or with pH 10.5 buffer resulted in the loss of protein and subsequent loosening of the DNA-protein complex such that the DNA could be visualized. The capsomere subunits released as a result of the EGTA-DTT treatment sedimented as 18S, 12S, and 5S subunits in sucrose gradients. Electrophoretic analysis of the isolated capsomeres demonstrated that VP,, VP2, and VP3 were present in each species, although the ratios of the proteins varied. In addition to the structural proteins, histones VP-7 were found to be predominantly associated with the 5S capsomere subunit.

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“…Given the consistent and severe assembly defects of the Region II mutants, we speculate that pentamer-pentamer cross-linking mediated by the distal parts of the invading C-arms and the Ca 2+ binding sites is a critical early step in the addition of pentamers to the minichromosome. Previous work from others has identified the importance of calcium in stabilizing the structure of preassembled particles (Brady et al 1977;Christiansen et al 1977;Stehle et al 1996). Furthermore, we have previously described a Ca 2+ -coordinating mutant, E330K, which forms particles but is unable to bind cells or penetrate into the cytoplasm (Li et al 2003), and proposed that the substituted lysine makes salt bridges with the side chains of the Ca 2+ -coordinating Glu 48 and Glu 216, thus displacing the Ca 2+ ion from site 1.…”

Section: Resultsmentioning

confidence: 91%

A structural rationale for SV40 Vp1 temperature‐sensitive mutants and their complementation

et al. 2006

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Two groups of temperature-sensitive (ts) mutants, termed ts B and ts C, have mutations in the major capsid protein of SV40, Vp1. These mutants have virion assembly defects at the nonpermissive temperature, but can complement one another when two mutants, one from each group, coinfect a cell. A third group of mutants, termed ts BC, have related phenotypes, but do not complement other mutants. We found that the mutations fall into two structural and functional classes. All ts C and one ts BC mutations map to the region close to the Ca 2+ binding sites, and are predicted to disrupt the insertion of the distal part of the C-terminal invading arm (C-arm) into the receiving clamp. They share a severe defect in assembly at the nonpermissive temperature, with few capsid proteins attached to the viral minichromosome. By contrast, all ts B and most ts BC mutations map to a contiguous region including acceptor sites for the proximal part of the C-arm and intrapentamer contacts. These mutants form assembly intermediates that carry substantial capsid proteins on the minichromosome. Thus, accurate virion assembly is prevented by mutations that disrupt interactions between the receiving pentamer and both the proximal and distal parts of the C-arms, with the latter having a greater effect. The distinct spatial localization and assembly defects of the two classes of mutants provide a rationale for their intracistronic complementation and suggest models of capsid assembly.

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Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions

Cited by 129 publications

References 24 publications

The Membrane M Protein Carboxy Terminus Binds to Transmissible Gastroenteritis Coronavirus Core and Contributes to Core Stability

The Membrane M Protein Carboxy Terminus Binds to Transmissible Gastroenteritis Coronavirus Core and Contributes to Core Stability

Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N'-tetraacetic acid and dithiothreitol

A structural rationale for SV40 Vp1 temperature‐sensitive mutants and their complementation

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