Crystallization and preliminary data analysis of flock house virus

Fisher, A.J.; McKinney, B. R.; Wery, J. P.; Johnson, John E.

doi:10.1107/s0108768192000053

Cited by 13 publications

(8 citation statements)

References 5 publications

(6 reference statements)

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“…This revealed that the number of virus particles per cell at 16 hours post-infection was greater than 6.0 × 10 4 , consistent with the large paracrystalline arrays of virions observed in cells by electron microscopy late in infection. 47,48 The growth curves of coat protein synthesis obtained by DIOS-MS and nanoLC-MS/MS were in good agreement. The DIOS-MS analysis was much more rapid, requiring only 3 hours, while nanoLC-MS/MS required 3 days of continuous automated analysis.…”

Section: Monitoring Viral Protein Expression Kinetics: Fhv-infected Dmentioning

confidence: 58%

Mass Spectrometry Reveals Specific and Global Molecular Transformations during Viral Infection

Wikoff

Shen

et al. 2006

J. Proteome Res.

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Mass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.

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Section: Monitoring Viral Protein Expression Kinetics: Fhv-infected Dmentioning

confidence: 58%

Mass Spectrometry Reveals Specific and Global Molecular Transformations during Viral Infection

Wikoff

Shen

et al. 2006

J. Proteome Res.

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“…Ever since it was isolated, PaV has been extensively studied using various techniques (3–6). The relatively small size (20nm diameter) and the ease with which it can be produced in various cell lines (7) make PaV and other members of the Nodaviridae family easy to characterize at the molecular level (8–10). …”

mentioning

confidence: 99%

Structural and electrostatic characterization of Pariacoto virus: Implications for viral assembly

et al. 2009

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We present the first all-atom model for the structure of a T=3 virus, pariacoto virus (PaV), which is a non-enveloped, icosahedral RNA virus and a member of the Nodaviridae family. The model is an extension of the crystal structure, which reveals about 88% of the protein structure but only about 35% of the RNA structure. Evaluation of alternative models confirms our earlier observation that the polycationic protein tails must penetrate deeply into the core of the virus, where they stabilize the structure by neutralizing a substantial fraction of the RNA charge. This leads us to propose a model for the assembly of small icosahedral RNA viruses: nonspecific binding of the protein tails to the RNA leads to a collapse of the complex, in a fashion reminiscent of DNA condensation. The globular protein domains are excluded from the condensed phase but are tethered to it, so they accumulate in a shell around the condensed phase, where their concentration is high enough to trigger oligomerization and formation of the mature virus.Pariacoto virus (PaV), a T=3, non-enveloped, icosahedral virus, is a member of the Nodaviridae family. It was originally isolated in Peru from the Southern Armyworm, Spodoptera eridania (1). Its genome consists of two positive-sense ssRNAs (2). RNA1 (3011 nucleotides) codes for protein A, the catalytic subunit for the host RNA replicase, which enables the RNAdependent RNA replicase to start replicating the viral RNA. RNA2 (1311 nucleotides) codes for capsid precursor protein α. 180 of these α proteins and the genome assemble together to make up the virus. Ever since it was isolated, PaV has been extensively studied using various techniques (3-6). The relatively small size (20nm diameter) and the ease with which it can be produced in various cell lines (7) make PaV and other members of the Nodaviridae family easy to characterize at the molecular level (8-10).Structural studies of viruses are very important to understand protein-protein and protein-RNA interactions as well as to understand assembly pathways in RNA viruses (11)(12)(13)(14). In the last few years, many studies have been done on RNA viruses using molecular modeling as supplementary method when other methods such as x-ray crystallography and cryo-electron microscopy (cryo-EM) do not give sufficient structural information. An all-atom model was derived for Satellite Tobacco Mosaic Virus (STMV), a T=1 virus, using molecular modeling (15). Those authors also carried out molecular dynamics simulations on the model to study the stability of the protein capsid and the RNA genome (15 RNA and the protein capsid were studied in Cowpea Chlorotic Mottle Virus (CCMV) by modeling the virus using coarse-grained modeling and representing RNA nucleotides by unconnected spheres that were distributed using the Monte Carlo method (16). In that study, no attempt was made to model the RNA structure. Other electrostatic studies of RNA viruses have also been aimed at understanding the molecular interactions and their effects on virus structure (17,1...

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“…The volume per molecular weight (V,,) (Matthews, 1968) implied two provirion-like paricles per unit cell with a value of 2.74 A 3 Da-~. This compares with 2.70 A 3 Da-1 for the trigonal native FHV crystals indicating equivalent solvent content in the crystals (Fisher et al, 1992).…”

Section: Resultsmentioning

confidence: 85%

“…This difference may result from surface changes caused by the uncleaved capsid protein that could alter crystal packing or from growing baculoexpressed particles in a different cell line from native FHV . Native FHV crystallized in the trigonal space group R3 (Fisher, McKinney, Wery & Johnson, 1992) while the 'synthetic' particles crystallized in the monoclinic space group with cell parameters: a = 464.8, b=333.9, c=325.2A and fl=91.9 ° . The volume per molecular weight (V,,) (Matthews, 1968) implied two provirion-like paricles per unit cell with a value of 2.74 A 3 Da-~.…”

Section: Resultsmentioning

confidence: 99%

Production and crystallization of virus-like particles assembled in a heterologous protein expression system

McKinney¹,

Agrawal²,

Fisher³

et al. 1994

Acta Cryst D

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It is of considerable interest to separate the processes of viral infectivity and virion assembly. Until recently this has only been possible with viruses that could be disassembled and reassembled in vitro. Even in these cases it was difficult to establish the authenticity of reassembled capsid protein because of possible irreversible damage that may have occurred to the protein during disassembly. An ideal method for the study of virus assembly is a protein expression system in which conditions are appropriate for spontaneous particle formation from freshly synthesized polypeptides. The baculovirus expression system has proven to be an excellent means to this end. Recently, this approach has been used to study the T = 3 Flock House insect virus and it has been demonstrated that subunits with the wild-type protein sequence, and with site-specific mutations that prevent particle maturation, will assemble and crystallize. This same approach has now been used at Purdue to study the T = 4 Nudaurelia to capensis insect virus. There is no cell culture system currently available for the study of NtoV, thus the expression system provides the first opportunity to study assembly under controlled conditions. Abbreviations ADA, N-(a-acetamido)-2-iminodiacetic acid, N-(carbamoylmethyl)iminodiacetic acid (from Sigma Chemicals). BME, /3-mercaptoethanol. HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid.

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Crystallization and preliminary data analysis of flock house virus

Cited by 13 publications

References 5 publications

Mass Spectrometry Reveals Specific and Global Molecular Transformations during Viral Infection

Mass Spectrometry Reveals Specific and Global Molecular Transformations during Viral Infection

Structural and electrostatic characterization of Pariacoto virus: Implications for viral assembly

Production and crystallization of virus-like particles assembled in a heterologous protein expression system

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