scholarly journals Norwalk Virus Assembly and Stability Monitored by Mass Spectrometry

2010 ◽  
Vol 9 (8) ◽  
pp. 1742-1751 ◽  
Author(s):  
Glen K. Shoemaker ◽  
Esther van Duijn ◽  
Sue E. Crawford ◽  
Charlotte Uetrecht ◽  
Marian Baclayon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Virus Assembly ◽  
Norwalk Virus

scholarly journals Proteomics Analysis Unravels the Functional Repertoire of Coronavirus Nonstructural Protein 3

2008 ◽  
Vol 82 (11) ◽  
pp. 5279-5294 ◽  
Author(s):  
Benjamin W. Neuman ◽  
Jeremiah S. Joseph ◽  
Kumar S. Saikatendu ◽  
Pedro Serrano ◽  
Amarnath Chatterjee ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Metal Ion ◽  
Rna Binding ◽  
Virus Assembly ◽  
Nonstructural Protein ◽  
Domain Architecture ◽  
Complete Characterization ◽  
Functional Domain ◽  
Metal Ion Binding ◽  
Nonstructural Protein 3

ABSTRACT Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructural protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

scholarly journals Two Nonoverlapping Domains on the Norwalk Virus Open Reading Frame 3 (ORF3) Protein Are Involved in the Formation of the Phosphorylated 35K Protein and in ORF3-Capsid Protein Interactions

2003 ◽  
Vol 77 (6) ◽  
pp. 3569-3577 ◽  
Author(s):  
Pamela J. Glass ◽  
Carl Q. Zeng ◽  
Mary K. Estes
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Capsid Protein ◽  
Full Length ◽  
Open Reading Frame ◽  
Norwalk Virus ◽  
Two Dimensional Gel Electrophoresis ◽  
Reading Frame ◽  
Orf3 Protein ◽  
Orf2 Protein

ABSTRACT Expression of the Norwalk virus open reading frame 3 (ORF3) in Spodoptera frugiperda (Sf9) cells yields two major forms, the predicted 23,000-molecular-weight (23K) form and a larger 35K form. The 23K form is able to interact with the ORF2 capsid protein and be incorporated into virus-like particles. In this paper, we provide mass spectrometry evidence that both the 23K and 35K forms are composed only of the ORF3 protein. Two-dimensional gel electrophoresis and phosphatase treatment showed that the 35K form results solely from phosphorylation and that the 35K band is composed of several different phosphorylated forms with distinct isoelectric points. Furthermore, we analyzed deletion and point mutants of the ORF3 protein. Mutants that lacked the C-terminal 33 amino acids (ORF31-179, ORF31-152, and ORF31-107) no longer produced the 35K form. An N-terminal truncation mutant (ORF351-212) and a site-directed mutant (ORF3T201V) were capable of producing the larger form, which was converted to the smaller form by treatment with protein phosphatase. These data suggest that the region between amino acids 180 and 212 is phosphorylated, and mass spectrometry showed that amino acids Arg196 to Arg211 are not phosphorylated; thus, phosphorylation of the serine-threonine-rich region from Thr181 to Ser193 must be involved in the generation of the 35K form. Studies of the interaction between the ORF2 protein and full-length and mutated ORF3 proteins showed that the full-length ORF3 protein (ORF3FL), ORF31-179, ORF31-152, and ORF351-212 interacted with the ORF2 protein, while an ORF31-107 protein did not. These results indicate that the region of the ORF3 protein between amino acids 108 and 152 is responsible for interaction with the ORF2 protein.

scholarly journals Determining the topology of virus assembly intermediates using ion mobility spectrometry-mass spectrometry

2010 ◽  
Vol 24 (20) ◽  
pp. 3033-3042 ◽  
Author(s):  
Tom W. Knapman ◽  
Victoria L. Morton ◽  
Nicola J. Stonehouse ◽  
Peter G. Stockley ◽  
Alison E. Ashcroft
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility Spectrometry ◽  
Ion Mobility ◽  
Virus Assembly

scholarly journals The Lipidomes of Vesicular Stomatitis Virus, Semliki Forest Virus, and the Host Plasma Membrane Analyzed by Quantitative Shotgun Mass Spectrometry

2009 ◽  
Vol 83 (16) ◽  
pp. 7996-8003 ◽  
Author(s):  
Lucie Kalvodova ◽  
Julio L. Sampaio ◽  
Sandra Cordo ◽  
Christer S. Ejsing ◽  
Andrej Shevchenko ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Host Cells ◽  
Enveloped Virus ◽  
Host Plasma Membrane ◽  
Vesicular Stomatitis ◽  
Shotgun Mass Spectrometry

ABSTRACT Although enveloped virus assembly in the host cell is a crucial step in the virus life cycle, it remains poorly understood. One issue is how viruses include lipids in their membranes during budding from infected host cells. To analyze this issue, we took advantage of the fact that baby hamster kidney cells can be infected by two different viruses, namely, vesicular stomatitis virus and Semliki Forest virus, from the Rhabdoviridae and Togaviridae families, respectively. We purified the host plasma membrane and the two different viruses after exit from the host cells and analyzed the lipid compositions of the membranes by quantitative shotgun mass spectrometry. We observed that the lipid compositions of these otherwise structurally different viruses are virtually indistinguishable, and only slight differences were detected between the viral lipid composition and that of the plasma membrane. Taken together, the facts that the lipid compositions of the two viruses are so similar and that they strongly resemble the composition of the plasma membrane suggest that these viruses exert little selection in including lipids in their envelopes.

How SIMS microscopy can be used in medicine

1992 ◽  
Vol 50 (2) ◽  
pp. 1602-1603
Author(s):  
Philippe Fragu
Keyword(s):  
Mass Spectrometry ◽  
Biomedical Applications ◽  
Secondary Ion Mass Spectrometry ◽  
Chemical Elements ◽  
Biological Applications ◽  
The Past ◽  
Potential Source ◽  
Secondary Ion ◽  
Chemical Integrity ◽  
Scientific Endeavour

The identification, localization and quantification of intracellular chemical elements is an area of scientific endeavour which has not ceased to develop over the past 30 years. Secondary Ion Mass Spectrometry (SIMS) microscopy is widely used for elemental localization problems in geochemistry, metallurgy and electronics. Although the first commercial instruments were available in 1968, biological applications have been gradual as investigators have systematically examined the potential source of artefacts inherent in the method and sought to develop strategies for the analysis of soft biological material with a lateral resolution equivalent to that of the light microscope. In 1992, the prospects offered by this technique are even more encouraging as prototypes of new ion probes appear capable of achieving the ultimate goal, namely the quantitative analysis of micron and submicron regions. The purpose of this review is to underline the requirements for biomedical applications of SIMS microscopy.Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue.

Imaging of Al-Li-Cu alloys with a Scanning Ion Microprobe

1990 ◽  
Vol 48 (2) ◽  
pp. 314-315
Author(s):  
K.K. Soni ◽  
D.B. Williams ◽  
J.M. Chabala ◽  
R. Levi-Setti ◽  
D.E. Newbury
Keyword(s):  
Mass Spectrometry ◽  
Secondary Ion Mass Spectrometry ◽  
Equilibrium Phase ◽  
Icosahedral Symmetry ◽  
Ion Microprobe ◽  
X Ray ◽  
Cu Alloys ◽  
Two Phases ◽  
The University ◽  
Secondary Ion

In contrast to the inability of x-ray microanalysis to detect Li, secondary ion mass spectrometry (SIMS) generates a very strong Li+ signal. The latter’s potential was recently exploited by Williams et al. in the study of binary Al-Li alloys. The present study of Al-Li-Cu was done using the high resolution scanning ion microprobe (SIM) at the University of Chicago (UC). The UC SIM employs a 40 keV, ∼70 nm diameter Ga+ probe extracted from a liquid Ga source, which is scanned over areas smaller than 160×160 μm2 using a 512×512 raster. During this experiment, the sample was held at 2 × 10-8 torr.In the Al-Li-Cu system, two phases of major importance are T1 and T2, with nominal compositions of Al2LiCu and Al6Li3Cu respectively. In commercial alloys, T1 develops a plate-like structure with a thickness <∼2 nm and is therefore inaccessible to conventional microanalytical techniques. T2 is the equilibrium phase with apparent icosahedral symmetry and its presence is undesirable in industrial alloys.

Applications of Time-OF-Flight Secondary Ion Mass Spectrometry (TOF-SIMS)

1990 ◽  
Vol 48 (2) ◽  
pp. 308-309
Author(s):  
Bruno Schueler ◽  
Robert W. Odom
Keyword(s):  
Mass Spectrometry ◽  
Secondary Ion Mass Spectrometry ◽  
Structural Information ◽  
Time Of Flight ◽  
Biological Tissues ◽  
Polymer Surfaces ◽  
Tof Sims ◽  
Secondary Ions ◽  
Ion Mass Spectrometry ◽  
Secondary Ion

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) provides unique capabilities for elemental and molecular compositional analysis of a wide variety of surfaces. This relatively new technique is finding increasing applications in analyses concerned with determining the chemical composition of various polymer surfaces, identifying the composition of organic and inorganic residues on surfaces and the localization of molecular or structurally significant secondary ions signals from biological tissues. TOF-SIMS analyses are typically performed under low primary ion dose (static SIMS) conditions and hence the secondary ions formed often contain significant structural information.This paper will present an overview of current TOF-SIMS instrumentation with particular emphasis on the stigmatic imaging ion microscope developed in the authors’ laboratory. This discussion will be followed by a presentation of several useful applications of the technique for the characterization of polymer surfaces and biological tissues specimens. Particular attention in these applications will focus on how the analytical problem impacts the performance requirements of the mass spectrometer and vice-versa.

Involvement of GER in Friend leukemia virus assembly in high-pressure frozen cells

1990 ◽  
Vol 48 (3) ◽  
pp. 590-591
Author(s):  
W. Djaczenko ◽  
M. Müller ◽  
A. Benedetto ◽  
G. Carbone
Keyword(s):  
High Pressure ◽  
Virus Assembly ◽  
Leukemia Virus ◽  
Leukemia Cells ◽  
Serial Sections ◽  
Myeloma Cells ◽  
Virus Particles ◽  
Friend Leukemia ◽  
Carbon Coated ◽  
Friend Leukemia Virus

A thickening of ER membranes in murine myeloma cells was attributed by de Harven to the assembly of intracisternal virus particles. We observed similar thickening of GER membranes in Friend leukemia cells (FLC) apparently associated with Friend leukemia virus (FLV) assembly. We reinvestigated the problem of GER involvement in FLV assembly using high pressure cryofixed FLC.FLC (745A clone growing in suspension and FF clone growing in monolayer) were immersed in Hexadecene (Fluka, Switzerland) and rapidly frozen in Balzers HPM 010 freezing machine working at 2200 bar. All cells were freeze substituted at -90°C in 2% OsO4 in absolute acetone. Serial sections cut to avoid misinterpretations due to the geometry of sections, were collected on carbon coated 100 mesh grids.

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