Study of vaccinia virus binding and entry using confocal microscopy, an original approach

1998 ◽  
Vol 90 (3) ◽  
pp. 256-256
Author(s):  
Alain Vanderplasschen ◽  
Geoffrey L. Smith
Keyword(s):  
Confocal Microscopy ◽  
Vaccinia Virus ◽  
Virus Binding ◽  
Original Approach

scholarly journals Observations on photodynamic inactivation of vaccinia virus and its effect on immunogenicity

1965 ◽  
Vol 63 (3) ◽  
pp. 395-410 ◽  
Author(s):  
G. S. Turner ◽  
C. Kaplan
Keyword(s):  
Methylene Blue ◽  
Nucleic Acid ◽  
Vaccinia Virus ◽  
Neutralizing Antibody ◽  
Hydrogen Ions ◽  
High Concentration ◽  
Virus Binding ◽  
Wide Range ◽  
Effects Of Ph ◽  
Recombination Experiments

Photoinactivation of vaccinia virus sensitized by methylene blue had parameters similar to those observed with other viruses. Thus inactivation proceeded exponentially to completion, was irreversible, independent of temperature and the intensity of illumination. Inactivation was dependent on the dose of illumination and the concentrations of both methylene blue and hydrogen ions. The effects of pH appeared to be primarily concerned with the tenacity of dye-virus binding. Inactivation was inhibited by small amounts of nucleic acid but not by their bases or pentoses. Inactivation was only affected by the presence of extraneous protein in relatively high concentration: it was not affected by the enzymes catalase or peroxidase. Attempts to obtain direct chemical evidence of the participation of viral nucleic acid in photoinactivation were unsuccessful.Recombination experiments strongly indicated the involvement of viral protein in photoinactivation. Immunogenicity was not impaired since good responses of neutralizing antibody were obtained in rabbits immunized with vaccines photoinactivated over a wide range of exposure times.

scholarly journals One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

2007 ◽  
Vol 7 (1) ◽  
pp. 48 ◽  
Author(s):  
Andreas Nitsche ◽  
Andreas Kurth ◽  
Anna Dunkhorst ◽  
Oliver Pänke ◽  
Hendrik Sielaff ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Dna Aptamers ◽  
Virus Binding ◽  
One Step ◽  
Step Selection ◽  
Selection Of

scholarly journals Cell Surface Proteoglycans Are Necessary for A27L Protein-Mediated Cell Fusion: Identification of the N-Terminal Region of A27L Protein as the Glycosaminoglycan-Binding Domain

1998 ◽  
Vol 72 (10) ◽  
pp. 8374-8379 ◽  
Author(s):  
Jye-Chian Hsiao ◽  
Che-Sheng Chung ◽  
Wen Chang
Keyword(s):  
Virus Infection ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Cell Fusion ◽  
Binding Domain ◽  
Terminal Region ◽  
Virus Binding ◽  
Vaccinia Virus Infection

ABSTRACT We previously showed that vaccinia virus infection of BSC40 cells was blocked by soluble heparin, suggesting that cell surface heparan sulfate mediates vaccinia virus binding (C.-S. Chung, J.-C. Hsiao, Y.-S. Chang, and W. Chang, J. Virol. 72:1577–1585, 1998). In this study, we extended our previous work and demonstrated that soluble A27L protein bound to heparan sulfate on cells and interfered with vaccinia virus infection at a postbinding step. In addition, we investigated the structure of A27L protein that provides for its binding to heparan sulfate on cells. A mutant of A27L protein, named D-A27L, devoid of a cluster of 12 amino acids rich in basic residues, was constructed. In contrast to the soluble A27L protein, purified D-A27L protein was inactive in all of our assays, including binding to heparin in vitro, binding to heparan sulfate on cells, and the ability to block virus infection. These data demonstrated that the N-terminal region acts as a glycosaminoglycan (GAG)-binding domain critical for A27L protein binding to cells. Previously A27L protein was thought to be involved in fusion of virus-infected cells induced by acid treatment. When we investigated whether cell surface GAGs also participate in A27L-dependent fusion, our results indicated that soluble A27L protein blocked cell fusion, whereas D-A27L protein did not. Taken together, the results therefore demonstrated that A27L-mediated cell fusion is triggered by its interaction with cell surface GAGs through the N-terminal domain.

scholarly journals Redistribution of Cyclophilin A to Viral Factories during Vaccinia Virus Infection and Its Incorporation into Mature Particles

2003 ◽  
Vol 77 (16) ◽  
pp. 9052-9068 ◽  
Author(s):  
Ana Paula V. Castro ◽  
Técia M. U. Carvalho ◽  
Nissin Moussatché ◽  
Clarissa R. A. Damaso
Keyword(s):  
Confocal Microscopy ◽  
Virus Infection ◽  
Vaccinia Virus ◽  
Conformational Changes ◽  
Cyclophilin A ◽  
Cellular Systems ◽  
Cell Cytoplasm ◽  
Electron Microscopic ◽  
The Core ◽  
Infected Cells

ABSTRACT Cyclophilins are peptidyl-prolyl cis-trans isomerases involved in catalyzing conformational changes and accelerating the rate of protein folding and refolding in several cellular systems. In the present study, we analyzed the expression pattern and intracellular distribution of the cellular isomerase cyclophilin A (CypA) during vaccinia virus (VV) infection. An impressive increase in CypA stability was observed, leading to a practically unchanged accumulation of CypA during infection, although its synthesis was completely inhibited at late times. By confocal microscopy, we observed that CypA went through an intense reorganization in the cell cytoplasm and colocalized with the virosomes late in infection. CypA relocation to viral factories required the synthesis of viral postreplicative proteins, and treatment of infected cells with cyclosporine (CsA) prevented CypA relocation, clearly excluding the virosomes from CypA staining. Immunoelectron microscopy of VV-infected cells showed that CypA was incorporated into VV particles during morphogenesis. Biochemical and electron microscopic assays with purified virions confirmed that CypA was encapsidated within the virus particle and localized specifically in the core. This work suggests that CypA may develop an important role in VV replication.

scholarly journals A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

1998 ◽  
Vol 72 (2) ◽  
pp. 1577-1585 ◽  
Author(s):  
Che-Sheng Chung ◽  
Jye-Chian Hsiao ◽  
Yuan-Shau Chang ◽  
Wen Chang
Keyword(s):  
Virus Infection ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Mammalian Cells ◽  
Myxoma Virus ◽  
Cowpox Virus ◽  
Virus Binding ◽  
Vaccinia Virus Infection

ABSTRACT Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.

Two-photon excitation microscopy in cellular biophysics

1995 ◽  
Vol 53 ◽  
pp. 62-63
Author(s):  
David W. Piston ◽  
Brian D. Bennett ◽  
Robert G. Summers
Keyword(s):  
Confocal Microscopy ◽  
Laser Beam ◽  
Duty Cycle ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10-5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

Layered and ion implantation specimens as possible reference materials for the electron-probe microanalysis

1992 ◽  
Vol 50 (2) ◽  
pp. 1652-1653
Author(s):  
G. Remond ◽  
R.H. Packwood ◽  
C. Gilles ◽  
S. Chryssoulis
Keyword(s):  
Ion Implantation ◽  
Reference Materials ◽  
Analytical Techniques ◽  
Thin Film Deposition ◽  
Film Deposition ◽  
X Ray ◽  
Spatially Resolved ◽  
Analytical Expressions ◽  
Original Approach ◽  
Depth Concentration

Merits and limitations of layered and ion implanted specimens as possible reference materials to calibrate spatially resolved analytical techniques are discussed and illustrated for the case of gold analysis in minerals by means of x-ray spectrometry with the EPMA. To overcome the random heterogeneities of minerals, thin film deposition and ion implantation may offer an original approach to the manufacture of controlled concentration/ distribution reference materials for quantification of trace elements with the same matrix as the unknown.In order to evaluate the accuracy of data obtained by EPMA we have compared measured and calculated x-ray intensities for homogeneous and heterogeneous specimens. Au Lα and Au Mα x-ray intensities were recorded at various electron beam energies, and hence at various sampling depths, for gold coated and gold implanted specimens. X-ray intensity calculations are based on the use of analytical expressions for both the depth ionization Φ (ρz) and the depth concentration C (ρz) distributions respectively.

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