Changes in membrane permeability during semliki forest virus induced cell fusion

1992 ◽  
Vol 12 (3) ◽  
pp. 221-236 ◽  
Author(s):  
Markus Lanzrein ◽  
Nicole Käsermann ◽  
Christoph Kempf
Keyword(s):  
Membrane Permeability ◽  
Cell Fusion ◽  
Semliki Forest Virus ◽  
Divalent Cations ◽  
Ph Dependence ◽  
Syncytium Formation ◽  
Permeability Change ◽  
Infected Cells ◽  
Ph Dependent ◽  
Radiolabeled Compounds

The infection of Aedes albopictus cells by Semliki Forest virus (SFV) is a non lytic event. Exposure of infected cells to mildly acidic pH (<6.2) leads to syncytium formation. This polykaryon formation is accompanied by an influex of protons into the cells (Kempf et al. Biosci. Rep. 7, 761–769, 1987). We have further investigated this permeability change using various fluorescent or radiolabeled compounds. A significant, pH dependent increase of the membrane permeability to low molecular weight compounds (Mr<1000) was observed when infected cells were exposed to a pH<6.2. The pH dependence of the peremability change was very similar to the pH dependence of cell-cell fusion. The permeability change was sensitive to divalent cations, protons and anionic antiviral drugs such as trypan blue. The nature of this virus induced, pH dependent permeability change is discussed.

Semliki Forest virus induced cell-cell fusion at neutral extracellular pH

1990 ◽  
Vol 10 (4) ◽  
pp. 363-374 ◽  
Author(s):  
Christoph Kempf ◽  
Marcel R. Michel ◽  
Adames Omar ◽  
Pia Jentsch ◽  
Andreas Morell
Keyword(s):  
Cell Fusion ◽  
Semliki Forest Virus ◽  
Covalent Modification ◽  
Surface Proteins ◽  
Acidic Ph ◽  
Fusion Event ◽  
Cell Surface Proteins ◽  
Infected Cells ◽  
Peptide Segments ◽  
Cell Cell

Semliki Forest virus-induced cell-cell fusion from within was considered to exclusively occur at mildly acidic pH (<6.2). Data of this study show that such cell fusion can also be triggered by transient acidification of the cytoplasm of infected cells at an extracellular, neutral pH. Results were obtained by utilizing NH4Cl pulses combined with covalent modification of cell surface proteins. The observation implies a revision of the current consensus regarding the mechanism of Semliki Forest virus induced cell-cell fusion. We propose a model in which at least two peptide segments of the viral spike protein E1 may be involved in triggering the fusion event.

scholarly journals Expression of the A56 and K2 Proteins Is Sufficient To Inhibit Vaccinia Virus Entry and Cell Fusion

2008 ◽  
Vol 83 (4) ◽  
pp. 1546-1554 ◽  
Author(s):  
Timothy R. Wagenaar ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Cell Fusion ◽  
Virus Entry ◽  
Virus Production ◽  
Syncytium Formation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Strong Connection ◽  
Early Expression ◽  
Infected Cells

ABSTRACT Many animal viruses induce cells to fuse and form syncytia. For vaccinia virus, this phenomenon is associated with mutations affecting the A56 and K2 proteins, which form a multimer (A56/K2) on the surface of infected cells. Recent evidence that A56/K2 interacts with the entry/fusion complex (EFC) and that the EFC is necessary for syncytium formation furnishes a strong connection between virus entry and cell fusion. Among the important remaining questions are whether A56/K2 can prevent virus entry as well as cell-cell fusion and whether these two viral proteins are sufficient as well as necessary for this. To answer these questions, we transiently and stably expressed A56 and K2 in uninfected cells. Uninfected cells expressing A56 and K2 exhibited resistance to fusing with A56 mutant virus-infected cells, whereas expression of A56 or K2 alone induced little or no resistance, which fits with the need for both proteins to bind the EFC. Furthermore, transient or stable expression of A56/K2 interfered with virus entry and replication as determined by inhibition of early expression of a luciferase reporter gene, virus production, and plaque formation. The specificity of this effect was demonstrated by restoring entry after enzymatically removing a chimeric glycophosphatidylinositol-anchored A56/K2 or by binding a monoclonal antibody to A56. Importantly, the antibody disrupted the interaction between A56/K2 and the EFC without disrupting the A56-K2 interaction itself. Thus, we have shown that A56/K2 is sufficient to prevent virus entry and fusion as well as formation of syncytia through interaction with the EFC.

Dynamic changes in plasma membrane properties of semliki forest virus infected cells related to cell fusion

1988 ◽  
Vol 8 (3) ◽  
pp. 241-254 ◽  
Author(s):  
C. Kempf ◽  
M. R. Michel ◽  
U. Kohler ◽  
H. Koblet ◽  
H. Oetliker
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Semliki Forest Virus ◽  
Membrane Properties ◽  
Acidic Ph ◽  
Dynamic Changes ◽  
Infected Cells

The mechanism of the processes leading to membrane fusion is as yet unknown. In this report we demonstrate that changes in membrane potential and potassium fluxes correlate with Semliki Forest virus induced cell-cell fusion at mildly acidic pH. The changes observed occur only at pH's below 6.2 corresponding to values required to trigger the fusion process. A possible role of these alterations of the plasma membrane related to membrane fusion phenomena is discussed.

scholarly journals RhoA Signaling Is Required for Respiratory Syncytial Virus-Induced Syncytium Formation and Filamentous Virion Morphology

2005 ◽  
Vol 79 (9) ◽  
pp. 5326-5336 ◽  
Author(s):  
Tara L. Gower ◽  
Manoj K. Pastey ◽  
Mark E. Peeples ◽  
Peter L. Collins ◽  
Lewis H. McCurdy ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Respiratory Syncytial Virus ◽  
Cell Fusion ◽  
Infectious Virus ◽  
Syncytium Formation ◽  
Velocity Sedimentation ◽  
Rsv Infection ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Rhoa Signaling

ABSTRACT Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants, the elderly, and immunocompromised adults. RSV infection of HEp-2 cells induces the activation of RhoA, a small GTPase. We therefore asked whether RhoA signaling is important for RSV replication or syncytium formation. The treatment of HEp-2 cells with Clostridium botulinum C3, an enzyme that ADP-ribosylates and specifically inactivates RhoA, inhibited RSV-induced syncytium formation and cell-to-cell fusion, although similar levels of PFU were released into the medium and viral protein expression levels were equivalent. Treatment with another inhibitor of RhoA signaling, the Rho kinase inhibitor Y-27632, yielded similar results. Scanning electron microscopy of C3-treated infected cells showed reduced numbers of single blunted filaments, in contrast to the large clumps of long filaments in untreated infected cells. These data suggest that RhoA signaling is associated with filamentous virus morphology, cell-to-cell fusion, and syncytium formation but is dispensable for the efficient infection and production of infectious virus in vitro. Next, we developed a semiquantitative method to measure spherical and filamentous virus particles by using sucrose gradient velocity sedimentation. Fluorescence and transmission electron microscopy confirmed the separation of spherical and filamentous forms of infectious virus into two identifiable peaks. The C3 treatment of RSV-infected cells resulted in a shift to relatively more spherical virions than those from untreated cells. These data suggest that viral filamentous protuberances characteristic of RSV infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play a role in initiating cell-to-cell fusion.

Rationalizing the pH-Activity Response for Escherichia Coli’s Glycinamide Ribonucleotidetransformylase Through Computational Methods

2019 ◽  
Author(s):  
Adrian Roitberg ◽  
Pancham Lal Gupta
Keyword(s):  
Transfer Reaction ◽  
Catalytic Mechanism ◽  
Ph Dependence ◽  
Ph Effects ◽  
Dependent Manner ◽  
E Coli ◽  
Gar Tfase ◽  
Activity Curve ◽  
Ph Dependent ◽  
Formyl Transfer

<div>Human Glycinamide ribonucleotide transformylase (GAR Tfase), a regulatory enzyme in the de novo purine biosynthesis pathway, has been established as an anti-cancer target. GAR Tfase catalyzes the formyl transfer reaction from the folate cofactor to the GAR ligand. In the present work, we study E. coli GAR Tfase, which has high sequence similarity with the human GAR Tfase with most functional residues conserved. E. coli GAR Tfase exhibits structural changes and the binding of ligands that varies with pH which leads to change the rate of the formyl transfer reaction in a pH-dependent manner. Thus, the inclusion of pH becomes essential for the study of its catalytic mechanism. Experimentally, the pH-dependence of the kinetic parameter kcat is measured to evaluate the pH-range of enzymatic activity. However, insufficient information about residues governing the pH-effects on the catalytic activity leads to ambiguous assignments of the general acid and base catalysts and consequently its catalytic mechanism. In the present work, we use pH-replica exchange molecular dynamics (pH-REMD) simulations to study the effects of pH on E. coli GAR Tfase enzyme. We identify the titratable residues governing the pH-dependent conformational changes in the system. Furthermore, we filter out the protonation states which are essential in maintaining the structural integrity, keeping the ligands bound and assisting the catalysis. We reproduce the experimental pH-activity curve by computing the population of key protonation states. Moreover, we provide a detailed description of residues governing the acidic and basic limbs of the pH-activity curve.</div>

pH dependence of facilitation by neurotransmitters and divalent cations of P2X2 purinoceptor/channels

1997 ◽  
Vol 337 (2-3) ◽  
pp. 309-314 ◽  
Author(s):  
Ken Nakazawa ◽  
Min Liu ◽  
Kazuhide Inoue ◽  
Yasuo Ohno
Keyword(s):  
Divalent Cations ◽  
Ph Dependence

scholarly journals The CD46 transmembrane domain is required for efficient formation of measles-virus-mediated syncytium

1997 ◽  
Vol 322 (1) ◽  
pp. 135-144 ◽  
Author(s):  
Tsukasa SEYA ◽  
Mitsue KURITA ◽  
Kazunori IWATA ◽  
Yusuke YANAGI ◽  
Kazuhiko TANAKA ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Measles Virus ◽  
Deletion Mutant ◽  
Cytopathic Effect ◽  
Transmembrane Domain ◽  
Binding Capacity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Critical Factor ◽  
Syncytium Formation

Two phosphatidylinositol (PI)-anchored versions of a measles virus (MV) receptor membrane cofactor protein (MCP; CD46) were generated by fusing the extracellular domain of MCP to the decay-accelerating factor (DAF; CD55) or its PI anchor. The PI-anchored forms of MCP expressed on Chinese hamster ovary cells, otherwise non-permissive to MV, conferred a smaller MV cytopathic effect than a wild-type MCP, a Ser/Thr-rich domain-deletion mutant and a cytoplasmic tail-deletion mutant of MCP. Therefore the differences in MV receptor properties between the two PI-anchored and three transmembrane forms were investigated. The PI-anchored forms were predominantly expressed on microvilli as in DAF, whereas the other transmembrane forms were found on intracellular membranes. The PI-anchored forms conferred high MV-binding capacity compared with the transmembrane versions. MV replication was, however, severely suppressed in cells expressing the PI-anchored forms, resulting in ineffective syncytium formation. In contrast, cell-to-cell fusion occurred efficiently after co-transfection of cDNA species encoding MV-H, MV-F and any version of MCP. Thus the PI-anchored forms, despite showing sufficient MV binding and cell-to-cell fusion competence together with MV-H and MV-F, mediate inefficient MV entry or replication, which causes severe suppression of the MV cytopathic effect. A biased receptor distribution on microvilli might participate in the selection of a low MV uptake pathway in the PI-anchored forms of MCP. Taken together, the transmembrane portion of MCP is a critical factor for effective virusŐcell fusion and the subsequent MV replication.

Enhanced Analytic Approach for Describing pH Triggered Fast Drug Release Systems

2021 ◽  
Vol 18 ◽  
Author(s):  
Aykut Elmas ◽  
Guliz Akyuz ◽  
Ayhan Bergal ◽  
Muberra Andac ◽  
Omer Andac
Keyword(s):  
Drug Release ◽  
Ph Dependence ◽  
Slope Angle ◽  
Ph Sensitive ◽  
Analytic Model ◽  
Two Dimensional ◽  
Analytic Equation ◽  
One Dimensional ◽  
Ph Dependent ◽  
Fast Release

Background: pH sensitive dendrimers attached to nanocarriers, as one of the drug release systems, has become quite popular due to their ease of manufacture in experimental conditions and ability to generate fast drug release in the targeted area. This kind of fast release behavior cannot be represented properly most of the existing kinetic mathematical models. Besides, these models have either no pH dependence or pH dependence added separately. So, they have remained one dimensional. Objective: The aim of this study was to establish the proper analytic equation to describe the fast release of drugs from pH sensitive nanocarrier systems. Then, to combine it with the pH dependent equation for establishing a two-dimensional model for whole system. Methods: We used four common kinetic models for comparison and we fitted them to the release data. Finding that, only Higuchi and Korsmeyer-Peppas models show acceptable fit results. None of these models have pH dependence. To get a better description for pH triggered fast release, we observed the behavior of the slope angle of the release curve. Then we puroposed a new analytic equation by using relation between the slope angle and time. Result: To add a pH dependent equation, we assumed the drug release is “on” or “off” above/below specific pH value and we modified a step function to get a desired behavior. Conclusion: Our new analytic model shows good fitting, not only one-dimensional time dependent release, but also two-dimensional pH dependent release, that provides a useful analytic model to represent release profiles of pH sensitive fast drug release systems.

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