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A Vanderplasschen

Publications and source records attributed to A Vanderplasschen.

At least 37 records · Page 2Linked to original sources
PubMed1999

Vaccinia virus intracellular mature virions contain only one lipid membrane.

Vaccinia virus (VV) morphogenesis commences with the formation of lipid crescents that grow into spherical immature virus (IV) and then infectious intracellular mature virus (IMV) particles. Early studies proposed that the lipid crescents were synthesized de novo and matured into IMV particles that contained a single lipid bilayer (S. Dales and E. H. Mosbach, Virology 35:564-583, 1968), but a more recent study reported that the lipid crescent was derived from membranes of the intermediate compartment (IC) and contained a double lipid bilayer (B. Sodiek et al., J. Cell Biol. 121:521-541, 1993). In the present study, we used high-resolution electron microscopy to reinvestigate the structures of the lipid crescents, IV, and IMV particles in order to determine if they contain one or two membranes. Examination of thin sections of Epon-embedded, VV-infected cells by use of a high-angular-tilt series of single sections, serial-section analysis, and high-resolution digital-image analysis detected only a single, 5-nm-thick lipid bilayer in virus crescents, IV, and IMV particles that is covered by a 8-nm-thick protein coat. In contrast, it was possible to discern tightly apposed cellular membranes, each 5 nm thick, in junctions between cells and in the myelin sheath of Schwann cells around neurons. Serial-section analysis and angular tilt analysis of sections detected no continuity between virus lipid crescents or IV particles and cellular membrane cisternae. Moreover, crescents were found to form at sites remote from IC membranes-namely, within the center of virus factories and within the nucleus-demonstrating that crescent formation can occur independently of IC membranes. These data leave unexplained the mechanism of single-membrane formation, but they have important implications with regard to the mechanism of entry of IMV and extracellular enveloped virus into cells; topologically, a one-to-one membrane fusion suffices for delivery of the IMV core into the cytoplasm. Consistent with this, we have demonstrated previously by confocal microscopy that uncoated virus cores within the cytoplasm lack the IMV surface protein D8L, and we show here that intracellular cores lack the surface protein coat and lipid membrane.

Epoxy Resins↗
PubMed1999

Susceptibility of bovine antigen-presenting cells to infection by bovine herpesvirus 1 and in vitro presentation to T cells: two independent events.

The aim of the present study was to develop an in vitro system for presentation of bovine herpesvirus 1 (BHV-1) antigens to bovine T lymphocytes and to characterize the antigen-presenting cells (APC) which efficiently activate CD4(+) T cells. Two approaches were used to monitor the infection of APC by BHV-1 as follows: (i) detection of viral glycoproteins at the cell surface by immunofluorescence staining and (ii) detection of UL26 transcripts by reverse transcription-PCR. The monocytes were infected, while dendritic cells (DC) did not demonstrate any detectable viral expression. These data suggest that monocytes are one site of replication, while DC are not. The capacities of monocytes and DC to present BHV-1 viral antigens in vitro were compared. T lymphocytes (CD2(+) or CD4(+)) from BHV-1 immune cattle were stimulated in the presence of APC previously incubated with live or inactivated wild-type BHV-1. DC stimulated strong proliferation of Ag-specific T cells, while monocytes were poor stimulators of T-cell proliferation. When viral attachment to the surface of the APC was inhibited by virus pretreatment with soluble heparin, T-cell proliferation was dramatically decreased. Unexpectedly, incubation of DC and monocytes with the deletion mutant BHV-1 gD-/-, which displays impaired fusion capacity, resulted in strong activation of T lymphocytes by both APC types. Collectively, these results indicate that presentation of BHV-1 antigens to immune T cells is effective in the absence of productive infection and suggest that BHV-1 gD-/- mutant virus could be used to induce virus-specific immune responses in cattle.

Animals↗
PubMed1999

[In vitro study of the interactions between bovine herpesvirus 4 and the bovine host cells].

This work was devoted to the study of the interactions between bovine herpesvirus 4 (BHV-4) and bovine cells in vitro. It led to the discovery of two interesting properties of BVH-4 replication cycle: first, the cellular receptor heparan sulfate was proven to mediate BVH-4 binding to target cells. This is the first description of the implication of heparan sulfate in the binding process of a gammaherpesvirus. Second, using synchronised cells, the replication of BVH-4 DNA was proven to be dependent on the S phase of the cell cycle. This dependence could explain some properties of BVH-4 infection in vitro and could play an important role in the biology of the infection in vivo. Finally, in order to produce monoclonal antibodies against BVH-4 IE1 and IE2 proteins, the genes coding for these proteins were cloned and expressed in prokaryotic cells.

Animals↗
PubMed1999

[Comparison of intracellular and extracellular particles of the vaccinia virus].

The construction of recombinant vaccinia viruses (VV) is definitely a promising approach to develop vaccines for pets. To produce safer VV recombinant vaccines, it is necessary to understand the pathogenesis of VV infection. The goal of this study was to compare the biological properties of the extracellular and intracellular infectious particles of VV. Four biological properties were investigated: (i) the binding into cellular receptor(s); (ii) the mechanism of entry; (iii) the resistance to neutralisation by antibodies; (iv) the resistance to complement lysis. The results obtained in this study emphasize the differences existing between the two infectious forms of VV and demonstrate the adaptation of the extracellular particle to its role of virus dissemination in vivo. The results of this study implicate that VV strains used as expression vector in vaccinology should be deleted for genes required for EEV morphogenesis.

Animals↗
PubMed1998

Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope.

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Here we have investigated the resistance of EEV and IMV to neutralization by complement in the absence of immune antibodies. When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV resistance was not a result of EEV protein B5R, despite its similarity to proteins of the regulators of complement activation (RCA) family, or to any of the other EEV proteins tested (A34R, A36R, and A56R gene products). EEV was sensitive to complement when the virus was grown in one species and challenged with complement from a different species, suggesting that complement resistance might be mediated by host RCA incorporated into the EEV outer envelope. This hypothesis was confirmed by several observations: (i) immunoblot analysis revealed that cellular membrane proteins CD46, CD55, CD59, CD71, CD81, and major histocompatibility complex class I antigen were detected in purified EEV but not IMV; (ii) immunoelectron microscopy revealed cellular RCA on the surface of EEV retained on the cell surface; and (iii) EEV derived from rat cells expressing the human RCA CD55 or CD55 and CD59 were more resistant to human complement than EEV derived from control rat cells that expressed neither CD55 nor CD59. These data justify further analysis of the roles of these (and possible other) cellular proteins in EEV biology.

Animals↗
PubMed1998

Intracellular and extracellular vaccinia virions enter cells by different mechanisms.

Vaccinia virus (VV) produces two antigenically distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Structurally, EEV consists of an IMV with an additional outer membrane containing proteins that are absent from IMV. EEV is important for virus dissemination both in vitro and in vivo. Studies of EEV entry have been hampered by having two infectious virions and by the rupture of the EEV outer membrane in the majority of EEV virions during their purification. To overcome these problems, we have developed a novel approach to study VV entry that is based on confocal microscopy and does not require EEV purification. This assay relies on immunofluorescent staining and detection of individual, intracellular, uncoated virus cores. By this method, we show that EEV entry, in contrast to IMV, is dependent on a low-pH pathway and that the IMV enwrapped inside the EEV exhibits a low-pH fusogenic activity. Together with neutralization data demonstrating that exposure to low pH disrupts the EEV outer membrane, this study strongly supports a model for EEV entry which consists of binding, endocytosis, low-pH-induced disruption of the EEV outer membrane and fusion of the exposed IMV with the endosomal membrane releasing the core into the cytosol. The roles of the EEV outer membrane in virus dissemination and virus entry are discussed in relation to this model.

Animals↗
PubMed1998

Extracellular enveloped vaccinia virus. Entry, egress, and evasion.

Vaccinia virus is a large and complex virus that produces two types of infectious virus particles, termed intracellular mature virus (IMV) and extracellular enveloped virus (EEV). EEV contains an extra lipid envelope and ten associated proteins that are absent from IMV. Although EEV represents less than 1% of infectious progeny it is very important biologically. First, it mediates virus dissemination and second, it is the virus against which protective immune responses are directed. This article reviews the genes known to encode EEV proteins and their functions, describes recent data showing that the cellular receptors for IMV and EEV are different, and demonstrates that EEV, in contrast to IMV, is resistant to neutralisation by antibody.

Antibodies, Viral↗
PubMed1997

Bovine herpesvirus 1-induced apoptosis occurs at the G0/G1 phase of the cell cycle.

We have previously shown that bovine herpesvirus 1 (BHV-1), even when inactivated, induces apoptotic cell death in mitogen-stimulated bovine peripheral blood mononuclear cells (PBMCs) (Hanon et al., 1996, J. Virol. 70, 4116-4120). In order to gain insight into this process, we have investigated the cell cycle phase at which BHV-1 induces apoptosis in PBMCs. Our results show that the percentage of cells that progress through the S phase was always lower in BHV-1-infected PBMCs than in control cells. This effect was not due to a defective activation of mitogen-stimulated PBMCs since BHV-1 only slightly affected the percentage of cells expressing BoCD25, a well-known lymphocyte activation marker. Furthermore, mimosine and cyclosporine A, two chemicals that inhibit entry into the S phase of the cell cycle by different pathways, did not affect the ability of BHV-1 to induce apoptosis. BHV-1-induced apoptosis also occurred in unstimulated PBMCs and interestingly, this was associated with the expression of c-myc and BoCD25 proteins both of which are related to cell cycle progression. All together, these data provide evidence demonstrating that BHV-1-induced apoptosis occurs at the G0/G1 phase of the cell cycle.

Animals↗
PubMed1997

Optimization of murine CD8+ cytotoxic T-lymphocyte responses to pseudorabies virus.

The aim of this work was to optimize the procedures used to elicit a cellular immune response to pseudorabies virus (PrV) in mice, using various immunization schedules and routes. An Eu-labeling-based cytotoxic T-lymphocyte (CTL) test was developed to measure the response. This necessitated optimization of numerous steps. In suspension, Eu labeling required high concentrations of dextran-sulfate (DXS) and Eu with a 30-min labeling time at room temperature. For anchored cells, the labeling time was 1 to 48 h, and the labeling efficiency depended strongly on the Eu concentration, but only marginally on the DXS concentration. In vivo and in vitro stimulation protocols were also optimized for the CTL test. For in vitro stimulation, spleen cells were cultured in T-25 flasks at a multiplicity of infection (m.o.i.) of 2. The CTL test was validated by specific depletion of CD8+ CTL, FACS analysis, and by comparing Eu and 51 Cr labeling. Then groups of mice were vaccinated once or twice by various routes (intraperitoneal (i.p.), intravenous (i.v.), subcutaneous (s.c.) and in the rear footpads (FP)) and according to different time schedules. CTL activity was detected only in boosted animals immunized FP, i.p. or i.v. That the cellular immune response contributes to protection was further suggested by the observation that i.p. immunization conferred better protection against challenge than s.c. immunization.

3T3 Cells↗
PubMed1997

Antibodies against vaccinia virus do not neutralize extracellular enveloped virus but prevent virus release from infected cells and comet formation.

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). EEV is important for the efficient dissemination of virus both in vivo and in vitro where it causes formation of comet-shaped virus plaques. Here, we show that EEV, in contrast to IMV, is resistant to neutralization by antibodies bound to its surface. However, antibodies against EEV can prevent comet formation in cell culture. To explain this apparent paradox, we investigated the mechanism by which antibodies inhibit comet formation and demonstrated that antibodies prevent EEV release from infected cells, and consequently comet formation, by agglutination of the virus on the cell surface. Two complementary observations allow this conclusion: first, electron microscopy showed that infected cells incubated with medium containing anti-vaccinia virus antibodies have virus aggregates on their surface; second, culture medium from these cells contained a 4 log10 fold reduction in the physical particle/ml titre in comparison with control culture. A mechanism by which antibodies to EEV proteins provide immunological protection is thus restriction of EEV release rather than neutralization of free EEV particles.

Agglutination↗
PubMed1997

Vaccinia virus immune evasion.

Vaccinia virus and other poxviruses express a wide variety of proteins which are non-essential for virus replication in culture but help the virus to evade the host response to infection. Examples include proteins which oppose apoptosis, synthesise steroids, capture chemokines, counteract complement, interfere with interferon and intercept interleukins. This review provides an overview of such proteins, with an emphasis on work from our laboratory, and illustrates how the study of these proteins can increase our understanding of virus pathogenesis, the function of the immune system and how to make safer and more immunogenic poxvirus-based vaccines.

Animals↗
PubMed1997

A novel virus binding assay using confocal microscopy: demonstration that the intracellular and extracellular vaccinia virions bind to different cellular receptors.

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV), which bind to unidentified and possibly different cellular receptors. Studies of VV binding have been hampered by having two infectious virions and by the rupture of the EEV outer membrane in the majority of EEV virions during purification. To overcome these problems, we have developed a novel approach to study VV binding that is based on confocal microscopy and does not require EEV purification. In this assay, individual virus particles adsorbed to the cell are simultaneously distinguished and quantified by double immunofluorescence labelling with antibody markers for EEV and IMV. By this method, we show unequivocally that IMV and EEV bind to different cellular receptors. Three independent observations allow this conclusion. First, the efficiencies with which IMV and EEV bind to different cell lines are unrelated; second, cell surface digestion with some enzymes affects IMV and EEV binding differently; and third, the binding of a monoclonal antibody to cells prevents IMV binding but not EEV binding. This technique may be widely applicable for studying the binding of different viruses.

Animals↗
PubMed1995

The replication in vitro of the gammaherpesvirus bovine herpesvirus 4 is restricted by its DNA synthesis dependence on the S phase of the cell cycle.

Because several observations have suggested that replication of the gammaherpesvirus bovine herpesvirus 4 (BHV-4) is influenced by the physiological state of the host cell, a study was carried out to determine the relationship between BHV-4 infection and the cell cycle. The temporal expression of BHV-4 late (L) proteins in unsynchronized cell cultures was first investigated by flow cytometry. Interestingly, L protein expression occurred in a limited number of cells infected with a high multiplicity of infection, and a reciprocal correlation between the percentage of positive cells and the cell density at the time of infection was demonstrated. Moreover, the finding that a BHV-4 early-late protein was expressed in nearly all the cells suggested that a blockage in the viral replication cycle occurred in some infected cells at the stage of viral DNA synthesis or L protein expression. Because this blockage could be the consequence of the dependence of one or both of these events on the cell cycle, they were investigated after infection of synchronized cell cultures. The following findings were made. (i) Cell transition through the S phase quantitatively increased the rate of BHV-4 DNA replication. (ii) BHV-4 DNA synthesis could not be detected in cells arrested in G0. (iii) Synchronization of MDBK cells with Lovastatin before infection increased the percentage of cells expressing L proteins. (iv) In contrast, infection of cells arrested in G0 led to few positive cells. Taken together these results showed that BHV-4 DNA replication and consequently the expression of L proteins are dependent on the S phase of the cell cycle. This dependence could be of importance for several biological properties of BHV-4 infection in vitro and might have implications for the biology of the virus in vivo.

Animals↗
PubMed1994

Madin Darby bovine kidney cell synchronization by lovastatin: application to bovine herpesvirus-1 gene expression.

The number of investigations involving cell proliferation has increased rapidly in the last years. One of the major difficulties in studying cell-cycle-related events is obtaining highly synchronous cell populations without metabolic imbalance. This study demonstrates that the Madin Darby bovine kidney (MDBK) cells, a commonly used cell line in veterinary research, can be effectively synchronized using lovastatin (Lov), a drug used to treat hypercholesteremia in humans. This was demonstrated by the following results: (i) Lov inhibits cell proliferation in a dose-dependent manner; (ii) Lov synchronizes MDBK cells mainly in the G1 and secondarily in the G2+M cell-cycle phases; (iii) the cytostatic effect of Lov can be specifically inhibited by addition of mevalonate (Mev) (Lov inhibits the synthesis of Mev); (iv) removal of Lov from G1-arrested cultures, followed by addition of Mev, resulted in the synchronous recovery of DNA synthesis; and (v) 5-bromo2'-deoxyuridine incorporation experiments revealed that MDBK cells synchronization by Lov can be followed for at least 3 cycles after removal of Lov and addition of Mev. Furthermore, as an application of investigations based on the availability of synchronized MDBK, we showed that bovine herpesvirus-1 gene expression is independent on the cell cycle.

Animals↗
PubMed1993

Attachment of the gammaherpesvirus bovine herpesvirus 4 is mediated by the interaction of gp8 glycoprotein with heparinlike moieties on the cell surface.

Cell surface heparan sulfate serves as the initial receptor for several alphaherpesviruses and at least one betaherpesvirus. This study shows that during the process of adsorption of the gammaherpesvirus bovine herpesvirus 4 (BHV-4), the viral glycoprotein gp8 interacts with heparinlike moieties of cell surface. This conclusion is based on the following findings. (i) Soluble heparin was capable of blocking BHV-4 infection of Georgia bovine kidney cells by inhibition of viral attachment. (ii) Nevertheless, after virus adsorption to Georgia bovine kidney cells, heparin was partially capable of removing adsorbed virus. (iii) Enzymatic digestion of cell surface heparan sulfate but not of chondroitin sulfates A, B, and C reduced the binding of the virus to the cells, and rendered the cells partially resistant to infection. (iv) Radiolabeled purified BHV-4 bound to wild-type Chinese hamster ovary cells, whereas binding of the virus to mutant Chinese hamster ovary cell lines that where deficient in either all glycosaminoglycans or only heparan sulfate was significantly impaired. (v) Using heparin-affinity chromatography, gp8 glycoprotein was shown to bind specifically to immobilized heparin and to elute in the presence of soluble heparin. These data together showed that the gammaherpesvirus BHV-4, like alphaherpesviruses and one betaherpesvirus, adsorbs to cells by binding to cell surface heparin-like moieties. Therefore, this study extends the group of herpesviruses interacting with heparinlike moieties at the cell surface to a member of the gammaherpesvirinae subfamily.

Adsorption↗