Biomedical subjects

Herman W Favoreel

Publications and source records attributed to Herman W Favoreel.

At least 19 recordsLinked to original sources

PubMed2006

Alpha-herpesvirus glycoprotein D interaction with sensory neurons triggers formation of varicosities that serve as virus exit sites.

Alpha-herpesviruses constitute closely related neurotropic viruses, including herpes simplex virus in man and pseudorabies virus (PRV) in pigs. Peripheral sensory neurons, such as trigeminal ganglion (TG) neurons, are predominant target cells for virus spread and lifelong latent infections. We report that in vitro infection of swine TG neurons with the homologous swine alpha-herpesvirus PRV results in the appearance of numerous synaptophysin-positive synaptic boutons (varicosities) along the axons. Nonneuronal cells that were juxtaposed to these varicosities became preferentially infected with PRV, suggesting that varicosities serve as axonal exit sites for the virus. Viral envelope glycoprotein D (gD) was found to be necessary and sufficient for the induction of varicosities. Inhibition of Cdc42 Rho GTPase and p38 mitogen-activated protein kinase signaling pathways strongly suppressed gD-induced varicosity formation. These data represent a novel aspect of the cell biology of alpha-herpesvirus infections of sensory neurons, demonstrating that virus attachment/entry is associated with signaling events and neuronal changes that may prepare efficient egress of progeny virus.

Animals↗

PubMed2006

Immune escape of equine herpesvirus 1 and other herpesviruses of veterinary importance.

Equine herpesvirus (EHV)-1 is a pathogen of horses, well known for its ability to induce abortion and nervous system disorders. Clinical signs may occur despite the presence of a virus-specific immune response in the horse. The current review will summarize the research, on how, EHV-1-infected cells can hide from recognition by the immune system. Research findings on immune evasion of EHV-1 will be compared with those of other herpesviruses of veterinary importance.

Animals↗

PubMed2006

The why's of Y-based motifs in alphaherpesvirus envelope proteins.

The Alphaherpesvirinae are large DNA viruses and represent the largest subfamily of the Herpesviridae with closely related members of man and animal, including herpes simplex virus, varicella-zoster virus, pseudorabies virus, bovine herpesvirus 1, and many others. The viral envelope proteins of alphaherpesviruses are remarkably diverse and are incorporated in the ER, Golgi, and plasma membrane of infected cells. The cytoplasmic domain of many of these envelope proteins contain specific tyrosine-based amino acids. During recent years, accumulating evidence indicates that these tyrosine-based motifs serve different important functions during the virus life cycle, and are implicated in endocytosis processes, intracellular trafficking, basolateral and axonal sorting, and signal transduction events. The current minireview will discuss the functions associated with these tyrosine-based motifs in alphaherpesvirus envelope proteins.

Alphaherpesvirinae↗

PubMed2005

Herpesvirus interference with virus-specific antibodies: bridging antibodies, internalizing antibodies, and hiding from antibodies.

Herpesviruses have developed different tools to thwart efficient antibody-dependent neutralisation and lysis of virions and elimination of infected cells. This overview will briefly summarize different of these tools, including (i) viral Fc receptors and the resulting process of antibody bridging, (ii) internalization of individual viral proteins and clustered antibody-antigen complexes from the plasma membrane of infected cells, and (iii) directed egress of virus particles to sites of intimate cell-cell contact that are difficult to access for antibodies.

Animals↗

PubMed2005

Cell type-specific resistance of trigeminal ganglion neurons towards apoptotic stimuli.

Trigeminal ganglion (TG) neurons are important target cells for many alphaherpesviruses and constitute a major site of virus latency and reactivation. Earlier we showed that porcine TG neurons are remarkably more resistant towards (apoptotic) cell death resulting from infection by the swine alphaherpesvirus pseudorabies virus (PRV) compared to a broad range of other primary porcine cell types and that this resistance does not depend on the strongly anti-apoptotic US3 viral protein kinase (Geenen, K., Favoreel, H.W., Nauwynck, H.J., 2005a. Higher resistance of porcine trigeminal ganglion neurons towards pseudorabies virus-induced cell death compared with other porcine cell types in vitro. J. Gen. Virol. 86, 1251-1260). Although other viral anti-apoptotic proteins may be involved in survival of TG neurons during PRV infection, an additional factor may be that TG neurons possess a cell type-dependent capacity to withstand apoptosis compared to other cell types. To investigate this, we treated uninfected porcine TG cultures, swine kidney cells, and porcine superior cervical ganglion (SCG) neurons with several apoptosis-inducing reagents (staurosporine, camptothecin and genistein). None of these reagents were able to trigger substantial apoptotic cell death in TG neurons, whereas non-neuronal TG cells, swine kidney cells, and SCG neurons showed a clear dose-dependent increase in apoptosis using either of these reagents. In conclusion, sensory TG neurons may contain a cell type-specific capacity to withstand different apoptotic assaults, including infection with an alphaherpesvirus.

Animals↗

PubMed2005

Cytoskeletal rearrangements and cell extensions induced by the US3 kinase of an alphaherpesvirus are associated with enhanced spread.

The US3 protein is a viral kinase that is conserved among the Alphaherpesvirinae. Here, we show that US3 of the swine alphaherpesvirus pseudorabies virus causes dramatic alterations in the cytoskeleton, resulting in the formation of long actin- and microtubule-containing cell projections in infected and transfected cells. Analysis with a GFP-labeled virus showed that multiple virus particles move inside the projections toward the tip. GFP-labeled virus could also be found in the cytoplasm of neighboring cells that were in contact with the projections. In addition, projection formation could be inhibited by using the actin-stabilizing drug jasplakinolide and could be induced by using the Rho kinase inhibitor Y27632. Analyzing the effect of these drugs on intercellular virus spread indicated that the observed US3-induced alterations in the host cytoskeleton are associated with enhanced intercellular virus spread, thereby suggesting a previously undescribed aspect of alphaherpesvirus spread.

Amides↗

PubMed2005

The pseudorabies virus US3 protein kinase possesses anti-apoptotic activity that protects cells from apoptosis during infection and after treatment with sorbitol or staurosporine.

Most large DNA viruses, like herpesviruses, encode anti-apoptotic proteins to interfere with the apoptotic cellular response to infection. Previous studies have shown that the US3 protein kinase of herpes simplex virus, in contrast to US3 of bovine herpes virus 1, is very potent in protecting cells from apoptosis induced by the virus itself or by a broad range of exogenous apoptotic stimuli. Here, we demonstrate that US3 of the swine alphaherpesvirus pseudorabies virus (PRV) suppresses PRV-induced apoptosis in swine-testicle (ST) cells at late stages in infection, and that it protects ST cells from apoptosis induced by either sorbitol or staurosporine. Interestingly, PRV US3 encodes a short and a long isoform, the latter of which contains a functional mitochondrial localization sequence. Transient transfections showed that the PRV US3 long isoform is more efficient in protecting ST cells from PRV- or staurosporine-induced apoptosis, suggesting a potential advantage for the mitochondrial localization of PRV US3 in implementing its anti-apoptotic function.

Animals↗

PubMed2005

Higher resistance of porcine trigeminal ganglion neurons towards pseudorabies virus-induced cell death compared with other porcine cell types in vitro.

Trigeminal ganglion (TG) neurons are important target cells for many alphaherpesviruses, constituting major sites for latency/reactivation events. Here, the in vitro kinetics of productive infection of the swine alphaherpesvirus pseudorabies virus (PRV) and resulting cell death in primary porcine TG neurons were determined, and these were compared with similar kinetics in many other porcine cell types. Confocal microscopy showed that all TG neurons expressed late genes such as viral glycoproteins, and that these glycoproteins were processed through the Golgi and reached the cell surface as in other cell types, albeit with a delay of +/-2-6 h. However, TG neurons were much more resistant towards PRV-induced cell death compared with all other porcine cell types tested (non-neuronal TG cells, superior cervical ganglion neurons, epithelial kidney cells, arterial endothelial cells, dermal fibroblasts and cells derived from a porcine swine kidney cell line). About half of the TG neurons survived up to 96 h post-inoculation (end of experiment), whereas all other cell types almost completely succumbed within 2 days post-inoculation. In addition, infection with a strongly pro-apoptotic PRV strain that misses the anti-apoptotic US3 protein did not lead to substantial apoptosis in TG neurons, even at 72 h post-inoculation. Thus, primary porcine TG neurons can be infected with PRV in vitro, and are remarkably more resistant to PRV-induced cell death compared with other porcine cell types, suggesting a cell type-specific resistance to alphaherpesvirus-induced cell death that may have important implications for different aspects of the virus life cycle, including latency/reactivation events.

Animals↗

PubMed2005

Pseudorabies virus glycoprotein gD contains a functional endocytosis motif that acts in concert with an endocytosis motif in gB to drive internalization of antibody-antigen complexes from the surface of infected monocytes.

Viral glycoproteins gB and gD of the swine alphaherpesvirus pseudorabies virus (PRV), which is closely related to human herpes simplex virus and varicella-zoster virus, are able to drive internalization of antibody-antigen complexes that may form at the cell surface of infected monocytes, thereby protecting these cells from efficient antibody-mediated lysis. We found earlier that gB relies on an endocytosis motif in its cytoplasmic domain for its function during this internalization process. Here, we report that the PRV gD protein also contains a functional endocytosis motif (YRLL) in its cytoplasmic domain that drives spontaneous endocytosis of gD from the cell surface early in infection and that acts in concert with the endocytosis motif in gB to contribute to efficient internalization of antibody-antigen complexes in PRV-infected monocytes.

Amino Acid Motifs↗

PubMed2004

Copatching and lipid raft association of different viral glycoproteins expressed on the surfaces of pseudorabies virus-infected cells.

Pseudorabies virus (PRV) is a swine alphaherpesvirus that is closely related to human herpes simplex virus (HSV). Both PRV and HSV express a variety of viral envelope glycoproteins in the plasma membranes of infected cells. Here we show that at least four major PRV glycoproteins (gB, gC, gD, and gE) in the plasma membrane of infected swine kidney cells and monocytes seem to be linked, since monospecific antibody-induced patching of any one of these proteins results in copatching of the others. Further, for all four PRV glycoproteins, monospecific antibody-induced patches were enriched in GM1, a typical marker of lipid raft microdomains, but were excluded for transferrin receptor, a nonraft marker, suggesting that these viral proteins may associate with lipid rafts. However, only gB and, to a lesser extent, gE were found in lipid raft fractions by using detergent floatation assays, indicating that gC and gD do not show strong lipid raft association. Addition of methyl-beta-cyclodextrin (MCD), a cholesterol-depleting agent that is commonly used to disrupt lipid rafts, only slightly reduced copatching efficiency between the different viral proteins, indicating that other factors, perhaps tegument-glycoprotein interactions, may be important for the observed copatching events. On the other hand, MCD strongly reduced polarization of the antibody-induced viral glycoprotein patches to a cap structure, a gE-dependent process that has been described for specific PRV- and HSV-infected cells. Therefore, we hypothesize that efficient gE-mediated capping of antibody-antigen patches may require the lipid raft-associated signal transduction machinery.

Animals↗

PubMed2004

Internalization of pseudorabies virus glycoprotein B is mediated by an interaction between the YQRL motif in its cytoplasmic domain and the clathrin-associated AP-2 adaptor complex.

The cytoplasmic domain of pseudorabies virus (PRV) glycoprotein B (gB) contains three putative internalization motifs. Previously, we demonstrated that the tyrosine-based YQRL motif at positions 902 to 905, but not the YMSI motif at positions 864 to 867 or the LL doublet at positions 887 and 888, is required for correct functioning of gB during antibody-mediated internalization of PRV cell surface-bound glycoproteins. In the present study, we demonstrate that the YQRL motif is also crucial to allow spontaneous internalization of PRV gB, and thus, that spontaneous and antibody-mediated internalizations of PRV gB occur through closely related mechanisms. Furthermore, we found that PRV gB colocalizes with the cellular clathrin-associated AP-2 adaptor complex and that this colocalization depends on the YQRL motif. In addition, by coimmunoprecipitation assays, we found that during both spontaneous and antibody-dependent internalization, PRV gB physically interacts with AP-2, and that efficient interaction between gB and AP-2 required an intact YQRL motif. Collectively, these findings demonstrate for the first time that during internalization of an alphaherpesvirus envelope protein, i.e., PRV gB, a specific amino acid sequence in the cytoplasmic tail of the protein interacts with AP-2 and may constitute a common AP-2-mediated mechanism of internalization of alphaherpesvirus envelope proteins.

Adaptor Protein Complex 2↗

PubMed2003

Antibody-induced internalization of viral glycoproteins and gE-gI Fc receptor activity protect pseudorabies virus-infected monocytes from efficient complement-mediated lysis.

Pseudorabies virus (PRV)-infected blood monocytes are able to transport virus throughout the body of vaccination-immune pigs. PRV-infected monocytes express viral glycoproteins in their plasma membrane that can be recognized by virus-specific antibodies. Recently, it has been shown that addition of PRV-specific polyclonal immunoglobulins to PRV-infected monocytes at 37 degrees C induces internalization of the majority of plasma membrane-expressed viral glycoproteins. This study investigated whether this process may interfere with efficient antibody-dependent complement-mediated lysis (ADCML) of infected monocytes. Therefore, an ADCML assay was set up in vitro. A significant decrease in the percentage of cells lysed by ADCML was observed when antibody-induced internalization of PRV glycoproteins occurred (P<0.005). Furthermore, it is shown (i) that the PRV gE-gI complex, which, like certain other alpha herpesvirus orthologues, possesses IgG-binding capacity, aids in avoiding efficient ADCML of PRV-infected monocytes and (ii) that the efficiency of PRV gE-gI-mediated evasion of ADCML can be decreased by the presence of gE-gI-specific antibodies.

Animals↗

PubMed2003

Virus complement evasion strategies.

The immune system has a variety of tools at its disposal to combat virus infections. These can be subdivided roughly into two categories: 'first line defence', consisting of the non-specific, innate immune system, and 'adaptive immune response', acquired over time following virus infection or vaccination. During evolution, viruses have developed numerous, and often very ingenious, strategies to counteract efficient recognition of virions or virus-infected cells by both innate and adaptive immunity. This review will focus on the different strategies that viruses use to avoid recognition by one of the components of the immune system: the complement system. Complement evasion is of particular importance for viruses, since complement activation is a crucial component of innate immunity (alternative and mannan-binding lectin activation pathway) as well as of adaptive immunity (classical, antibody-dependent complement activation).

Animals↗

PubMed2003

Transmission of pseudorabies virus from immune-masked blood monocytes to endothelial cells.

Pseudorabies virus (PRV) may cause abortion, even in the presence of vaccination-induced immunity. Blood monocytes are essential to transport the virus in these immune animals, including transport to the pregnant uterus. Infected monocytes express viral proteins on their cell surface. Specific antibodies recognize these proteins and should activate antibody-dependent cell lysis. Previous work showed that addition of PRV-specific polyclonal antibodies to PRV-infected monocytes induced internalization of viral cell surface proteins, protecting the cells from efficient antibody-dependent lysis in vitro (immune-masked monocytes). As a first step to reach the pregnant uterus, PRV has to cross the endothelial cell barrier of the maternal blood vessels. The current aim was to investigate in vitro whether immune-masked PRV-infected monocytes can transmit PRV in the presence of virus-neutralizing antibodies via adhesion and fusion of these monocytes with endothelial cells. Porcine blood monocytes, infected with a lacZ-carrying PRV strain, were incubated with PRV-specific antibodies to induce internalization. Then, cells were co-cultivated with endothelial cells for different periods of time. Only PRV-infected monocytes with internalized viral cell surface proteins adhered efficiently to endothelial cells. LacZ transmission to endothelial cells, as a measure for monocyte-endothelial cell fusion, could be detected after co-cultivation from 30 min onwards. Virus transmission was confirmed by the appearance of plaques. Adhesion of immune-masked PRV-infected monocytes to endothelial cells was mediated by cellular adhesion complex CD11b-CD18 and subsequent fusion was mediated by the virus. In conclusion, immune-masked PRV-infected monocytes can adhere and subsequently transmit virus to endothelial cells in the presence of PRV-neutralizing antibodies.

Animals↗

PubMed2003

Pseudorabies virus (PRV)-specific antibodies suppress intracellular viral protein levels in PRV-infected monocytes.

Blood monocytes infected with pseudorabies virus (PRV), a swine alphaherpesvirus, are not eliminated efficiently by antibody-dependent immunity and may occasionally transport PRV to the pregnant uterus of vaccinated animals. This study examines in vitro the long-term fate of PRV-infected monocytes cultivated in the presence of porcine PRV-specific antibodies. All monocytes were infected and expressed viral late proteins, and 30 % of PRV-infected monocytes cultivated with PRV-specific antibodies survived up to 194 h post-infection (p.i.), the end of the experiment (compared to 0 % for cells cultivated with PRV-negative antibodies). Of these surviving cells, +/-75 % no longer expressed microscopically detectable viral late proteins from 144 h p.i. onwards. Remarkably, monocytes infected with a PRV gB-null virus did not survive in the presence of PRV-specific antibodies. These data suggest that PRV-specific antibodies suppress viral protein levels in infected monocytes, perhaps helping the virus to persist and reach internal organs in vaccinated animals.

Animals↗

PubMed2003

Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.

Amino Acid Sequence↗

PubMed2003

Pseudorabies virus US3 protein kinase mediates actin stress fiber breakdown.

Disruption of specific components of the host cytoskeleton has been reported for several viruses and is thought to be beneficial for viral replication and spread. Our previous work demonstrated that infection of swine kidney (SK-6) cells with pseudorabies virus (PRV), a swine alphaherpesvirus, induced actin stress fiber breakdown. In the present study, using several PRV deletion mutants, we found that the US3 serine/threonine (S/T) protein kinase is involved in breakdown of actin stress fibers in different PRV-infected cell lines. Further, by transfection assays, we showed that PRV US3 itself, in the absence of other viral proteins, is able to trigger actin stress fiber breakdown when it is localized in sufficient amounts in the nucleus.

Actins↗

PubMed2002

Antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes and role of the cytoskeleton: a confocal study.

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane anchored viral glycoproteins. This process may interfere with antibody-dependent cell lysis and resembles the well-studied physiological endocytosis process. A confocal study was designed to investigate whether the major cellular components, involved in physiological endocytosis (clathrin, actin, dynein and microtubules), play a role in this virological internalization process. In order to visualize the interaction of endosomes, which contain the internalized viral glycoproteins, with clathrin, actin, dynein and microtubules, a double labeling of viral glycoproteins and different cellular proteins was performed. Porcine monocytes were inoculated with the PrV-strain 89V87 at a multiplicity of infection of 50 for 13h. After the addition of FITC-labeled porcine polyclonal PrV-specific antibodies, cells were fixed with para-formaldehyde at different time points and afterwards permeabilized. The different cellular components were visualized with monoclonal antibodies and a Texas Red-conjugate, with the exception of actin, which was stained with phalloidin-Texas Red. The cells were analyzed by confocal microscopy. A clear co-localization was observed between the viral glycoproteins and clathrin and dynein during the internalization process. The microtubules were in close contact with the internalized vesicles. For actin no co-localization could be observed. It can be stated that clathrin, dynein and microtubules, important components during physiological endocytosis, are also of importance during the antibody-induced internalization of viral glycoproteins.

Actins↗