Pseudorabies Virus US3 Protein Kinase Protects Infected Cells from NK Cell-Mediated Lysis via Increased Binding of the Inhibitory NK Cell Receptor CD300a.

UNLABELLED: Several reports have indicated that natural killer (NK) cells are of particular importance in the innate response against herpesvirus infections. As a consequence, herpesviruses have developed diverse mechanisms for evading NK cells, although few such mechanisms have been identified for the largest herpesvirus subfamily, the alphaherpesviruses. The antiviral activity of NK cells is regulated by a complex array of interactions between activating/inhibitory receptors on the NK cell surface and the corresponding ligands on the surfaces of virus-infected cells. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) displays previously uncharacterized immune evasion properties: it triggers the binding of the inhibitory NK cell receptor CD300a to the surface of the infected cell, thereby providing increased CD300a-mediated protection of infected cells against NK cell-mediated lysis. US3-mediated CD300a binding was found to depend on aminophospholipid ligands of CD300a and on group I p21-activated kinases. These data identify a novel alphaherpesvirus strategy for evading NK cells and demonstrate, for the first time, a role for CD300a in regulating NK cell activity upon contact with virus-infected target cells. IMPORTANCE: Herpesviruses have developed fascinating mechanisms to evade elimination by key elements of the host immune system, contributing to their ability to cause lifelong infections with recurrent reactivation events. Natural killer (NK) cells are central in the innate antiviral response. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus displays a previously uncharacterized capacity for evasion of NK cells. Expression of US3 protects infected cells from NK cell-mediated lysis via increased binding of the inhibitory NK cell receptor CD300a. We show that this US3-mediated increase in CD300a binding depends on aminophospholipids and on cellular p21-activated kinases (PAKs). The identification of this novel NK cell evasion strategy may contribute to the design of improved herpesvirus vaccines and may also have significance for other PAK- and CD300a-modulating viruses and cancer cells.

Identification of target cells of a European equine arteritis virus strain in experimentally infected ponies.

Currently, little is known on the cellular pathogenesis of equine arteritis virus (EAV). The purpose of the present study was to identify the target cells in ponies experimentally inoculated with EAV 08P178 (EU, clade-1). EAV-target organs (respiratory tissues with associated lymphoid tissues and large intestines), collected at 3 and 7 days post inoculation (dpi) and with virus titers≥10(5.0) TCID50/g, were processed with double immunofluorescence staining for the simultaneous detection of EAV N-protein and one of the following cell markers: CD172a (myeloid cells), CD3 (T lymphocytes), IgM (B lymphocytes) and von Willebrand factor (endothelial cells). In the different analyzed organs, 31-58% and 47-63% of the EAV-positive cells were mononuclear leukocytes (mainly CD172a(+) followed by CD3(+)) at 3 and 7 dpi, respectively. EAV-positive endothelial cells were not detected in 3.200 large blood vessels (≥3 endothelial cells/vessel cross section). However, in terminal capillaries (1-2 endothelial cells/vessel cross section) of the different organs, 15-51% of the endothelial cells were EAV-positive. In conclusion, the present study demonstrates that EAV 08P178 (i) has a main tropism for CD172a(+) and CD3(+) mononuclear leukocytes and (ii) infects a large number of endothelial cells in terminal capillaries. EAV 08P178 infection in capillaries is most probably the cause of an increased vascular permeability leading to leakage of fluid (edema-serous exudate) but not to severe vasculitis and hemorrhages.

Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea in human and animal. In piglets, ETEC having F4 fimbriae (F4(+) ETEC) induce severe diarrhea, dependent on the presence of receptors for F4 (F4R). In this study, porcine aminopeptidase N (pAPN) was identified as an F4R by comparative proteomic analysis of brush border proteins of F4R(+) and F4R(-) pigs and by adherence/internalization experiments on pAPN-transfected cells. Binding of F4 fimbriae to pAPN depended on sialic acid containing carbohydrate moieties, and resulted in clathrin-mediated endocytosis of the fimbriae. Endocytosis via pAPN was not restricted to F4 fimbriae, but was also observed for anti-pAPN antibodies. Both F4 fimbriae- and pAPN-specific antibodies were taken up in vivo by porcine enterocytes and induced subsequently a rapid immunoglobulin A and G response. In conclusion, we identified pAPN as an endocytotic receptor for F4 fimbriae and highlight the opportunity to target vaccine antigens to this epithelial receptor.

The IE180 protein of pseudorabies virus suppresses phosphorylation of translation initiation factor eIF2α.

We have previously shown that the porcine alphaherpesvirus pseudorabies virus (PRV) efficiently interferes with phosphorylation of the eukaryotic translation initiation factor eIF2α. Inhibition of phosphorylation of eIF2α has been reported earlier for the closely related alphaherpesvirus herpes simplex virus 1 (HSV-1) through its ICP34.5 and US11 proteins. PRV, however, does not encode an ICP34.5 or US11 orthologue. Assays using cycloheximide, UV-inactivated PRV, or phosphonoacetic acid (PAA) showed that de novo expression of one or more (immediate) early viral protein(s) is required for interference with eIF2α phosphorylation. In line with this, a time course assay showed that eIF2α phosphorylation was abolished within 2 h after PRV inoculation. PRV encodes only one immediate-early protein, IE180, the orthologue of HSV-1 ICP4. As reported earlier, a combinational treatment of cells with cycloheximide and actinomycin D allowed expression of IE180 without detectable expression of the US3 early protein in PRV-infected cells. This led to a substantial reduction in eIF2α phosphorylation levels, indicative for an involvement of IE180. In support of this, transfection of IE180 also potently reduced eIF2α phosphorylation. IE180-mediated interference with eIF2α phosphorylation was not cell type dependent, as it occurred both in rat neuronal 50B11 cells and in swine testicle cells. Inhibition of the cellular phosphatase PP1 impaired PRV-mediated interference with eIF2α phosphorylation, indicating that PP1 is involved in this process. In conclusion, the immediate-early IE180 protein of PRV has the previously uncharacterized ability to suppress phosphorylation levels of the eukaryotic translation initiation factor eIF2α.

Assessment of the efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and Tetrahymena spp.

The efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and two Tetrahymena spp. was determined based on the European Standard EN 1276:2009 suspension test. Trophozoite viability was assessed by determination of the membrane integrity using flow cytometry as a fast screening technique. Bovine serum albumin was added to simulate clean (0.3 g/liter) and dirty (3 g/liter) conditions. Benzalkonium chloride caused cell lysis at concentrations above 50 mg/liter under clean and dirty conditions. A concentration of 50 mg of free chlorine per liter had a strong biocidal effect on acanthamoebae and tetrahymenae after 15 min under clean and dirty conditions. Our results suggest that benzalkonium chloride and sodium hypochlorite were effective against the three microorganisms at concentrations commonly applied in the food industry.

Bacterial host interaction of GFP-labelled Vibrio anguillarum HI-610 with gnotobiotic sea bass, Dicentrarchus labrax (L.), larvae.

The location and cell damage caused by Vibrio anguillarum, the causative agent of classical vibriosis, within the developing gut of the newly hatched sea bass, Dicentrarchus labrax (L.), is unknown. A gnotobiotic sea bass model was used to investigate the early interactions of V. anguillarum with sea bass larvae. In the present study, germ-free sea bass larvae were orally exposed to a V. anguillarum HI-610 pathogen labelled with the green fluorescent protein (GFP-HI-610) and sampled at regular intervals. Pathogenic colonization of gut enterocytes was observed 2 h post-exposure (p.e.) and onwards, whereas bacteria within the swim bladder were visualized 48 h p.e and onwards. Ultrastructural findings demonstrated direct bacterial contact with the host cell in the oesophageal mucosa and putative attachment to microvilli of mid- and hindgut enterocytes. The present findings form a starting point for studies assessing the impact of potential candidates (probiotics, prebiotics, antimicrobial peptides) to mitigate bacterial virulence.

Keep it in the subfamily: the conserved alphaherpesvirus US3 protein kinase.

The US3 protein kinase is conserved over the alphaherpesvirus subfamily. Increasing evidence shows that, although the kinase is generally not required for virus replication in cell culture, it plays a pivotal and in some cases an essential role in virus virulence in vivo. The US3 protein is a multifunctional serine/threonine kinase that is involved in viral gene expression, virion morphogenesis, remodelling the actin cytoskeleton and the evasion of several antiviral host responses. In the current review, both the well conserved and virus-specific functions of alphaherpesvirus US3 protein kinase orthologues will be discussed.

Role of group A p21-activated kinases in the anti-apoptotic activity of the pseudorabies virus US3 protein kinase.

The alphaherpesvirus US3 kinase is a conserved multifunctional serine/threonine kinase that plays a role in several processes, including modulation of the actin cytoskeleton, egress of virus particles from the nucleus and inhibition of apoptosis. However, the mechanisms used by the US3 protein to exert its functions remain poorly understood. Recently, we identified the group A p21-activated kinases PAK1 and PAK2 as important effectors in the US3-mediated cytoskeletal rearrangements. Here, we investigated if group A PAKs are also involved in the anti-apoptotic properties of US3. Infection experiments using a group A PAK inhibitor pointed at a moderate role for group A PAKs in the anti-apoptotic properties of US3. Furthermore, infection assays using wild type and US3null PRV in wild type MEF, PAK1(-/-) MEF and PAK2(-/-) MEF indicated that PAK2 does not play a role in US3-mediated inhibition of apoptosis during infection, whereas PAK1 plays a significant, yet limited role. Experiments in US3-transfected MEF using staurosporine as apoptosis trigger confirmed these observations. These results show that PAK1 plays a significant, yet limited, role in the anti-apoptotic activity of US3.

Different replication characteristics of historical pseudorabies virus strains in porcine respiratory nasal mucosa explants.

Different alphaherpesviruses, including pseudorabies virus (PRV), are able to cross the basement membrane barrier in nasal respiratory epithelium. As a first step in investigating this invasion process, a detailed quantitative analysis system was set up to determine the kinetics of horizontal and vertical virus spread in nasal explants, using the virulent PRV strain 89V87. Plaque latitudes, total depths, depths measured from the basement membrane and volumes were determined at 0, 12, 24 and 36h post inoculation (pi). PRV 89V87 was found to spread in a plaquewise manner and to cross the basement membrane between 12 and 24hpi. During the 1960s-1970s, an increase in PRV virulence has been reported. To analyse potential differences in efficiency of infection and spread for different historical PRV strains, single infected cells and plaques of infected cells were quantified at 12 and 36hpi in nasal mucosa explants for seven European PRV strains, isolated in the 1960s (Becker, NIA1), the 1970s (NS374, NIA3, 75V19) and later (89V87, 00V72). All viruses were used at second passage in cell culture, except for the Becker strain, which had an unknown passage history. Older strains, Becker, NIA1 and/or NS374, showed lower numbers of primary infectious centers, lower capacity to form plaques and/or lower capacity to cross the basement membrane. The observed differences in virus-mucosa interactions may aid in understanding the virulence increase of PRV. The quantitative assay established here will be of use in unravelling the mechanism of alphaherpesvirus-mediated invasion through the basement membrane.

[Immune evasion of alphaherpesviruses]. / Immuno evasie van alfaherpesvirussen.

Alphaherpesviruses represent the largest subfamily of the herpesviruses and comprise many different, closely related pathogens of man and animal, including herpes simplex virus (cold sores, genital lesions) and varicella-zoster virus (chickenpox, shingles) in man, pseudorabies virus orAujeszky's disease virus in pigs (neurological and respiratory symptoms, abortion), equine herpesvirus type 1 (neurological and respiratory symptoms, abortion), and bovine herpesvirus type 1 (respiratory symptoms, abortion). Typical for alphaherpesviruses, and for herpesviruses in general, is their ability to persist in a non-replicative, latent state in their host during its entire lifetime. Specific stimuli can lead to reactivation of these viruses from their latent state, which can lead to renewed spread within and between hosts and recurrent symptoms. This recurrent replication and spread implies that herpesviruses have evolved techniques to delay and avoid recognition and elimination by the immune system, so-called immune evasion mechanisms. In the current manuscript, different alphaherpesvirus immune evasion mechanisms will be reviewed that have been discovered and elucidated at our research group based on pseudorabies virus and that interfere with the antiviral activity of virus-specific antibodies. Investigating immune evasion mechanisms leads to novel insights in the interactions between viruses, host cells, and the immunity, but can also lead to novel avenues in the design of strategies to interfere with viral infections.

In vitro culture of porcine respiratory nasal mucosa explants for studying the interaction of porcine viruses with the respiratory tract.

The mucosal surface of the respiratory tract is a common site of entry of many viruses. Molecular and cellular aspects of the interactions of respiratory viruses with the respiratory nasal mucosa are largely unknown. In order to be able to study those interactions in depth, an in vitro model was set up. This model consists of porcine respiratory nasal mucosa explants, cultured at an air-liquid interface. Light microscopy, scanning electron microscopy and transmission electron microscopy, combined with morphometric analysis and a fluorescent Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labelling (TUNEL) staining were used to evaluate the effects of in vitro culture on the integrity and viability of the explants. The explants were maintained in culture for up to 60 h post-sampling without significant morphometric (epithelial thickness, epithelial morphology, thickness of the lamina reticularis, continuity of the lamina densa, relative amounts of collagen and nuclei) changes and changes in viability. The potential to infect the explants was demonstrated for two porcine respiratory viruses of major importance: suid herpesvirus 1 and swine influenza virus H1N1. In conclusion, this in vitro model represents an ideal tool to study interactions between infectious agents and porcine respiratory nasal mucosa.

Receptor-determined susceptibility of preimplantation embryos to pseudorabies virus and porcine reproductive and respiratory syndrome virus.

In the present study, the in vitro interaction of embryos with pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) was investigated by viral antigen detection and by evaluating the expression of virus receptors, namely, poliovirus receptor-related 1 (PVRL1; formerly known as nectin 1) for PRV and sialoadhesin for PRRSV. Embryonic cells of zona pellucida intact embryos incubated with PRV remained negative for viral antigens. Also, no antigen-positive cells could be detected after PRV incubation of protease-treated embryos, since the protease disrupted the expression of PRVL1. However, starting from the five-cell-stage onwards, viral antigen-positive cells were detected after subzonal microinjection of PRV. At this stage, the first foci of PVRL1, also a known cell adhesion molecule, were expressed. At the expanded blastocyst stage, a lining pattern of PVRL1 in the apicolateral border of trophectoderm cells was present, whereas the expression in the inner cell mass was low. Furthermore, PVRL1-specific monoclonal antibody CK41 significantly blocked PRV infection of trophectoderm cells of hatched blastocysts, while the infection of the inner cell mass was only partly inhibited. Viral antigen-positive cells were never detected after PRRSV exposure of preimplantation embryos up to the hatched blastocyst stage. Also, expression of sialoadhesin in these embryonic stages was not detected. We conclude that the use of protease to investigate the virus embryo interaction can lead to misinterpretation of results. Results also show that blastomeres of five-cell embryos up to the hatched blastocysts can become infected with PRV, but there is no risk of a PRRSV infection.

A homologous in vitro model to study interactions between alphaherpesviruses and trigeminal ganglion neurons.

A key aspect in the life cycle of alphaherpesviruses is their neurotropic behaviour. Sensory neurons of the trigeminal ganglion (TG) are important target cells for many alphaherpesviruses (including herpes simplex virus 1, pseudorabies virus (PRV), bovine herpesvirus 1) and constitute major sites for latent infections. The aim of this study was to develop an in vitro model that simulates the in vivo infection pattern of TG neurons by alphaherpesviruses. To this end, we developed a homologous in vitro two-chamber model using PRV and porcine TG neurons. TG of 4- to 6-week-old piglets were dissociated and cultured in the inner chamber of the in vitro model, which is separated from the outer chamber by a medium- and virus-impermeable silicon barrier. Outgrowth of axons from neuronal cell bodies in the inner chamber through the silicon barrier into the outer chamber could be observed after 2-3 weeks of cultivation. Subsequent addition of PRV to the outer chamber resulted in exclusive infection of the TG neurons by transport of virus through the axons, subsequently giving rise to productively infected TG neurons that transmitted virus to contacting neurons and non-neuronal cells in the inner chamber. Thus, we established a homologous in vitro model that mimics the natural route of alphaherpesvirus infection of TG neurons that can be used to study interactions between these viruses and this pathogenetically very important cell type.

Aujeszky's disease vaccination and differentiation of vaccinated from infected pigs.

In this paper, the use of a glycoprotein E (gE)-deleted vaccine to eradicate Aujeszky's disease virus (ADV) or pseudorabies virus in swine is described. In regions where there is a dense population of swine, ADV is highly prevalent and intensive vaccination with such a marker vaccine has resulted, in several countries, in a decrease of the field virus prevalence to a sufficiently low level so that culling becomes economically feasible. The evolution in the reduction of circulating field virus can be followed at herd and/or population level by serological monitoring for antibodies against gE, using commercially available enzyme-linked immunosorbent assays. To be successful, other measures besides the vaccination in itself are important, such as the proper use and administration of the vaccine, repeated boosters, prevention of introduction of gE-positive animals and restriction in the movement of animals from gE-positive herds. When pockets of infection remain, intensifying the vaccination may be needed. Culling of remaining gE-positive animals, particularly breeders, is usually necessary. Some European Union (EU) member states have become ADV-free using this approach. Others are still working at it. The success is often dependent on whether or not a good discipline exists. The disease situation in the different EU member states is described. In ADV-free regions with dense swine populations at risk, it is advisable not to stop the vaccination too soon, particularly when the infection status in neighbouring countries or trade partners is not equally good.

Temporary disturbance of actin stress fibers in swine kidney cells during pseudorabies virus infection.

Rounding and loosening of cells is a consequence of infection with pseudorabies virus (PrV), both in vitro and in vivo. These changes in the normal structure of the cell may be the result of cytoskeletal changes. Immunofluorescence staining of actin filaments and microtubule bundles was performed to examine whether PrV induces a reorganization of these cytoskeletal components in infected swine kidney (SK) cells. Every 2h until 12h post-inoculation (p.i.), cells were washed in cytoskeleton stabilizing buffer (CSB), fixed with paraformaldehyde and washed again with CSB. Cells were permeabilized with a 1/1000 dilution of Triton X-100 and actin filaments were stained by incubating cells with phalloidin-Texas Red. Staining of microtubules was done by incubating the cells subsequently with mouse monoclonal anti-alpha-tubulin and goat anti-mouse IgG-FITC. During the course of infection, actin fibers of SK cells were rearranged in the following sequence: (1) disappearance of thick actin stress fibers between 4 and 6h p.i., (2) complete loss of stress fibers between 6 and 8h p.i., and (3) reappearance of thin stress fibers starting from 10h p.i. In contrast to herpes simplex virus 1 (HSV1) or equine herpesvirus 1 (EHV1), PrV infection did not induce changes in the cellular microtubule network. PrV infection induces a temporary disassembly of actin stress fibers.

Involvement of cellular cytoskeleton components in antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes.

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane-anchored viral glycoproteins and this may interfere with antibody-dependent cell lysis. We investigated the role of actin, microtubules, clathrin, and dynein, the major cellular components involved in physiological endocytosis during this virological internalization. Porcine monocytes were infected in vitro for 13 h and afterward treated with different concentrations of colchicine, cytochalasin D, latrunculin B, and amantadine-HCl, which inhibit polymerization of microtubules, actin/clathrin, actin, and clathrin, respectively. This resulted in a significant reduction of internalization compared to the nontreated control, indicating that these components are involved in the process. A double labeling was performed during the internalization process and a clear colocalization of actin, microtubules, clathrin, and dynein with the viral glycoproteins was observed at different stages during the internalization process. We conclude that these cellular components are used by PrV to generate the antibody-induced internalization of viral glycoproteins.

Immunological hiding of herpesvirus-infected cells.

Over the past years, numerous research groups have discovered various strategies that herpesviruses use to hide themselves from recognition by the immune system of their hosts. The current review gives a summary of this research, with emphasis on the mechanisms by which herpesvirus-infected cells escape from elimination by complement, phagocytes, cytotoxic T-lymphocytes and/or natural killer cells.

Role of anti-gB and -gD antibodies in antibody-induced endocytosis of viral and cellular cell surface glycoproteins expressed on pseudorabies virus-infected monocytes.

The addition of porcine pseudorabies virus (PrV)-specific polyclonal IgG antibodies to PrV-infected monocytes induces internalization of plasma membrane-anchored viral glycoproteins and major histocompatibility complex (MHC) class I. Using PrV deletion strains, it was shown that gB and gD are essential for the process to occur. The purpose of the current study was to evaluate whether antibodies directed against single viral glycoproteins are able to induce endocytosis. It was shown that monoclonal antibodies directed against viral glycoprotein gB and gD, but not against gC and gE, are able to induce internalization of their respective ligand. Adding a combination of monoclonal antibodies against gB and gD resulted in endocytosis levels, comparable to the endocytosis levels observed when adding porcine PrV-specific polyclonal antibodies. The addition of genistein and tyrphostin 25, two inhibitors of tyrosine kinase activity, abolished endocytosis induced by monoclonal anti-gB and -gD antibodies in a concentration-dependent manner. The addition of similar concentrations of tyrphostin 1, an inactive tyrphostin, had no effect on endocytosis. It was also shown that a mixture of polyclonal, but not monoclonal, antibodies against gB and gD is able to induce cointernalization of MHC class I. This indicates that MHC class I cointernalization results from a passive catching of the molecules rather than from a specific interaction of the MHC class I molecules with one or more viral glycoproteins. In conclusion, it can be stated that antibody-induced crosslinking of gB and gD induces the activation of a tyrosine phosphorylation-dependent signal transduction pathway, leading to their endocytosis. Cointernalization of other viral glycoproteins and MHC class I is most likely caused by a passive catching of these molecules in the gB and gD aggregates.

Antibody-induced endocytosis of viral glycoproteins and major histocompatibility complex class I on pseudorabies virus-infected monocytes.

Purified porcine monocytes, the natural carrier cells of pseudorabies virus (PrV) in the pig, were inoculated in vitro with PrV. At different time-points post-inoculation (p.i.) (from 7 to 17 h p.i.), the cells were washed and incubated with fluorescein isothiocyanate-labelled porcine PrV-specific polyclonal antibodies (IgG) at 37 degrees C. At all time-points tested p.i., 1 h of antibody incubation induced passive patching and subsequent internalization of the plasma membrane-anchored viral glycoproteins in approximately 65% of the infected monocytes. This endocytosis process is antibody-dependent, since biotinylated glycoproteins did not undergo spontaneous endocytosis. The process is fast and efficient, since only very low amounts of viral glycoproteins on the plasma membrane (7 h p.i.) and a minimal concentration of antibodies (0.04 mg IgG/ml) were needed to induce endocytosis. Experiments with PrV strains carrying deletions in the genes encoding the 11 different viral glycoproteins showed that viral glycoproteins gB and gD play a very important role in endocytosis (80% reduction with deletion mutants, P < 0.001), while the gE:gI Fc receptor complex, but not gE or gI alone, has a significant but lesser effect (45% reduction, P < 0.05). Double staining of viral glycoproteins and major histocompatibility complex class I (MHC I) showed a clear co-localization and co-endocytosis of MHC I with the viral glycoproteins, suggesting a possible role of the process in immune evasion of the virus.

Role of the cytoplasmic tail of gE in antibody-induced redistribution of viral glycoproteins expressed on pseudorabies-virus-infected cells.

Pseudorabies virus (PrV) glycoprotein gE is a nonessential glycoprotein involved in virulence and spread of the virus. It also has an important, yet unknown, function during antibody-induced capping of viral glycoproteins on the plasma membrane of PrV-infected swine kidney cells. In the present study, it was shown, by the use of a PrV strain expressing a truncated gE glycoprotein, that the cytoplasmic tail of gE is of significant importance for viral glycoprotein capping to occur. In addition, using PrV strains carrying point mutations in the cytoplasmic tail of gE, it was demonstrated that two tyrosine-based motifs are very important for correct functioning of gE during viral glycoprotein capping. Furthermore it was shown that genistein and tyrphostin, two tyrosine kinase activity inhibitors, inhibit viral glycoprotein capping in a concentration-dependent manner. In conclusion, it can be stated that efficient antibody-induced viral glycoprotein capping requires the presence of two YxxL sequences in the cytoplasmic tail of glycoprotein gE, as well as the activation of a tyrosine phosphorylation signal transduction pathway.