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See detailVaricella-zoster virus induces the formation of dynamic nuclear capsid aggregates.
Lebrun, Marielle ULg; Thelen, Nicolas ULg; Thiry, Marc ULg et al

in Virology (2014), 454-455

The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains ... [more ▼]

The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. [less ▲]

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See detailHigh levels of p105 (NFKB1) and p100 (NFKB2) proteins in HPV16-transformed keratinocytes: role of E6 and E7 oncoproteins.
Havard, Laurence; Rahmouni, Souad ULg; Boniver, Jacques ULg et al

in Virology (2005), 331(2), 357-66

We have previously shown that functional components of the NF-kappaB signaling pathway are up-regulated and sequestered in the cytoplasm of human papillomavirus 16 (HPV16)-transformed cell lines leading ... [more ▼]

We have previously shown that functional components of the NF-kappaB signaling pathway are up-regulated and sequestered in the cytoplasm of human papillomavirus 16 (HPV16)-transformed cell lines leading to a reduced activity of NF-kappaB. In this study, we examined the expression of the NF-kappaB precursors p100 and p105 in keratinocytes transformed or not by HPV16. Western immunoblotting experiments demonstrated high levels of p100 and p105 proteins not only in HPV16+ cervical carcinoma-derived keratinocytes but also in keratinocytes stably transfected by HPV16 E6 or E7 oncogenes. Moreover, p100 and p105 proteins were predominantly cytoplasmic and nuclear in keratinocytes expressing E7 and E6, respectively. A predominantly cytoplasmic localization of E7 protein was also detected in all keratinocytes expressing E7. Our results suggest that HPV16 E6 and E7 proteins modulate the expression and the subcellular localization of p100 and p105 NF-kappaB precursors. [less ▲]

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See detailVaricella-Zoster virus proteins encoded by open reading frames 14 and 67 are both dispensable for the establishment of latency in a rat model.
Grinfeld, Esther; Sadzot-Delvaux, Catherine ULg; Kennedy, Peter G E

in Virology (2004), 323(1), 85-90

A rat model of Varicella-Zoster virus (VZV) provides a system in which to investigate the molecular determinants of viral latency in dorsal root ganglia (DRG). In this study, we determined whether the VZV ... [more ▼]

A rat model of Varicella-Zoster virus (VZV) provides a system in which to investigate the molecular determinants of viral latency in dorsal root ganglia (DRG). In this study, we determined whether the VZV glycoproteins gC and gI, corresponding to VZV open reading frames (ORFs) 14 and 67, respectively, were required for the establishment of latency in this model. A VZV gI deletion mutant (DeltagI) derived from a recombinant Oka (rOka) cosmid and a gC null mutant obtained from a clinical isolate were inoculated into the footpads of 6-week-old rats, and the presence of viral DNA and eight different VZV RNA transcripts corresponding to the three classes of genes was investigated by in situ RT-PCR amplification and in situ hybridization (ISH) in the DRG at 1 week, 1 month, and 18-24 months after infection. VZV DNA and restricted RNA expression was established with both deletion mutants as well as the parental rOka virus. Both VZV DNA and RNA were detected in neurons and non-neuronal cells. The pattern of viral RNA expression detected with both gC and gI mutants was restricted with transcripts for VZV genes 62 and 63 most frequently expressed 18-24 months after infection. Transcripts for VZV genes 18, 28, and 29 were also detected at these time points but at a slightly lower frequency. Transcripts for the late gene 40 were never detected. We conclude that VZV ORFs 14 and 67 are dispensable for the establishment of a latent infection in this model. [less ▲]

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See detailThe structures of bovine herpesvirus 1 virion and concatemeric DNA: implications for cleavage and packaging of herpesvirus genomes
Schynts, F.; McVoy, M. A.; Meurens, F. et al

in Virology (2003), 314(1), 326-335

Herpesvirus genomes are often characterized by the presence of direct and inverted repeats that delineate their grouping into six structural classes. Class D genomes consist of a long (L) segment and a ... [more ▼]

Herpesvirus genomes are often characterized by the presence of direct and inverted repeats that delineate their grouping into six structural classes. Class D genomes consist of a long (L) segment and a short (S) segment. The latter is flanked by large inverted repeats. DNA replication produces concatemers of head-to-tail linked genomes that are cleaved into unit genomes during the process of packaging DNA into capsids. Packaged class D genomes are an equimolar mixture of two isomers in which S is in either of two orientations, presumably a consequence of homologous recombination between the inverted repeats. The L segment remains predominantly fixed in a prototype (P) orientation; however, low levels of genomes having inverted L (I-L) segments have been reported for some class D herpesviruses. Inefficient formation of class D I-L genomes has been attributed to infrequent L segment inversion, but recent detection of frequent inverted L segments in equine herpesvirus 1 concatemers [Virology 229 (1997) 415-420] suggests that the defect may be at the level of cleavage and packaging rather than inversion. In this study, the structures of virion and concatemeric DNA of another class D herpesvirus, bovine herpesvirus 1, were determined. Virion DNA contained low levels of I-L genomes, whereas concatemeric DNA contained significant amounts of L segments in both P and I-L orientations. However, concatemeric termini exhibited a preponderance of L termini derived from P isomers which was comparable to the preponderance of P genomes found in virion DNA. Thus, the defect in formation of I-L genomes appears to lie at the level of concatemer cleavage. These results have important implications for the mechanisms by which herpesvirus DNA cleavage and packaging occur. (C) 2003 Elsevier Inc. All rights reserved. [less ▲]

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See detailLack of trans-activation function for Maedi Visna virus and Caprine arthritis encephalitis virus Tat proteins
Villet, S.; Faure, C.; Bouzar, Amel ULg et al

in Virology (2003)

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See detailDifferential production of cytokines and activation of NF-kappa B in HPV-transformed keratinocytes
Havard, L.; Delvenne, Philippe ULg; Frare, P. et al

in Virology (2002), 298(2), 271-285

We have proposed that chronic infection of keratinocytes by HPV modifies the expression of potentially important cytokines by interfering with the NF-kappaB signal pathway We evaluated the constitutive ... [more ▼]

We have proposed that chronic infection of keratinocytes by HPV modifies the expression of potentially important cytokines by interfering with the NF-kappaB signal pathway We evaluated the constitutive and IL-1beta-induced expression of GM-CSF and TNF-alpha and the expression/activity of NF-kappaB in HPV+ and HPV- cell lines. Despite the enhanced expression of the functional components of the NF-kappaB signaling pathway in HPV+ cell lines by a mechanism implicating the HPV oncoprotein E6, the constitutive activity of NF-kappaB and the expression of GM-CSF/TNF-alpha were significantly reduced relative to the HPV- cell line and normal keratinocytes. In contrast, we observed a superactivation of NF-kappaB activity after IL-1beta stimulation, a strong and transient induction of GM-CSF/TNF-alpha mRNA, but undetectable levels of secreted proteins in HPV+ cell lines. Our data demonstrate that E6 modulates the NF-kappaB signaling pathway and suggest that other HPV proteins also interfere with GM-CSF/TNF-alpha expression by transcriptional and/or posttranscriptional mechanisms. (C) 2002 Elsevier Science (USA). [less ▲]

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See detailVaricella-Zoster virus gene expression in latently infected rat dorsal root ganglia.
Kennedy, P. G.; Grinfeld, E.; Bontems, Sébastien ULg et al

in Virology (2001), 289(2), 218-23

Latent infection of human ganglia with Varicella-Zoster virus (VZV) is characterized by a highly restricted pattern of viral gene expression. To enhance understanding of this process we used in situ ... [more ▼]

Latent infection of human ganglia with Varicella-Zoster virus (VZV) is characterized by a highly restricted pattern of viral gene expression. To enhance understanding of this process we used in situ hybridization (ISH) in a rat model of VZV latency to examine expression of RNA corresponding to eight different VZV genes in rat dorsal root ganglia (DRG) at various times after footpad inoculation with wild-type VZV. PCR in situ amplification was also used to determine the cell specificity of latent VZV DNA. It was found that the pattern of viral gene expression at 1 week after infection was different from that observed at the later times of 1 and 18 months after infection. Whereas multiple genes were expressed at 1 week after infection, gene expression was restricted at the later time points when latency had been established. At the later time points after infection the RNA transcripts expressed most frequently were those for VZV genes 21, 62, and 63. Gene 63 was expressed more than any other gene studied. While VZV DNA was detected almost exclusively in 5-10% of neurons, VZV RNA was detected in both neurons and nonneuronal cells at an approximate ratio of 3:1. A newly described monoclonal antibody to VZV gene 63-encoded protein was used to detect this protein in neuronal nuclei and cytoplasm in almost half of the DRG studied. These results demonstrate that (1) this rat model of latency has close similarities in terms of viral gene expression to human VZV latency which makes it a useful tool for studying this process and its experimental modulation and (2) expression of VZV gene 63 appears to be the single most consistent feature of VZV latency. [less ▲]

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See detailChannel catfish virus gene 50 encodes a secreted, mucin-like glycoprotein
Vanderheijden, Nathalie; Hanson, Larry A.; Thiry, Etienne ULg et al

in Virology (1999), 257(1), 220-7

Cells infected with the wild-type (WT) strain of channel catfish virus (CCV) secreted a glycoprotein with an apparent molecular mass (MM) superior to 200 kDa into the culture medium. This protein ... [more ▼]

Cells infected with the wild-type (WT) strain of channel catfish virus (CCV) secreted a glycoprotein with an apparent molecular mass (MM) superior to 200 kDa into the culture medium. This protein, designated gp250, was the sole viral glycoprotein detected in the culture medium after [3H]mannose labeling of the infected cells. When cells were infected with the attenuated V60 strain, a glycoprotein of 135 kDa (designated gp135) was detected instead of gp250. Because WT gene 50 is predicted to encode a secreted, mucin-type glycoprotein, we expressed this gene transiently and detected a glycoprotein of the same apparent MM as gp250 in the culture medium of transfected catfish cells. The increased mobility in SDS-PAGE of the secreted V60 glycoprotein correlated with the presence of a major deletion in V60 gene 50. Therefore, we concluded that gp250 in the WT and gp135 in the V60 strains are both likely encoded by gene 50. An important shift in the relative mobility of gp250 in SDS-PAGE was observed after tunicamycin treatment of infected cells labeled with [3H]glucosamine, confirming the presence of N-linked sugars on gp250. We observed variations in the size of PCR products derived from gene 50 amplification in three different field isolates. Such genetic variations are a characteristic feature of mucin genes and are linked to crossing-over events between internal repeated sequences, such as those present in gene 50. [less ▲]

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See detailBovine herpesvirus 1-induced apoptotic cell death: role of glycoprotein D
Hanon, Emmanuel; Keil, Günther; Van Drunen Littel-Van Den Hurk, Sylvia et al

in Virology (1999), 257

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in peripheral blood mononuclear cells and in bovine B lymphoma (BL-3) cells. Attachment but not penetration of BHV-1 is necessary to induce ... [more ▼]

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in peripheral blood mononuclear cells and in bovine B lymphoma (BL-3) cells. Attachment but not penetration of BHV-1 is necessary to induce apoptosis in target cells, suggesting that one or more BHV-1 envelope glycoproteins could be involved in the activation of the apoptotic process. In the present study, we demonstrate that, although BHV-1 virions devoid of glycoprotein D (BHV-1 gD-/-) still bind to BL-3 cells, they are no longer able to induce apoptosis. In contrast, virions that contain glycoprotein D (gD) in the viral envelope but do not genetically encode gD (BHV-1 gD-/+) induce a level of apoptosis comparable to that produced by wild-type (wt) BHV-1. In addition, monoclonal antibodies directed against gD, but not against gB or gC, strongly reduced the high levels of apoptosis induced by BHV-1. These observations demonstrate that the induction of apoptosis is directly due to BHV-1 viral particles harboring gD in the viral envelope. Interestingly, binding of affinity-purified gD to BL-3 cells did not induce apoptosis but inhibited the ability of wt BHV-1 to induce apoptosis. Altogether, these results provide evidence for the direct or indirect involvement of gD in the mechanism by which BHV-1 induces apoptosis. [less ▲]

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See detailBovine Herpesvirus 1-Induced Apoptosis Occurs At The G0/G1 Phase Of The Cell Cycle
Hanon, Emilien ULg; Hoornaert, S.; Dequiedt, Franck ULg et al

in Virology (1997), 232(2), 351-358

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 ... [more ▼]

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. [less ▲]

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See detailThe attenuated V60 strain of channel catfish virus possesses a deletion in ORF50 coding for a potentially secreted glycoprotein
Vanderheijden, NATHALIE; Alard, PHILIPPE; Lecomte, CORINE et al

in Virology (1996), 218(2), 422-6

A wild-type strain of channel catfish virus was compared at the genomic level with the attenuated strain V60. In addition to several minor differences, restriction mapping revealed one major deletion ... [more ▼]

A wild-type strain of channel catfish virus was compared at the genomic level with the attenuated strain V60. In addition to several minor differences, restriction mapping revealed one major deletion (approximately 1200 bp) in ORF50 of the V60 strain. Cloning and sequencing of part of this ORF confirmed the presence of a 1164-bp deletion. It should result in a protein of 282 amino acids instead of 670. The predicted truncated protein lacks most of a threonine-rich, highly repetitive region in its central part. Since the protein encoded by ORF50 possesses a hydrophobic N-terminal leader sequence and no membrane anchor sequence, we suggest that it could be a secreted glycoprotein. This protein might be N-glycosylated (35 potential sites) and, given the repetitive arrangement of its residues (mainly threonines), also heavily O-glycosylated like the mucin-type glycoproteins. The deletion observed in ORF50 of the V60 strain implies the loss of 24 potential N-glycosylation sites and should considerably reduce the extent of O-glycosylation. [less ▲]

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See detailThe Replication in Vitro of the Gammaherpesvirus Bovine Herpesvirus 4 Is Restricted by Its DNA Synthesis Dependence on the S Phase of the Cell Cycle
Vanderplasschen, Alain ULg; Goltz, M.; Lyaku, J. et al

in Virology (1995), 213(2), 328-40

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 ... [more ▼]

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. [less ▲]

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See detailSynthesis and Processing of Bovine Herpesvirus-1 Glycoprotein H
Baranowski, Eric; Dubuisson, Jean; van Drunen Little-van den Hurk, Sylvia et al

in Virology (1995), 206(1), 651-4

The translation product of the bovine herpesvirus-1 (BHV-1) gH gene was identified and characterized. Synthetic peptides were used to generate specific antisera and a glycoprotein of 108K was precipitated ... [more ▼]

The translation product of the bovine herpesvirus-1 (BHV-1) gH gene was identified and characterized. Synthetic peptides were used to generate specific antisera and a glycoprotein of 108K was precipitated by one of the antisera. Cross-immunoprecipitations with monoclonal antibodies to BHV-1 glycoprotein gp108 and the anti-gH peptide antiserum demonstrated that gp108 is the translation product of the gH open reading frame. Glycoprotein gH synthesis and intracellular processing was analyzed in infected Madin-Darby bovine kidney cells using anti-gp 108 monoclonal antibodies. Glycoprotein gH is expressed as a beta-gamma protein and could be detected by radioimmunoprecipitation as early as 2 hr postinfection. Cotranslational N-glycosylation of gH is essential for the recognition by monoclonal antibodies, suggesting that N-linked glycans are involved in protein folding or that they are targets for most of monoclonal antibodies used in this study. [less ▲]

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See detailLack of LTR and ENV genetic variation during bovine leukemia virus-induced leukemogenesis.
Willems, Luc ULg; Kerkhofs, P.; Burny, A. et al

in Virology (1995), 206(1),

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See detailAttachment of the Gammaherpesvirus Bovine Herpesvirus 4 Is Mediated by the Interaction of Gp8 Glycoprotein with Heparinlike Moieties on the Cell Surface
Vanderplasschen, Alain ULg; Bublot, M.; Dubuisson, J. et al

in Virology (1993), 196(1), 232-40

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 ... [more ▼]

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. [less ▲]

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