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See detailProteomic characterization of murid herpesvirus 4 extracellular virions.
Vidick, Sarah ULg; Leroy, Baptiste; Gonon Rodrigues Palmeira, Leonor ULg et al

in PloS one (2013), 8(12), 83842

Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi's sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic ... [more ▼]

Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi's sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic diseases. As EBV and KSHV are host-range specific and replicate poorly in vitro, animal counterparts such as Murid herpesvirus-4 (MuHV-4) have been widely used as models. In this study, we used MuHV-4 in order to improve the knowledge about proteins that compose gammaherpesviruses virions. To this end, MuHV-4 extracellular virions were isolated and structural proteins were identified using liquid chromatography tandem mass spectrometry-based proteomic approaches. These analyses allowed the identification of 31 structural proteins encoded by the MuHV-4 genome which were classified as capsid (8), envelope (9), tegument (13) and unclassified (1) structural proteins. In addition, we estimated the relative abundance of the identified proteins in MuHV-4 virions by using exponentially modified protein abundance index analyses. In parallel, several host proteins were found in purified MuHV-4 virions including Annexin A2. Although Annexin A2 has previously been detected in different virions from various families, its role in the virion remains controversial. Interestingly, despite its relatively high abundance in virions, Annexin A2 was not essential for the growth of MuHV-4 in vitro. Altogether, these results extend previous work aimed at determining the composition of gammaherpesvirus virions and provide novel insights for understanding MuHV-4 biology. [less ▲]

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See detailAlternative splicing switches tropism of a gammaherpesvirus
Machiels, Bénédicte ULg; Gillet, Laurent ULg

Conference (2012, November)

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See detailBovine Herpesvirus Type 4 Glycoprotein L Is Nonessential for Infectivity but Triggers Virion Endocytosis during Entry
Lété, Céline ULg; Machiels, Bénédicte ULg; Stevenson, P. G. et al

in Journal of Virology (2012)

The core entry machinery of mammalian herpesviruses comprises glycoproteins B, H and L (gB, gH and gL). gH and gL form a heterodimer with a central role in viral membrane fusion. When archetypal alpha- or ... [more ▼]

The core entry machinery of mammalian herpesviruses comprises glycoproteins B, H and L (gB, gH and gL). gH and gL form a heterodimer with a central role in viral membrane fusion. When archetypal alpha- or beta-herpesviruses lack gL, gH misfolds and progeny virions are non-infectious. However, the gL of the rhadinovirus Murid herpesvirus 4 (MuHV-4) is non-essential for infection. In order to define more generally what role gL plays in rhadinovirus infections, we disrupted its coding sequence in Bovine herpesvirus-4 (BoHV-4). BoHV-4 lacking gL showed altered gH glycosylation and incorporated somewhat less gH into virions but remained infectious. However, gL- virions showed poor growth associated with an entry deficit. Moreover a major part of their entry defect appeared to reflect impaired endocytosis, which occurs upstream of membrane fusion itself. Thus, the rhadinovirus gL may be more important for driving virion endocytosis than for incorporating gH into virions, and is non-essential for membrane fusion. [less ▲]

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See detailProteomic characterization of bovine herpesvirus 4 extracellular virions.
Lété, Céline ULg; Palmeira, Leonor ULg; Leroy, Baptiste et al

in Journal of Virology (2012), 86(21), 11567-80

Gammaherpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful ... [more ▼]

Gammaherpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful infection. The different proteins that compose viral particles are therefore likely to have critical functions not only in viral structures and in entry into target cell but also in evasion of the host's antiviral response. In this study, we analyzed the protein composition of bovine herpesvirus 4 (BoHV-4), a close relative of the human Kaposi's sarcoma-associated herpesvirus. Using mass spectrometry-based approaches, we identified 37 viral proteins associated with extracellular virions, among which 24 were resistant to proteinase K treatment of intact virions. Analysis of proteins associated with purified capsid-tegument preparations allowed us to define protein localization. In parallel, in order to identify some previously undefined open reading frames, we mapped peptides detected in whole virion lysates onto the six frames of the BoHV-4 genome to generate a proteogenomic map of BoHV-4 virions. Furthermore, we detected important glycosylation of three envelope proteins: gB, gH, and gp180. Finally, we identified 38 host proteins associated with BoHV-4 virions; 15 of these proteins were resistant to proteinase K treatment of intact virions. Many of these have important functions in different cellular pathways involved in virus infection. This study extends our knowledge of gammaherpesvirus virions composition and provides new insights for understanding the life cycle of these viruses. [less ▲]

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See detailAntibody evasion by a gammaherpesvirus O-glycan shield.
Machiels, Bénédicte ULg; Gillet, Laurent ULg

Conference (2011, November)

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See detailAntibody evasion by a gammaherpesvirus o-glycan shield.
Machiels, Bénédicte ULg; Lété, Céline ULg; Guillaume, Antoine ULg et al

in PLoS Pathogens (2011), 7(11), 1002387

All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus gp350. These glycoproteins are often involved in cell binding, and some provide neutralization targets. However, the ... [more ▼]

All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus gp350. These glycoproteins are often involved in cell binding, and some provide neutralization targets. However, the capacity of gammaherpesviruses for long-term transmission from immune hosts implies that in vivo neutralization is incomplete. In this study, we used Bovine Herpesvirus 4 (BoHV-4) to determine how its gp350 homolog - gp180 - contributes to virus replication and neutralization. A lack of gp180 had no impact on the establishment and maintenance of BoHV-4 latency, but markedly sensitized virions to neutralization by immune sera. Antibody had greater access to gB, gH and gL on gp180-deficient virions, including neutralization epitopes. Gp180 appears to be highly O-glycosylated, and removing O-linked glycans from virions also sensitized them to neutralization. It therefore appeared that gp180 provides part of a glycan shield for otherwise vulnerable viral epitopes. Interestingly, this O-glycan shield could be exploited for neutralization by lectins and carbohydrate-specific antibody. The conservation of O-glycosylation sites in all gp350 homologs suggests that this is a general evasion mechanism that may also provide a therapeutic target. [less ▲]

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See detailSequencing of Bovine herpesvirus 4 V.test strain reveals important genome features
Palmeira, Leonor ULg; Machiels, Bénédicte ULg; Lété, Céline ULg et al

in Virology Journal (2011), 8(1), 406

Background Bovine herpesvirus 4 (BoHV-4) is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this ... [more ▼]

Background Bovine herpesvirus 4 (BoHV-4) is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this virus have been greatly facilitated by the cloning of the BoHV-4 V.test strain as a Bacterial Artificial Chromosome (BAC), the lack of a complete genome sequence for this strain limits its experimental use. Methods In this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach. Results The long unique coding region (LUR) consists of 108,241 bp encoding at least 79 open reading frames and is flanked by several polyrepetitive DNA units (prDNA). As previously suggested, we showed that the prDNA unit located at the left prDNA-LUR junction (prDNA-G) differs from the other prDNA units (prDNA-inner). Namely, the prDNA-G unit lacks the conserved pac-2 cleavage and packaging signal in its right terminal region. Based on the mechanisms of cleavage and packaging of herpesvirus genomes, this feature implies that only genomes bearing left and right end prDNA units are encapsulated into virions. Conclusions In this study, we have determined the complete genome sequence of the BAC-cloned BoHV-4 V.test strain and identified genome organization features that could be important in other herpesviruses. [less ▲]

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See detailBovine Herpesvirus 4 Bo10 gene encodes a nonessential viral envelope protein that regulates viral tropism through both positive and negative effects.
Machiels, Bénédicte ULg; Lété, Céline ULg; Defays, Katalin et al

in Journal of Virology (2011), 85(2), 1011-1024

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's Sarcoma associated herpesvirus (KSHV) K8.1. In this study, we characterized that of ... [more ▼]

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's Sarcoma associated herpesvirus (KSHV) K8.1. In this study, we characterized that of Bovine Herpesvirus-4 (BoHV-4), encoded by the Bo10 gene. We identified a 180 kDa gene product, gp180, which was incorporated into the virion envelope. A Bo10 deletion virus was viable, but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since the Bo10 mutant was both less infectious for GAG(+) cells than the wild-type and more infectious for GAG(-) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the Murid Herpesvirus 4 gp150, and also to the Epstein-Barr virus gp350 that promotes CD21(+) cell infection and inhibits CD21(-) cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor. [less ▲]

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See detailBovine herpesvirus 4 ORF73 is dispensable for viral growth in vitro but is essential for viral persistence in vivo.
Thirion, M.; Machiels, Bénédicte ULg; Farnir, Frédéric ULg et al

in Journal of General Virology (The) (2010), 91(10), 2574-84

ORF73 orthologues encoded by different rhadinoviruses have been studied extensively. These studies revealed that the ORF73 expression product (pORF73) is a multifunctional protein essential for latency ... [more ▼]

ORF73 orthologues encoded by different rhadinoviruses have been studied extensively. These studies revealed that the ORF73 expression product (pORF73) is a multifunctional protein essential for latency that enables episome tethering to mitotic chromosomes and modulates cellular pathways implicated in growth and survival of latently infected cells. Comparison of pORF73 orthologues encoded by rhadinoviruses reveals important variations in amino acid sequence length and composition. Bovine herpesvirus 4 (BoHV-4) encodes by far the shortest ORF73 orthologue with a size equivalent to only 22% of the largest orthologues. The present study focused on determining if BoHV 4 ORF73 is a bona fide gene and investigating whether it is essential for latency as established for larger ORF73 orthologues. Our results demonstrate that BoHV-4 ORF73 is transcribed as immediate-early bicistronic mRNA together with ORF71. Using a BoHV-4 bacterial artificial chromosome clone, we produced a strain deleted for ORF73 and a revertant strain. Deletion of BoHV-4 ORF73 did not affect the capacity of the virus to replicate in vitro, but it prevented latent infection in vivo using a rabbit model. Interestingly, the strain deleted for ORF73 induced an anti-BoHV-4 humoral immune response comparable to that elicited by wild-type and revertant recombinants. Together, these results demonstrate that despite its relatively small size, BoHV-4 ORF73 is a functional homologue of larger rhadinovirus ORF73 orthologues, and highlight the potential of ORF73 deletion for the development of BoHV-4 as a vector in vaccinology. [less ▲]

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See detailNatural antibody--complement dependent neutralization of bovine herpesvirus 4 by human serum
Machiels, Bénédicte ULg; Gillet, Laurent ULg; Brito, Sieberth Do Nascimento et al

in Microbes & Infection (2007), 9(14-15), 1530-1537

In contrast to most gammaherpesviruses, Bovine herpesvirus 4 (BoHV-4) has a broad range of host species both in vitro and in vivo. Several in vitro studies demonstrated that some human cell lines are ... [more ▼]

In contrast to most gammaherpesviruses, Bovine herpesvirus 4 (BoHV-4) has a broad range of host species both in vitro and in vivo. Several in vitro studies demonstrated that some human cell lines are sensitive or even permissive to BoHV-4. These observations led to the hypothesis that cross-species transmission of BoHV-4 could lead to human infections. In the present study, we investigate the sensitivity of BoHV-4 to neutralization by naïve human sera in order to determine if humans exhibit innate anti-viral activities against this virus. Our results demonstrate that human sera from naïve individuals, in contrast to the sera of naïve subjects from various animal species, neutralize BoHV-4 efficiently. A series of complementary experiments were performed to unravel the mechanism(s) of this neutralization. The data obtained in this study demonstrates that human serum neutralizes BoHV-4 in a complement dependent manner activated by natural antibodies raised against the Galalpha1-3Galbeta1-4GlcNAc-R epitope expressed by bovine cells [less ▲]

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