Bénédicte Machiels

Antibody evasion by a gammaherpesvirus o-glycan shield.

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.

The bovine herpesvirus 4 Bo10 gene encodes a nonessential viral envelope protein that regulates viral tropism through both positive and negative effects.

ABSTRACT All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposis sarcoma-associated herpesvirus (KSHV) K8.1. In this study, we characterized the positional homologous glycoprotein of bovine herpesvirus 4 (BoHV-4), encoded by the Bo10 gene. We identified a 180-kDa gene product, gp180, that 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 compared to the wild-type virus, the Bo10 mutant virus was both less infectious for GAG-positive (GAG+) cells and more infectious for GAG-negative (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 that of 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.

Bovine Herpesvirus Type 4 Glycoprotein L Is Nonessential for Infectivity but Triggers Virion Endocytosis during Entry

ABSTRACT The core entry machinery of mammalian herpesviruses comprises glycoprotein B (gB), gH, and gL. gH and gL form a heterodimer with a central role in viral membrane fusion. When archetypal alpha- or betaherpesviruses lack gL, gH misfolds and progeny virions are noninfectious. However, the gL of the rhadinovirus murid herpesvirus 4 (MuHV-4) is nonessential 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 it is nonessential for membrane fusion.

Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions

ABSTRACT 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 hosts antiviral response. In this study, we analyzed the protein composition of bovine herpesvirus 4 (BoHV-4), a close relative of the human Kaposis 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.

Proteomic characterization of murid herpesvirus 4 extracellular virions.

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.

A gammaherpesvirus provides protection against allergic asthma by inducing the replacement of resident alveolar macrophages with regulatory monocytes

The hygiene hypothesis postulates that the recent increase in allergic diseases such as asthma and hay fever observed in Western countries is linked to reduced exposure to childhood infections. Here we investigated how infection with a gammaherpesvirus affected the subsequent development of allergic asthma. We found that murid herpesvirus 4 (MuHV-4) inhibited the development of house dust mite (HDM)-induced experimental asthma by modulating lung innate immune cells. Specifically, infection with MuHV-4 caused the replacement of resident alveolar macrophages (AMs) by monocytes with regulatory functions. Monocyte-derived AMs blocked the ability of dendritic cells to trigger a HDM-specific response by the TH2 subset of helper T cells. Our results indicate that replacement of embryonic AMs by regulatory monocytes is a major mechanism underlying the long-term training of lung immunity after infection.

Sequencing of bovine herpesvirus 4 v.test strain reveals important genome features

BackgroundBovine herpesvirus 4 (BoHV-4) is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposis 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.MethodsIn this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach.ResultsThe 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.ConclusionsIn 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.

A Gammaherpesvirus Uses Alternative Splicing to Regulate Its Tropism and Its Sensitivity to Neutralization.

Human gammaherpesviruses are associated with the development of lymphomas and epithelial malignancies. The heterogeneity of these tumors reflects the ability of these viruses to route infection to different cell types at various stages of their lifecycle. While the Epstein Barr virus uses gp42 – human leukocyte antigen class II interaction as a switch of cell tropism, the molecular mechanism that orientates tropism of rhadinoviruses is still poorly defined. Here, we used bovine herpesvirus 4 (BoHV-4) to further elucidate how rhadinoviruses regulate their infectivity. In the absence of any gp42 homolog, BoHV-4 exploits the alternative splicing of its Bo10 gene to produce distinct viral populations that behave differently based on the originating cell. While epithelial cells produce virions with high levels of the accessory envelope protein gp180, encoded by a Bo10 spliced product, myeloid cells express reduced levels of gp180. As a consequence, virions grown in epithelial cells are hardly infectious for CD14+ circulating cells, but are relatively resistant to antibody neutralization due to the shielding property of gp180 for vulnerable entry epitopes. In contrast, myeloid virions readily infect CD14+ circulating cells but are easily neutralized. This molecular switch could therefore allow BoHV-4 to promote either, on the one hand, its dissemination into the organism, or, on the other hand, its transmission between hosts.

Bovine herpesvirus 4 ORF73 is dispensable for virus growth in vitro, but is essential for virus persistence in vivo.

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 that of the largest orthologues. The present study focused on determining whether 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 polycistronic 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 the 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.

Multivalent binding of herpesvirus to living cells is tightly regulated during infection

We probe herpesvirus binding to living cells using atomic force microscopy. Viral infection, initiated by the landing of a virion on a cellular surface, is largely defined by the preliminary interactions established between viral particles and their receptors at the cell surface. While multiple parallel interactions would allow strong virus attachment, a low number of bonds could be preferred to allow lateral diffusion toward specific receptors and to promote efficient release of progeny virions from the cell surface. However, so far, the molecular mechanisms underlying the regulation of the multivalency in virus attachment to receptors are poorly understood. We introduce a new method to force-probe multivalent attachment directly on living cells, and we show, for the first time, direct evidence of a new mechanism by which a herpesvirus surface glycoprotein acts as a key negative regulator in the first step of herpesvirus binding. Using atomic force microscopy, we probe at the single-virion level the number and the strength of the bonds established with heparan sulfate both on model surfaces and on living cells. Our biophysical results, correlated with other techniques, show that the major envelope glycoprotein functions as a regulator of binding valency during both attachment and release steps, determining the binding, diffusion, and release potential of virions at the cellular surface.