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 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.Many viruses use a single glycoprotein for both cell binding and membrane fusion. Herpesviruses are more complex. Three proteins-gB, gH, and gL-form a core fusion machinery conserved in the Alpha-, Beta-, and Gammaherpesvirinae subfamilies (21). Most herpesviruses also encode at least one additional receptor-binding protein that is more specific for a given virus subfamily. For example, herpes simplex virus first attaches to cells by gB or gC binding to the heparan sulfate moieties of the cell surface proteoglycans. gD must then bind for fusion to occur (47).Our understanding of gammaherpesvirus glycoprotein functions is more limited. This is due to the fact that the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) show limited lytic growth in vitro. Nevertheless, understanding how viral glycoproteins function is important for vaccination and for the development of neutralizing antibodies. While gB, gH, and gL are probably the key partners of the gammaherpesvirus membrane fusion machinery (22,39,41), the function of accessory entry proteins is less clear. EBV gp350/220 is a highly glycosylated membrane protein that adopts its 2 differently sized forms by alternative splicing (23). It is the most abundant protein in the virion envelope, binds to complement receptor 2 (CD21) on B cells (40,49), and is a target for antibodies that neutralize B cell infection (53). Soluble recombinant gp350 can also inhibit EBV infection of CD21-positive (CD21 ϩ

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.

cGammaherpesviruses 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.

BackgroundBovine 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.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.

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.

ObjectiveThe link between BRCA1 and homologous recombination deficiency (HRD) in cancer has gained importance with the emergence of new targeted cancer treatments, while the available data on the role of the gene in colorectal cancer (CRC) remain contradictory. The aim of this case series was to elucidate the role of known pathogenic BRCA1 variants in the development of early-onset CRC.DesignPatients were evaluated using targeted next generation sequencing, exome sequencing and chromosomal microarray analysis of the paired germline and tumor samples. These results were used to calculate the HRD score and the frequency of mutational signatures in the tumors.ResultsThree patients with metastatic CRC were heterozygous for a previously known BRCA1 nonsense variant. All tumors showed remarkably high HRD scores, and the HRD-related signature 3 had the second highest contribution to the somatic pattern of variant accumulation in the samples (23% in 1 and 2, and 13% in sample 3).ConclusionsA BRCA1 germline pathogenic variant can be involved in CRC development through HRD. Thus, BRCA1 testing should be considered in young patients with a personal history of microsatellite stable CRC as this could further allow a personalized treatment approach.

Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, during coevolution with their hosts, viruses have developed sophisticated mechanisms to hijack for their profit different pathways of glycan synthesis. Thus, the Bo17 gene of Bovine herpesvirus 4 (BoHV-4) encodes a homologue of the cellular core 2 protein ␤-1,6-N-acetylglucosaminyltransferase-mucin type (C2GnT-M), which is a key player for the synthesis of complex O-glycans. Surprisingly, we show in this study that, as opposed to what is observed for the cellular enzyme, two different mRNAs are encoded by the Bo17 gene of all available BoHV-4 strains. While the first one corresponds to the entire coding sequence of the Bo17 gene, the second results from the splicing of a 138-bp intron encoding critical residues of the enzyme. Antibodies generated against the Bo17 C terminus showed that the two forms of Bo17 are expressed in BoHV-4 infected cells, but enzymatic assays revealed that the spliced form is not active. In order to reveal the function of these two forms, we then generated recombinant strains expressing only the long or the short form of Bo17. Although we did not highlight replication differences between these strains, glycomic analyses and lectin neutralization assays confirmed that the splicing of the Bo17 gene gives the potential to BoHV-4 to fine-tune the global level of core 2 branching activity in the infected cell. Altogether, these results suggest the existence of new mechanisms to regulate the activity of glycosyltransferases from the Golgi apparatus. IMPORTANCEViruses are masters of adaptation that hijack cellular pathways to allow their growth. Glycans play a central role in many biological processes, and several studies have highlighted mechanisms by which viruses can affect glycosylation. Glycan synthesis is a nontemplate process regulated by the availability of key glycosyltransferases. Interestingly, bovine herpesvirus 4 encodes one such enzyme which is a key enzyme for the synthesis of complex O-glycans. In this study, we show that, in contrast to cellular homologues, this virus has evolved to alternatively express two proteins from this gene. While the first one is enzymatically active, the second results from the alternative splicing of the region encoding the catalytic site of the enzyme. We postulate that this regulatory mechanism could allow the virus to modulate the synthesis of some particular glycans for function at the location and/or the moment of infection.