The human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated Herpesvirus (KSHV), are involved in several cancers. These viruses evolved to coexist with the host specific
antibodies since they are able to induce reactivation and reexcretion in an immune carrier. Understanding precisely how they enter the target cells is crucial to improve their control and to design
original vaccinal strategies. However, entry of gammaherpesviruses is a complex process that requires different cellular receptors and different viral glycoproteins. Among them, the EBV glycoprotein,
gp350, binds to CD21 on B cells and is a target for antibodies that neutralize B cell infection. Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that shares many common properties with EBV and KSHV.
Thus, the Bo10 gene potentially encodes the EBV gp350 homologous protein. In order to further explore the relationship between gp350 or its homologs, host cells and antibodies, this work focused on the
function of the BoHV-4 Bo10 gene in the biology of viral infection. The results obtained are presented in four studies.
1. Bovine Herpesvirus-4 Bo10 gene encodes a non-essential viral envelope protein that regulates viral tropism through both positive and negative effects. Machiels B., Lete C., de Fays K., Mast J., Dewals B., Stevenson P.G., Vanderplasschen A. and Gillet L. Journal of Virology, (2011), 85 (2), 1011-1024.
This study demonstrated that the Bo10 gene encodes a 180 kDa product (gp180), which is incorporated in the virion envelope. This protein is not essential for viral replication but is involved in
viral entry. Indeed, Bo10 deleted virus showed a growth deficit associated with a reduced binding to cellular glycosaminoglycans (GAGs). On the opposite, Bo10 deletion improves infection of GAG negative
cells. These results suggest that gp180 regulates BoHV-4 tropism both positively and negatively depending upon the presence or absence of its receptor. This mechanism could be shared by the other
gammaherpesviruses. We hypothesized that interaction with heparan sulfates of the cell surface displaces gp180 allowing the exposition of viral entry epitopes. A possible rationale for this arrangement
would be for gp180 and its homologs to protect an otherwise vulnerable virion protein from antibody neutralization. This hypothesis has been investigated in a second study.
2. Antibody evasion by a gammaherpesvirus O-glycan shield. Machiels B., Lété C., Guillaume A., Mast J., Stevenson P.G., Vanderplasschen A. and Gillet L. Submitted for publication.
This study showed that a lack of gp180 has no impact on the establishment and maintenance of BoHV-4 latency, but markedly sensitizes virions to neutralization by immune sera. The results demonstrated
that gp180 hides some different epitopes on gB, gH and gL, including epitopes targeted by neutralizing antibodies. It appeared that gp180 provides part of a glycan shield for otherwise vulnerable viral
epitopes and protects them from antibody neutralization. While these proteins are diverse in sequence, the conservation of O-glycosylation sites in all gp350 homologs suggests that this is a general
evasion mechanism in the gammaherpesvirinae subfamily.
3. « Natural antibody-complement dependent neutralization of bovine herpesvirus 4 by human serum ». Machiels B., Gillet L., Do Nascimento Brito S., Drion P., Delforge C., Nizet Y., Gianello P., Bona C., Costes B., Markine-Goriaynoff N. and Vanderplasschen A. Microbes and Infection, (2007), 9, 1530-1537.
The importance of human exposure to BoHV-4 and the lack of prophylactic scheme against this virus make human contamination possible. However, to date there is no reported case of human infection
by BoHV-4. This observation raises the question of a natural human resistance against this virus. The results of this study highlighted an efficient neutralization of BoHV-4 by human serum, in contrast
to sera of various animal species. The mechanism of this neutralization depends of the complement through activation of the classical pathway by natural antibodies raised against the cellular gal epitope.
A supplemental experiment revealed that gp180 represents the main backbone for gal epitope exposition. The results of this study allowed us, on one hand, to objectivize the risk of BoHV-4 transmission to
humans. On the other hand, this study illustrated a situation where glycosylation of viral proteins did not give a selective advantage to the virus but on the opposite, allowed neutralization. Therefore,
glycans that represent a crucial stategy for evasion of antibody response by gammaherpesviruses, could also become the target of original vaccine approaches.
4. Alternative splicing switches tropism of a gammaherpesvirus. Machiels B., Stevenson P.G., Vanderplasschen A. and Gillet L. In preparation.
Herpesviruses have complex lifecycles that involve infection of various cell types. Interestingly, some of them are able to route infection by the use of different cellular receptors and different
viral glycoprotein complexes. In this study, we showed that BoHV-4 could use alternative splicing of the Bo10 gene to orientate tropism of progeny virions. Indeed, the spliced transcript of Bo10 produces
gp180 while the absence of splicing leads to the reading of a stop codon in the intron. The traduction of this unspliced transcript could generate a truncated protein without transmembrane anchor.
Although only gp180 is involved in attachment to cellular GAGs, the function of this truncated protein is not still defined. As a similar gene organization is conserved in the KSHV K8.1 homologous gene,
we hypothesized that a similar mechanism could be conserved in different rhadinoviruses.
In conclusion, the study of BoHV-4 Bo10 gene products highlighted the complexity of gammaherpesviruses entry mechanism. This elaborate process could be required to protect vulnerable epitopes from
antibody neutralization. This work also demonstrated the importance of O-glycosylation in this mechanism and suggested that this immune evasion strategy is widespread within the gammaherpesvirinae
subfamily. The results of this thesis give the first basis for fundamental and applied researches.