During the last years, much progress has been made in determining the roles of different entry pathways of herpesviruses. It has been reported that HSV-1, the prototypical alphaherpesvirus, enters certain cells through fusion with the plasma membrane and others via endocytosis. Moreover, it was shown that the cell type and the respective receptors are the main determinants of whether the fusion process occurs at the plasma membrane or through an endocytic route of entry (13
). Recently, it was shown that EHV-1 can enter cells by these two different pathways as well (11
In the present study, we could demonstrate that αV integrins are important during the early steps of EHV-1 entry via endocytosis and, more specifically, through the interaction of the virus with an Arg-Gly-Asp (RGD) recognition site. The RGD motif is the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins, such as αVβ3, αVβ5, and α3β1 (2
). Such RGD motifs are also essential for integrin receptor binding of many enveloped and nonenveloped viruses, such as foot-and-mouth disease virus and coxsackievirus (5
). Like the case for other herpesviruses, human herpesvirus 8 has been shown to enter certain cell types via endocytosis upon binding to integrin α3β1, also mediated by an RGD sequence present in gB (3
). In the case of HSV-1, viral interaction with integrins was evaluated by employing peptides containing known integrin recognition sites and blocking antibodies. No effect on HSV-1-induced plaque formation in epithelial Hep-2 cells could be observed by using an RGD peptide or MAbs to the human β1 or β4 integrin subunit (19
). This is in contrast to our results where the RGD peptide and the function-blocking αVβ3 antibody had a significant effect on productive EHV-1 infection in CHO-K1 cells.
In subsequent experiments, we could demonstrate by mutational analysis that an RSD motif present in EHV-1 gD is involved in the early steps of EHV-1 uptake via endocytosis. Previously, mutational analyses of integrin binding motifs in viral glycoproteins were also done with HSV-1. In one study, a recombinant soluble form of HSV-1 gH was created and showed binding to Vero cells and CHO-K1 cells expressing human αVβ3 (33
). Binding was abolished upon mutation of an RGD motif in the glycoprotein, indicating that HSV-1 can bind to integrins via an RGD motif present in gH. In another study, however, a recombinant HSV-1 with a point mutation targeting the exact same motif showed no growth deficits in vitro and was still able to enter cells at rates equivalent to those of wild-type virus (12
), which raises questions about the functional significance of the gH RGD-mediated interaction with αVβ3 integrins. Based on the data obtained in this study, the RSD motif in EHV-1 gD does appear to be functionally important, as infection with the L11gD152N
virus was clearly less efficient than that with wild-type EHV-1. Recently, HSV-1 gD was shown to be important for entry into C10 melanoma cells via low-pH-independent endocytic entry, and the presence of gD was absolutely necessary for the ability of HSV-1 to successfully infect CHO-K1 cells by endocytosis (26
). The precise motif(s) in HSV-1 gD responsible for the virus-receptor interaction during endocytosis, however, has not been identified to date.
In the last part of this study, we extended our observations about the mechanisms by which EHV-1 enters permissive cells beyond hamster CHO-K1 cells to cell types that are more relevant to EHV-1 replication in vivo. We were able to show that EHV-1 entry in equine EC does not occur primarily via endocytosis but via fusion at the plasma membrane (Fig. ). Interestingly, when we used very low MOIs to infect EC, a significant difference in the percentage of infected cells became apparent (data not shown), indicating that endocytic entry of EHV-1 in this cell type might occur, albeit at a very low efficiency. The presence of enveloped EHV-1 particles within uncoated vesicles has been reported before for equine brain microvascular EC, suggesting the utilization of an endocytic entry pathway for equine cells of endothelial origin (16
). Similar experiments with equine monocytes and lymphocytes showed that in contrast to the case with EC, endocytosis appears to be a biologically relevant and important pathway for EHV-1 entry and infection in these cells (Fig. ). Very similar to our observations with EHV-1, it has been reported that Epstein-Barr virus infects epithelial cells via fusion at the plasma membrane but requires pH-independent endocytosis for entry into B cells (17
). In the case of HSV-1, viral entry was also analyzed in two human cell types that seem relevant for pathogenesis, and the virus was shown to enter human keratinocytes by a low-pH endocytic pathway, whereas human neurons were infected by an entry pathway that was pH independent (29
). The differential requirements for lymphotropic herpesviruses such as Epstein-Barr virus, human herpesvirus 8, and EHV-1 to enter their target cells via an endocytic pathway are interesting from a pathogenetic standpoint and may indicate that those cells are more readily amenable to accept large DNA viruses by the endocytic route.
FIG. 8. Putative model of the steps involved in the different EHV-1 entry pathways in CHO-K1 cells, equine EC, and equine PBMC. (A) Initial attachment of EHV-1 occurs via binding of gC to proteoglycans. (B1) Fusion at the plasma membrane involves interaction (more ...)
Although we clearly identified RGD-binding integrins such as αVβ5 or αVβ3 and an RSD motif in EHV-1 gD as important requirements for EHV-1 endocytosis, more research will be necessary to study this complex, multistep process of EHV-1 entry in more detail. In the experiments presented here, we could never completely abolish EHV-1 infection, suggesting that other receptors are likely involved in EHV-1 endocytic entry. In line with this observation, it is interesting that EHV-1 gB and gH, two glycoproteins important for herpesviral entry, each contain integrin binding motifs. These motifs, LDI in gB and YGL in gH, are sequences present in fibronectin and were shown to mediate binding to integrins α4β1 and α4β7 (22
). In addition, such motifs are present in the VP4 spike protein of rotaviruses and were shown to mediate cell entry and therefore infectivity of several rotaviruses (14
). In future experiments, we plan to target the LDI motif and/or the YGL motif, present in gB and gH, respectively, alone or in combination with the point mutation in the RSD gD motif. Evaluation of the efficiency of infection by such EHV-1 recombinant viruses, especially infection of equine PBMC, would be of interest since α4 integrins are dominantly and highly expressed on B and T lymphocytes and monocytes (18
Taken together, we provide evidence here that (i) EHV-1 entry via endocytosis involves at least an RSD-integrin interaction and (ii) EHV-1 uses at least two different cellular entry pathways to infect important target cell populations of the natural host. Viral entry via multiple pathways involving several receptors increases the chance of a virus to survive in the host. The different strategies for infection of different host cells seem necessary for this herpesvirus to be capable of establishing a successful infection and maintaining itself in the individual horse and the population.