The Ebola virus GP is synthesized in a secreted (sGP) or full-length transmembrane form, and each gene product has distinct biochemical and biological properties. For example, GP appears to form a trimeric complex (30
) and binds preferentially to endothelial cells, whereas sGP does not (49
). Preferential binding of Ebola virus GP to the endothelium was demonstrated by use of two independent methodologies as follows: direct binding was assessed by fluorescence-activated cell sorter analysis, and pseudotyping experiments were performed in which virus titers, cell numbers, and confluence were carefully determined so that the multiplicity of infection was controlled and equal in all cell types. Another study failed to demonstrate this preferential binding (17
), but direct binding of GP to endothelial cells was not measured and neither the multiplicity of infection, target cell numbers, nor cell confluence was reported in that study. The receptors required for cell binding and infection are not completely understood. A folate-related receptor can serve as a cofactor to facilitate infection (8
), but whether it serves as a receptor remains unclear. The cell surface lectin DC-SIGN can also facilitate GP binding to cells through viral carbohydrate determinants, but it does not appear to mediate entry by itself (1
). In contrast to GP, sGP gives rise to a dimeric protein (30
) that interacts with neutrophils (49
). sGP mediates neutrophil binding, directly or indirectly, through CD16b, the neutrophil-specific form of the Fcγ receptor III (49
). After the initial description of the neutrophil binding of sGP, it was shown that immunoglobulin G (IgG), but not an Fab fragment, against sGP was needed to detect neutrophil binding (T. Maruyama, M. J. Buchmeier, P. W. H. I. Parren, and D. R. Burton, Technical Comment, Science 282:
843-844, 1998). A subsequent study showed that the binding could also be seen if an irrelevant IgG was used with the Fab fragment against sGP (Z.-Y. Yang, R. Delgado, L. Xu, R. F. Todd, E. G. Nabel, A. Sanchez, and G. J. Nabel, Author's Reply, Science 282:
844-846, 1998). Though such binding could potentially arise from binding of immune complexes, additional studies using resonance energy transfer showed that neutrophils incubated with sGP showed a significant reduction in the CR3-Fcγ RIIIB RET signal (22
), demonstrating that sGP alters the physical and functional interaction between Fcγ RIIIB and CR3. Through this interaction, sGP may contribute to immune evasion by inhibiting early steps in neutrophil activation (as measured by the down-modulation of l
-selectin) that would ordinarily assist in virus clearance (49
Several lines of evidence suggest that the viral GP plays a key role in the manifestations of Ebola virus infection. The transmembrane form of GP targets the Ebola virus to cells that are relevant to its pathogenesis. Specifically, GP allows the virus to introduce its contents into monocytes and/or macrophages, where cell damage or exposure to viral particles may cause the release of cytokines (34
) associated with inflammation and fever, and into endothelial cells, which damages vascular integrity (48
) (Fig. ). Thus, sGP may alter the immune response by inhibiting neutrophil activation, while the transmembrane GP may contribute to the hemorrhagic fever symptoms by targeting virus to cells of the reticuloendothelial network and the lining of blood vessels.
FIG. 1. Host immune responses to Ebola virus and cell damage due to direct infection of monocytes and macrophages cause the release of cytokines associated with inflammation and fever (A). Infection of endothelial cells also induces a cytopathic effect and damage (more ...)
GP expression in cultured human endothelial and epithelial cells causes cell rounding and detachment (48
). GP is the only one of the seven Ebola virus gene products to exert this effect, and though GP from all four documented Ebola virus strains acts similarly, the highly pathogenic Zaire strain has the most potent activity in this cell culture assay (33
). These effects require the presence of the mucin-like, serine-and-threonine-rich domain of GP and correspond with the down-regulation of specific molecules on the cell surface (48
). Cytotoxicity appears to be precisely controlled by a mechanism involving down-regulation of GP expression through a transcriptional RNA editing event by the viral polymerase. The importance of this phenomenon was shown by use of a reverse genetics system for replicating Ebola virus in which a mutation that increases the level of full-length GP expression is significantly more cytotoxic than the wild-type virus (42
The in vivo relevance of GP-induced endothelial cell toxicity was explored in blood vessel explants (48
) in which human saphenous veins were infected with replication-defective adenoviral vectors carrying the gene for GP or sGP. Staining with horseradish peroxidase and scanning electron microscopy were used to observe severe damage to the endothelial cell lining in vessels that received the virus encoding full-length Ebola virus GP but not sGP or vectors in which the mucin domain of GP was removed. Cell damage in explant cultures paralleled the species specificity of different Ebola virus strains: no toxicity was observed when Reston strain GP was introduced into human vascular explants, whereas significant tissue damage was observed in vascular explants from nonhuman primates.
Further in vitro analyses have begun to elucidate the molecular mechanisms underlying GP-induced cytotoxicity. Critical mediators of cell adhesion to the matrix and immune signaling (e.g., integrins and major histocompatibility complex class I cell surface proteins) are among the cell surface molecules that are dysregulated (33
). Transient expression of Ebola virus GP in human kidney 293T cells caused a reduction of specific integrins (primary molecules responsible for cell adhesion to the extracellular matrix) on the cell surface. GP mutants lacking the membrane-spanning region of the ectodomain did not cause this down-regulation, suggesting that anchorage of GP to the cell membrane is required for this effect. Disruption of major histocompatibility complex class I expression on the cell surface is a mechanism for evading host immune responses that is shared by several pathogens, including cytomegalovirus, human immunodeficiency virus (HIV), and herpesviruses (27
). It is not known whether GP affects integrin levels by altering intracellular trafficking or by modulation of protein synthesis or degradation, but preliminary experiments suggest a role for cellular protein transport machinery in GP-mediated cytotoxicity (N. Sullivan, unpublished observations). In any event, the biologic effects of GP alone may account largely for the features of Ebola virus infection that lead to fatal disease, including inflammatory dysregulation, immune suppression, and loss of vascular integrity.
Structural analyses of GP have revealed features in common with other viral envelope proteins. The crystal structure of the GP ectodomain revealed a coiled-coil domain resembling a trimer of helical hairpin-like loops (23
). The hairpin structure is adjacent to the fusion-peptide region (16
) hypothesized to insert directly into the target cell membrane. Analogous coiled-coil regions have been defined for GPs of influenza virus, murine retroviruses, HIV, and simian immunodeficiency virus (SIV) as well as for some cellular proteins, called SNARES, that function in intracellular vesicle fusion (44
). For HIV gp160, it has been possible to identify peptides that bind to a transient intermediate form that precedes hairpin formation. Because of their potent inhibition of viral entry, these reagents have shown considerable promise in clinical trials (21
). The Ebola virus GP contains a homologous hairpin structure for which a possible inhibitory peptide has been identified (43
), a region that remains a potential therapeutic target.