Ebola virus typically avoids clearance by the host immune response in humans, resulting in uncontrolled replication, damage to host cells, and, ultimately, fatal organ destruction. Though some patients who mount a strong, early immune response can recover from infection (3
), the fatality rates remain high. An understanding of Ebola virus pathogenic mechanisms facilitates the development of antiviral and immune therapies, which are presently unavailable. Though previous reports have suggested that Ebola virus GP alters expression of cell surface proteins (15
), the specificity and mechanisms of these effects have not been defined. The findings presented here demonstrate that Ebola virus GP exerts this effect through a specific mechanism involving a pathway that is dependent upon the GTPase dynamin. Through its interaction with dynamin, GP disrupts the normal intracellular trafficking of cell surface proteins that are essential for cell attachment, viability, and immune signaling. The functional interactions shown herein appear to indicate a direct binding of GP to dynamin and a specific subset of cell surface molecules. However, Ebola virus GP might also influence trafficking in particular regions of the cell surface membrane in which these specific sets of molecules reside.
We now have several lines of evidence to suggest that αV down-regulation is highly dependent on GP-trafficking within the cell. For example, both transmembrane deletion and alternate soluble forms of GP have no effect on αV levels, implying that residence of GP at the plasma membrane (or at least within membrane compartments that are in equilibrium with the plasma membrane) is necessary for the effect. The brefeldin A results reinforce this interpretation, because brefeldin A acts between the endoplasmic reticulum and Golgi complex to inhibit membrane traffic and therefore likely prevents GP from accessing specific membrane compartments or, perhaps, impedes posttranslational modifications of GP that occur late in trafficking and that are required for the effect. It is noteworthy that the GP-induced cytopathic effects require cell surface expression of Ebola virus GP (16
). A previous report suggested that global down-regulation of cell surface molecules is exerted by GP (15
), but our present experiments and those of others (15
) demonstrate that the expression of at least some cell surface molecules is unaffected. With human kidney 293T cells, minimal to nonexistent down-regulation was observed for several α-integrins (16
), whereas β1 integrin is unaffected in HUVEC (15
) but is down-regulated in 293T cells (15
These disparate results suggest that Ebola virus GP does not cause global down-regulation but, rather, is linked to particular groups of molecules depending on cell type. Moreover, specific intracellular trafficking pathways that affect cell surface expression seem to be particularly relevant to down-regulation. Brefeldin A prevents Ebola virus GP from accessing cell surface microcompartments, and dynamin influences cycling to and from these same areas of the cell. We have observed that GP expression appears to be lost from the cell surface at time points when αV down-regulation is maximal. A likely explanation for this observation is that at late stages, down-regulation of GP is linked to removal of αV from the cell surface. Our biochemical and confocal results indicate that these two molecules reside in close proximity to one another. Therefore, a mechanism (e.g., increased endocytosis) that down-regulates αV may be expected to also influence cell surface levels of GP. The results presented herein provide a mechanism by which Ebola virus GP might influence groups of molecules, specified by particular cell trafficking pathways or by microlocalization at the cell membrane.
Unlike many enveloped viruses, the envelope glycoprotein gene of Ebola virus does not exhibit the high sequence variability that readily allows immune escape. These results suggest that Ebola virus has evolved in its natural host a mechanism that allows immune evasion in humans. Through its effects on specific cell surface molecules, Ebola virus disrupts several processes essential for immune activation and recognition, such as cell trafficking and antigen presentation. Adhesion and accessory proteins such as αV are involved in immune cell homing and signaling (2
), and down-regulation by GP may further affect homing, acute inflammation, and costimulation that would reduce both innate and acquired immune responses. At the same time, the cytotoxic effects of GP on macrophage and endothelial cell function affect the function of inflammatory cells (10
) and disrupt the integrity of the vasculature (18
). Together, these cytotoxic effects are likely responsible for the inflammatory dysregulation, immune suppression, and vascular dysfunction that are hallmarks of lethal Ebola virus infection.