The Ebola virus GP is a class I viral fusion protein that exists as a metastable complex of trimers on the virus surface (8
). Membrane fusion catalyzed by viral fusion proteins is characterized by a triggered series of dramatic structural rearrangements that culminate in merger of the host and viral membranes. Recognized triggers for these structural changes include binding to cellular receptors, as typified by HIV, and exposure to a low-pH environment encountered as virions traffic through the endocytic pathway, as demonstrated by the influenza virus hemagglutinin protein. Variations of these triggering schemes exist. Notably, the avian sarcoma and leukosis virus Env fusion complex has evolved a unique multistep process in which receptor binding at neutral pH triggers initial structural changes that drive viral fusion peptide insertion into the host membrane, while low pH is subsequently required to initiate hemifusion and fusion pore formation (1
). Another striking variation of these models was recently described in which an apparent pH trigger reflected the requirement for pH-dependent endosomal cathepsins for viral entry (3
The initial observation that CatL- and thermolysin-proteolysed Ebola virus GP pseudovirions were more infectious that untreated virions led us to question the role of GP proteolysis during entry (Fig. ). We found that virions associated with the 18-kDa GP1 fragment bound cells more efficiently than virions with full-length GP, and this is likely the explanation for the observed increase in infectivity (Fig. ). Our results are supported by work from Kuhn et al. (7
) in which an approximately 16-kDa N-terminal fragment of Ebola virus GP1 (residues 54 to 201), and not full-length GP1, was capable of binding VeroE6 cells and blocking infection with Ebola virus (7
). Taken together, these results suggest that truncation of Ebola virus GP1 activates the receptor binding potential of the protein, a process that likely occurs upon cathepsin proteolysis in vivo. Similar to Kuhn et al., we observed greatly enhanced cell surface binding of virions bearing a truncated GP1 fragment of nearly the same molecular weight. The idea that Ebola virus GP truncation enhances binding is further supported by studies of the SARS coronavirus S protein in which truncation of the S1 subunit yielded an independently folding receptor-binding domain that bound cells more efficiently than full-length S1 (23
). It is possible that a key role for CatL during Ebola virus and SARS coronavirus entry is to proteolytically truncate extraneous regions of the GP, thereby exposing the receptor binding domain or allowing higher affinity receptor binding.
Ebola virus entry appears to be a multistep process in which CatB and CatL cleave GP1 to yield a stable 18-kDa intermediate in a manner that was recapitulated in vitro using CatL alone (Fig. ). It remains unclear what role CatL plays during the second step of viral entry, yet several explanations are possible. An additional cellular factor dependent on the activity of CatL may be required for triggering the fusion peptide, as proposed by Schornberg et al. (15
). Alternatively, interaction with a cellular receptor within the endosome, or alteration of an existing receptor interaction after the initial proteolysis, may induce structural rearrangements in Ebola virus GP. Previously inaccessible regions in GP may then undergo further CatL proteolysis, thereby lowering the activation barrier and allowing the fusion machinery to trigger. The latter model assumes that our observations using in vitro proteolysed virions interacting with the cell surface recapitulate events that occur within the endosome. As many cell surface proteins are known to recycle through endosomal compartments, it seems reasonable to hypothesize that virions may encounter factors at the cell surface that are also present in CatL-enriched endosomes.
The mucin domain is involved in binding of Ebola virus GP to the C-type lectin hMGL (19
). The significance of this interaction remains unclear, but it may contribute to trafficking of virions from the cell surface to endosomes, where cathepsin proteolysis proceeds. Our results analyzing cells that do not express hMGL suggest that the mucin domain may inhibit receptor binding and that removal of the mucin domain by genetic removal or by cathepsin proteolysis promotes virus-cell interactions. This is most obvious in the experiments using Ebola virus GP-Δmuc virions, which bound cells more effectively than wild-type Ebola virus GP virions (Fig. ). While the mucin domain may contribute to hMGL interactions, it appears that the costs associated with the mucin domain may include reduced affinity for a cellular factor. It is unclear why the mucin domain may inhibit virus entry, but is possible that the bulky glycosylated region may sterically prevent interactions of the Ebola virus GP N terminus with cellular factors. Removal of the mucin region upon cathepsin proteolysis in the endosome may promote receptor engagement and permit virus entry to proceed. Analysis of mutants in which the carbohydrate addition sites are altered will be required to discriminate whether loss of the glycan residues or deletion of the protein sequence is responsible for increased binding of the GP-Δmuc virions.
Unlike Kuhn et al. (7
), who did not detect binding of a truncated GP1 construct to lymphocytes, we observed a significant increase in binding of CatL-proteolysed GP1 relative to virions possessing full-length GP (Fig. ). We believe that the discrepancy in these findings is due to differences among the Ebola virus GP substrates used in our binding experiments. CatL-proteolysed Ebola virus GP in our experiments was a higher-molecular-weight species than the minimal fragment used by Kuhn et al. Moreover, in our experiments binding was examined in the context of virions rather than with purified nonoligomeric protein. Avidity-driven virion binding would likely reveal interactions that are not detected with purified protein. Furthermore, it remains unclear whether other proteolysed GP products remain noncovalently associated with virions in our studies, further contributing to cell binding. The observation that Jurkat cells remained largely resistant to infection with CatL-proteolysed virions despite enhancement of virus binding at the cell surface may suggest that the block to infection in Jurkat cells is downstream of virus binding. Identifying the block in lymphocytes may reveal additional factors or steps involved in Ebola virus entry.