The high mortality rate associated with filovirus infection and the lack of any effective therapeutic strategy have led to their classification as biosafety level 4 pathogens. This extreme pathogenesis has greatly hindered the study of their replication, and thus they remain a very poorly characterized group of viruses. To study the process of Ebola virus entry, we produced retroviral particles pseudotyped with Ebo-GP. Production of high-titer MLV(Ebola) pseudotypes (up to 6 × 106
IU/ml) allowed analysis of the host range conferred by Ebo-GP. The host range conferred by Ebo-GP is very broad, enabling the infection by MLV(Ebola) of a variety of cell lines from diverse species and tissues. The infection of many of the cell lines described in Table is not unexpected, since humans, monkeys, and bats have all been reported to be infectible by the Ebola virus in vivo (1
). A recent report suggests that pigeons and mice may be refractory to Ebola virus infection (29
). However, our results suggest that the described block to infection of these animals is not due to an inability of Ebo-GP to mediate viral entry into cells from these species, since NIH 3T3 murine fibroblasts, primary CEF and TEF, and quail muscle sarcoma cells (QT6) were effectively infected by MLV(Ebola).
Interestingly, we found that both human and murine cells of the lymphoid lineage were completely resistant to infection by MLV(Ebola). This block to infection mediated by Ebo-GP correlates with observations made during the course of infections with the Ebola virus in laboratory animals, in which lymphoid cells are generally uninfected by the virus (24a
). In addition, both the HUT-78 and CEM(E) human T-cell lines have been shown to be resistant to infection by all subtypes of Ebola virus analyzed, as well as Marburg virus (22
). Together, these data suggest that cells of the lymphoid lineage either lack a functional Ebola virus receptor or somehow inhibit the Ebo-GP-mediated entry event. Cocultivation of 293T cells with a number of lymphoid cell lines did not reduce the susceptibility of these cells to infection by MLV(Ebola), indicating that a soluble inhibitory factor was not likely to be the cause of the block to Ebola virus infection of B and T cells. The demonstration that B and T lymphocytes are unable to support Ebola virus entry should enable the cloning of the cellular receptor for Ebola viruses through complementation with genomic DNA or a cDNA library from a cell line susceptible to MLV(Ebola) infection.
The production of high-titer MLV(Ebola) pseudotypes will also allow studies of the function of Ebo-GP to be done. It has been noted that the carboxyl-terminal regions of Ebo-GP and the ASLV envelope glycoprotein are very similar, containing a conserved CX6
CC motif, a potential internal fusion peptide domain, and a coiled-coil region (12
). These similarities between Ebo-GP and the well-characterized ASLV envelope provide a framework within which to analyze the function of specific regions of Ebo-GP through a targeted mutation strategy. The production of high-titer MLV(Ebola) pseudotypes will also allow studies of the overall function of the glycoprotein to be performed. Since Ebola virus must be handled as a biosafety level 4 pathogen, even relatively simple functional analyses, such as determining the pH dependence of entry, are laborious and expensive. In contrast, using MLV(Ebola) pseudotypes to address such questions is simple and straightforward.
The administration of neutralizing antibodies has been highly successful in mediating clearance of both rabies virus and hepatitis B virus from infected individuals (11
), and it has been suggested that the administration of neutralizing antibodies early in the course of an Ebola virus infection might increase survival rates (25
). Moreover, in animals recovering from filovirus infection, the development of high-titer antibodies specific for Ebola antigens coincides with viral clearance (9
), suggesting that neutralizing antibodies may play a role in the clearance of Ebola virus from infected individuals. Our preliminary results from an analysis of an antiserum made against the viral glycoprotein indicate that neutralizing epitopes may exist within Ebo-GP. Experiments performed with this antiserum and wild-type Ebola virus have yielded very similar neutralization activity (24a
). Therefore, MLV(Ebola) should be useful for rapid and effective screening of panels of antibodies for Ebola virus neutralization ability and for characterization of the humoral response to Ebo-GP in infected animals and individuals.
The results of experiments utilizing ammonium chloride and chloroquine show that MLV(Ebola) is very sensitive to pretreatment of target cells with weak bases, as are MLV(VSV) pseudotypes, while infection by MLV(ASLV-A), a pH-independent virus, is unaffected. These data suggest that entry of Ebola virus is pH dependent. The Ebola virus and ASLV glycoproteins exhibit extensive homology in a region postulated to be intimately involved with the fusion process (4
). However, despite these similarities, the mechanisms of activation of the fusogenic machinery of Ebola virus and ASLV appear to differ. Further studies to confirm the pH dependence of Ebola virus entry, such as determination of whether cells expressing Ebo-GP can be induced to form syncytia upon treatment with an acidic medium or whether MLV(Ebola) virions can be inactivated by treatment with acid prior to infection of target cells, are currently under way.
We have described a system in which Ebo-GP can be efficiently pseudotyped into murine leukemia virus particles to produce high-titer, infectious MLV(Ebola) pseudotypes. Our results suggest that MLV(Ebola) virions accurately mimic the entry process of the Ebola virus, both in cellular host range and ability to be neutralized by treatment with a polyclonal antiserum raised against Ebo-GP, and indicate that Ebola may be a pH-dependent virus. These pseudotypes thus provide an ideal system in which to analyze immune responses to Ebola virus infection as well as to examine the function of Ebo-GP, and they will be useful in the identification of the cellular receptor for this deadly virus.