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Ebola virus infects a wide variety of adherent cell types, while nonadherent cells are found to be refractory. To explore this correlation, we compared the ability of pairs of related adherent and nonadherent cells to bind a recombinant Ebola virus receptor binding domain (EboV RBD) and to be infected with Ebola virus glycoprotein (GP)-pseudotyped particles. Both human 293F and THP-1 cells can be propagated as adherent or nonadherent cultures, and in both cases adherent cells were found to be significantly more susceptible to both EboV RBD binding and GP-pseudotyped virus infection than their nonadherent counterparts. Furthermore, with 293F cells the acquisition of EboV RBD binding paralleled cell spreading and did not require new mRNA or protein synthesis.
The filovirus Ebola causes severe hemorrhagic fever in humans and nonhuman primates, with mortality rates reaching 90% (8, 15). Ebola virus encodes a single glycoprotein (GP) that is both necessary and sufficient for virus binding to and penetration of host cells. Ebola virus GP confers broad cellular tropism and binds to an as-yet unidentified proteinaceous host cell receptor (3, 9, 11, 18, 20, 22, 23). To date, a wide array of adherent cell types, including fibroblasts and epithelial cells, have been shown to be susceptible to Ebola virus GP-mediated infection (14, 20, 22, 23). These adherent cells are anchorage dependent, requiring adhesion to extracellular matrix components in order to survive and proliferate, with these processes being mediated at least in part by integrin signaling through extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Rac (17). Conversely, the cell types that have been shown to be refractory to Ebola virus infection are human B and T lymphocytes, which exhibit anchorage-independent growth and can survive and proliferate as nonadherent suspension cultures.
A receptor binding domain of Ebola virus GP (EboV RBD) has been defined (3, 11, 12). Human 293T and African green monkey Vero cells, both of which are adherent cells that are permissive for Ebola virus GP-mediated infection, were reported to bind an EboV RBD-Fc construct, whereas Jurkat T cells bound only minimal levels of the EboV RBD (11). These observations suggested that anchorage-dependent cells may, as a general rule, be more susceptible to Ebola virus GP-mediated RBD binding and infection than cells capable of anchorage-independent growth.
To begin to test this hypothesis, we first confirmed and extended basic observations on EboV RBD binding and GP-mediated infection. As seen in Fig. Fig.1A,1A, 293T and Vero cells (adherent) bind significant levels of EboV RBD (16 and 31% of cells, respectively). We examined the infection status of these cells using vesicular stomatis virus (VSV)-pseudotyped particles bearing a mucin domain-deleted Ebola virus GP (GPΔ) and encoding green fluorescent protein (GFP) (VSV-GPΔ) (16). As seen in Fig. Fig.1B,1B, 293T and Vero cells were well infected by VSV-GPΔ (48 and 56% of cells, respectively). In contrast, and as previously shown (11), Jurkat T cells (nonadherent) bind only low levels of the RBD (1% of cells) and are minimally susceptible to VSV-GPΔ (2% of cells) (Fig. (Fig.1).1). In addition to Jurkat cells, two other (nonadherent) lymphocyte cell lines, Hut78 and Daudi, bound minimal amounts of EboV RBD (4 and 1.6% of cells, respectively) (Fig. (Fig.1A)1A) and were poorly infected by VSV-GPΔ (<2% of cells) (Fig. (Fig.1B).1B). All cell lines were well infected with VSV-pseudotyped particles that bore the native VSV glycoprotein (VSV-G).
To test the hypothesis that there is a general block to Ebola virus entry into nonadherent cell lines, we compared the ability of two pairs of highly related cells, grown either in suspension or as adherent cells, to bind EboV RBD and to be infected with VSV-GPΔ. THP-1 cells are a human monocyte cell line that can be differentiated into macrophage-like cells with phorbol 12-myristate 13-acetate (PMA) or retinoic acid (RA) (7). Untreated (and mock-treated) THP-1 cells grow in suspension. RA-treated cells remain in suspension but express the macrophage differentiation markers CD11a and CD11b; CD11b is restricted to mature myeloid cells. PMA-treated cells express CD11a and CD11b, but in contrast to mock-treated or RA-treated THP-1 cells, they become adherent (10, 13). Mock-treated THP-1 cells displayed a low level of EboV RBD binding (4.7% of cells). RA-treated cells showed somewhat increased binding (9.3% of cells), but the difference was not statistically significant. In contrast, PMA-treated THP-1 cells bound significantly higher levels of EboV RBD (19% of cells) (Fig. (Fig.2A).2A). We next examined these cells for susceptibility to VSV-GPΔ. Both mock- and RA-treated THP-1 cells (which remain in suspension) were poorly infected with VSV-GPΔ (5 and 4% of cells, respectively), whereas PMA-treated THP-1 cells (which are adherent) were well infected (39% of cells) (Fig. (Fig.2B).2B). There was no significant difference in the susceptibility of these cells to infection with VSV-G. A viral entry assay that used human immunodeficiency virus pseudovirions harboring β-lactamase and bearing Ebola virus GPΔ or VSV-G supported these infection results; a significant increase of Ebola virus GP-mediated viral entry was seen in PMA-treated THP-1 cells compared to that of mock- or RA-treated cells (Fig. (Fig.2C2C).
293F cells are derivatives of 293 cells. They remain in suspension if grown on a shaking platform but will become adherent and spread if left in static culture (5, 6). We compared the EboV RBD binding ability and susceptibility to VSV-GPΔ infection of 293F cells in suspension to those of ones allowed to adhere for 18 h. As seen in Fig. Fig.3A,3A, there was an approximately fourfold increase in EboV RBD binding to adherent 293F cells compared to that of cells kept in suspension (29 and 7% of cells, respectively). Correspondingly, suspension 293F cells were poorly infected (4%), while those allowed to adhere were well infected (20%), representing a fivefold increase in susceptibility (Fig. (Fig.3B).3B). This increase in the susceptibility of the adherent cells was not seen with VSV particles bearing either VSV-G or measles virus F and H (Fig. 3B and C). A similar increase in susceptibility to GP-mediated viral entry was seen using the β-lactamase entry assay (Fig. (Fig.3D3D).
To further assess if EboV RBD binding correlated with the cell adhesion phenotype, we examined the ability of 293F cells to bind EboV RBD after various lengths of time in static culture. At time zero, suspension 293F cells displayed a low average cell area (a measure of cell adhesion and spreading) and low EboV RBD binding. However, within 2 h in static culture, both the average cell area and EboV RBD binding reached maximal values (Fig. 3E and F). For reasons that we do not understand, we saw a small decrease in cell area and RBD binding at the 4-h time point. Nevertheless, these results clearly demonstrate that, at least in the case of 293F cells, the ability to bind EboV RBD is acquired in parallel with cell spreading.
To examine the mechanism by which cell adhesion promotes the binding of the EboV RBD, we tested the effects of actinomycin D, an inhibitor of transcription, and cycloheximide, an inhibitor of protein translation (4, 6). Neither inhibitor significantly affected the binding of the EboV RBD to adherent 293F cells (Fig. (Fig.4).4). This suggests that the increase in EboV RBD binding that accompanies cell adhesion and spreading does not require the synthesis of new mRNA or protein.
In summary, we have shown that when two nonadherent human cell lines (THP-1 and 293F) are treated so as to render them adherent, they concomitantly acquire the abilities to bind the EboV RBD and to be infected with Ebola virus GP-pseudotyped viral particles. Previous work suggested that circulating monocytes can be infected by Ebola virus (19). However, infection occurred in concert with the activation and aggregation of the cells (cell-cell adhesion), and it is unclear whether infection occurred in isolated nonadherent monocytes. Thus, the inability of nonadherent cell lines (e.g., cells in addition to lymphocytes) to be infected by Ebola virus GP pseudotypes may be due, at least in part, to a deficiency in binding the RBD of the Ebola virus GP. Moreover, our findings suggest that the acquisition of RBD binding and Ebola virus GP pseudotype infection occur in parallel with cell adhesion and spreading, and that these properties arise due to posttranslational events.
Several possible explanations can account for these observations. For one, an Ebola virus receptor or binding cofactor(s) already present in the cell may relocate from the cell interior to the cell surface upon cell adhesion. Such relocation would allow virus binding and infection. Precedence for protein relocation in concert with cell adhesion has been shown for components of lipid raft domains (1), which have been proposed to be important for Ebola virus binding and entry (2). Our initial studies suggest that there are comparable levels of the lipid raft marker GM1 on the surfaces of adherent and nonadherent 293F cells (data not shown). It remains possible, however, that other raft or nonraft components involved in facilitating Ebola virus GP binding are inserted into the plasma membrane upon cell adhesion. Alternatively, cell adhesion may promote a conformational change or posttranslational modification in a cell surface protein(s) that confers the ability to bind the RBD of the Ebola virus GP. With respect to the correlation between cell adhesion and EboV RBD binding and infection, it is interesting that we (K. L. Schornberg, A. H. Bouton, and J. M. White, unpublished data) and others (21) have found that β1 integrins promote Ebola virus GP-mediated entry, since β1 integrins are central to cell adhesion and signaling for anchorage-dependent cell growth. Studies are in progress to determine how cell adhesion and spreading promote Ebola virus GP-mediated binding and infection.
Work in our laboratories was supported by grants from the NIH (AI22470 and U54AI57168 to J.M.W.; AI050733 to A.H.B.; and AI057168 to C.C.B.). D.D. and K.L.S. were supported in part by training grants 5T32 AI055432 and 5T32 AI0704627 to the University of Virginia.
We thank Edward Park for excellent technical assistance, Kirsten Kabsch for the production of the measles virus F and H pseudovirions, and Yusuke Yanagi for supplying the measles virus F and H cDNAs.
Published ahead of print on 30 April 2008.