Comparative analysis of Ebola virus glycoprotein interactions with human and bat cells

Abstract

Infection with Ebola virus (EBOV) causes hemorrhagic fever in humans with high case-fatality rates. The EBOV-glycoprotein (EBOV-GP) facilitates viral entry and promotes viral release from human cells. African fruit bats are believed not to develop disease upon EBOV infection and have been proposed as a natural reservoir of EBOV. We compared EBOV-GP interactions with human cells and cells from African fruit bats. We found that susceptibility to EBOV-GP-dependent infection was not limited to bat cells from potential reservoir species, and we observed that GP displayed similar biological properties in human and bat cells. The only exception was GP localization, which was to a greater extent intracellular in bat cells as compared to human cells. Collectively, our results suggest that GP interactions with fruit bat and human cells are similar and do not limit EBOV tropism for certain bat species.

Figure 1.

Cleavage of Ebola virus glycoproteins (EBOV-GPs) by subtilisin-like proteases in human and bat cells. We transfected 293T cells and EpoNi/22.1 cells with pCAGGS plasmids encoding the indicated V5-tagged EBOV-GPs or empty pCAGGS plasmid (Mock). Cells were harvested at 48 h after transfection. EBOV-GP expression in cell lysates was determined using anti-V5 antibody. Detection of β-actin served as loading control. Similar results were obtained in 3 independent experiments and with Hela, HypNi/1.1, and MyDauLu/47.1 cells. CIEBOV, Côte d'Ivoire ebolavirus; REBOV, Reston ebolavirus; SEBOV, Sudan ebolavirus; ZEBOV, Zaire ebolavirus.

Figure 2.

Ebola virus glycoproteins (EBOV-GPs) enhance budding of VP40-based virus-like particles (VLPs) in human and bat cells. We cotransfected 293T and EpoNi/22.1 cells with a VP40-c-myc expression plasmid and the indicated EBOV-GP-V5 expression plasmids or empty vector (Mock). Expression of VP40 in cell lysates and supernatants was detected using an anti-c-myc antibody. Detection of β-actin served as loading control. Similar results were obtained in 3 independent experiments. CIEBOV, Côte d'Ivoire ebolavirus; REBOV, Reston ebolavirus; SEBOV, Sudan ebolavirus; ZEBOV, Zaire ebolavirus.

Figure 3.

Expression of Ebola virus glycoproteins (EBOV-GPs) induces detachment of human and bat cells. A, 293T cells were transfected with pCAGGS plasmids encoding EBOV-GP-V5 or empty pCAGGS plasmid (Mock), and the number of cells in the culture supernatant was counted at 48 hours after transfection via fluorescence-activated cell sorting. The mean of 12 independent experiments is shown. Error bars indicate standard error of the mean (SEM). B, EpoNi/22.1 cells were transfected with pCAGGS plasmids encoding EBOV-GP-V5, and the number of cells in the supernatant was counted as described in A. The mean of 11 independent experiments is shown. Error bars indicate SEM. C, 293T cells were transfected with pCAGGS or pcDNA plasmids encoding EBOV-GPs with C-terminal V5 tag and treated as in Figure 1. D, 293T cells were transfected with pcDNA plasmids encoding EBOV-GP-V5, and the number of cells in the supernatant was counted as in A. The mean of 11 independent experiments is shown. Error bars indicate SEM. Asterisks indicate statistically significant differences (P < .05) relative to mock as determined by means of 2-tailed Student t test. CIEBOV, Côte d'Ivoire ebolavirus; REBOV, Reston ebolavirus; SEBOV, Sudan ebolavirus; ZEBOV, Zaire ebolavirus.

Figure 4.

Plasma membrane localization of Ebola virus glycoprotein (EBOV-GP) is more prominent in human than in bat cells. A, HeLa and EpoNi/22.1 cells were transfected with the indicated EBOV-GP-EYFP or VP40–enhanced green fluorescent protein expression plasmids. Cells were fixed at 48 hours after transfection, and cellular distribution of GP and VP40 was determined by fluorescence microscopy. Similar results were obtained in 3 independent experiments. White scale bar represents 20 μm. B, EpoNi/22.1, MyDauLu/47.1, and HypNi1.1 cells were transfected and analyzed as in A. Similar results were obtained in 2 independent experiments. White scale bar represents 20 μm. REBOV, Reston ebolavirus; ZEBOV, Zaire ebolavirus.

Figure 5.

The glycoproteins (GPs) of the Ebola virus (EBOV) species Zaire, Sudan, and Reston mediate entry into potential reservoir and nonreservoir cells. A, The indicated cell lines were infected with vesicular stomatitis virus (VSV)–based pseudotypes bearing either VSV-G or EBOV-GPs as envelope proteins. The different pseudotypes were normalized to comparable titers on Vero E6 cells prior to infection of bat cells. Pseudotypes bearing no GPs were used as negative control (mock) and not normalized. Infectivity was determined by microscopy, detecting green fluorescent protein signals of infected cells. One representative experiment of 3 independent experiments is shown. White scale bar represents 200 μm. B, The indicated cell lines were infected as in A. Infectivity was measured by luciferase readout. The signals obtained for samples infected with pseudotypes bearing no GP (mock) were set as 1. Infection was performed in duplicate. One representative experiment of 3 independent experiments is shown. Error bars indicate standard deviation (SD). REBOV, Reston ebolavirus; SEBOV, Sudan ebolavirus; ZEBOV, Zaire ebolavirus.

Figure 6.

Efficient replication of Ebola virus in EpoNi/22.1 and HypNi/1.1 cells. The indicated cell lines were seeded in 6-well plates; infected in duplicate with Zaire Ebola virus (ZEBOV), Kikwit strain (multiplicity of infection of 0.1); washed; and cultured. Samples were taken at the indicated time points after inoculation, and their titers were determined by endpoint titration in Vero E6 cells, using the development of cytopathic effects as readout (TCID₅₀, tissue culture infectious dose that leads to 50% cytopathic effects). Infectious titers were calculated from 3 replicates by the method of Spearman-Karber. The results represent the geometric mean titers obtained from 2 independent experiments. Error bars indicate standard deviation.