A system for functional analysis of Ebola virus glycoprotein

Abstract

Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green fluorescent protein gene instead of the receptor-binding G protein gene (VSVDeltaG*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSVDeltaG*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSVDeltaG* complemented with VSV G protein (VSVDeltaG*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSVDeltaG*-ResGP but not to VSVDeltaG*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and/or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured in vitro.

Figure 1

Incorporation of ResGP into VSV particles. Viral proteins were analyzed by SDS/PAGE in 10% gels under reducing conditions. 293T cells transfected with an expression vector plasmid containing the VSV G gene (lane 1), the ResGP gene (lane 2), or the vector plasmid only (lane 3) were labeled for 5 hr with [³⁵S]methionine 30 hr after transfection. Proteins in cell lysates were precipitated by a mAb to VSV G protein (lane 1) or ResGP (lanes 2 and 3). Recombinant VSVs, including VSVΔG* complemented with VSV G protein (lane 4, VSVΔG*-G) or with ResGP (lane 5, VSVΔG*-ResGP) or lacking complementation (lane 6, VSVΔG*), were labeled with [³⁵S]methionine and purified by differential centrifugation and sedimentation through 25–45% sucrose gradients.

Figure 2

Electron microscopy of recombinant VSV particles. VSVΔG* complemented with VSV G protein (VSVΔG*-G) or with ResGP (VSVΔG*-ResGP) or without complementation (VSVΔG*) was prepared and partially purified by centrifugation through 20% sucrose. Viruses were negatively stained (A) or labeled with a mouse mAb to ResGP (B) or VSV G protein (C) and anti-mouse IgG conjugated with colloidal gold, followed by negative staining.

Figure 3

Expression of GFP upon infection with recombinant VSVs. Vero cells were infected with VSVΔG*-G, VSVΔG*-ResGP, or VSVΔG*, and GFP expression was examined 12 hr after infection by fluorescence microscopy.

Figure 4

Infectivity of recombinant VSVs on chemically modified Vero cells. Vero cells were preincubated with indicated concentrations of reagents. Infectivities of VSVΔG*-G and VSVΔG*-ResGP were then examined. Each bar represents the percentage of infectious units calculated from 10 microscopic fields (mean ± SD).