Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines

Abstract

Studies analyzing Ebola virus replication have been severely hampered by the extreme pathogenicity of this virus. To permit analysis of the host range and function of the Ebola virus glycoprotein (Ebo-GP), we have developed a system for pseudotyping these glycoproteins into murine leukemia virus (MLV). This pseudotyped virus, MLV(Ebola), can be readily concentrated to titers which exceed 5 x 10(6) infectious units/ml and is effectively neutralized by antibodies specific for Ebo-GP. Analysis of MLV(Ebola) infection revealed that the host range conferred by Ebo-GP is very broad, extending to cells of a variety of species. Notably, all lymphoid cell lines tested were completely resistant to infection; we speculate that this is due to the absence of a cellular receptor for Ebo-GP on B and T cells. The generation of high-titer MLV(Ebola) pseudotypes will be useful for the analysis of immune responses to Ebola virus infection, development of neutralizing antibodies, analysis of glycoprotein function, and isolation of the cellular receptor(s) for the Ebola virus.

FIG. 1

Protein expression in virion and cell lysates. 293T cells were transiently transfected with MLV Gag-Pol and genome constructs with (+ EBO) or without (− EBO) pCB6-Ebo-GP, a Zaire subtype Ebo-GP expression construct. Viral supernatants were harvested from transfected cells and partially purified by pelleting through 20% sucrose. Cell lysates and lysed pellets were analyzed by Western blotting with anti-Ebo-GP serum and anti-MLV capsid serum. (A) Expression of Ebo-GP in cell lysates. Arrows indicate the two forms of Ebo-GP. (B) Incorporation of Ebo-GP into MLV particles. (C) Analysis of MLV capsid in viral pellets. Arrows indicate MLV Gag precursor (Pr65) and capsid (p30) proteins. The positions of molecular mass markers (in kilodaltons) are shown on the left of each gel.

FIG. 2

MLV(Ebola) neutralization by incubation with a polyclonal anti-Ebo-GP serum. Prior to infection of cells, MLV(Ebola) was incubated with either a control serum (⧫) or anti-Ebo-GP serum (□) for 30 min at room temperature.

FIG. 3

Inhibition of MLV(Ebola) infection by lysosomotropic agents. QT6 cells were treated with the indicated concentrations of either ammonium chloride (A) or chloroquine (B) for 1 h prior to infection with either MLV(Ebola) or MLV(ASLV-A). Cells were fed with fresh medium 16 h postinfection and stained 32 h later. ○, MLV(ASLV-A); □, MLV(Ebola); ◊, MLV(VSV).