Comparative Analysis of Host Cell Entry of Ebola Virus From Sierra Leone, 2014, and Zaire, 1976

Abstract

The ongoing Ebola virus (EBOV) disease (EVD) epidemic in Western Africa is the largest EVD outbreak recorded to date and requires the rapid development and deployment of antiviral measures. The viral glycoprotein (GP) facilitates host cell entry and, jointly with cellular interaction partners, constitutes a potential target for antiviral intervention. However, it is unknown whether the GPs of the currently and previously circulating EBOVs use the same mechanisms for cellular entry and are thus susceptible to inhibition by the same antivirals and cellular defenses. Here, we show that the GPs of the EBOVs circulating in 1976 and 2014 transduce the same spectrum of target cells, use the same cellular factors for host cell entry, and are comparably susceptible to blockade by antiviral interferon-induced transmembrane proteins and neutralizing antibody KZ52. Thus, the viruses responsible for the ongoing EVD epidemic should be fully susceptible to established antiviral strategies targeting GP and cellular entry factors.

Keywords: Ebola virus; West Africa; entry; glycoprotein.

Figure 1.

Pseudotypes bearing glycoprotein (GP) from ebolavirus (EBOV) strain Mayinga from 1976 (EBOV-GP 1976) and EBOV variant Makona from 2014 (EBOV-GP 2014) mediate entry into the same spectrum of cell lines. The indicated cell lines of human (A and B), monkey (C), and bat (D) origin were transduced with equal volumes of pseudotypes carrying the indicated GPs or no GP (pcDNA). Transduction efficiency was quantified by a luciferase assay 72 hours after transduction. Each experiment was performed with triplicate samples and repeated at least 3 times, using at least 2 independent pseudotype preparations. Error bars indicate standard deviations. HypLu/45.1, cell line derived from lung from H. monstrosus; HypNi/1.1, cell line derived from kidney from Hypsignathus monstrosus; RoNi/7.1, kidney cell line from Rousettus aegyptiacus.

Figure 2.

Comparable entry factor use by pseudotypes bearing glycoprotein (GP) from ebolavirus (EBOV) strain Mayinga from 1976 (EBOV-GP 1976) and EBOV variant Makona from 2014 (EBOV-GP 2014). A, 293T cells transfected to express the indicated lectins or transfected with empty plasmid (pcDNA3) were transduced with infectivity-normalized pseudotypes bearing the indicated viral GPs. Seventy-two hours after transduction, luciferase activities were determined in cell lysates. The results of a single, representative experiment performed with triplicate samples are shown. Transduction of control transfected cells (pcDNA) was set as 1. Similar results were obtained in 5 independent experiments, using different pseudotype preparations. B, Huh7 cells (left panel) and HeLa cells (right panel) underwent small interfering RNA (siRNA) transfection and were subsequently transduced with infectivity-normalized pseudotypes. Luciferase activities in cell lysates were measured 72 hours after transduction. The experiment was performed with triplicate samples and is representative of 3 independent experiments. C, 293T cells were preincubated with solvent (dimethyl sulfoxide [DMSO]) or the respective cationic amphiphiles in the indicated concentrations and subsequently transduced with pseudotypes bearing the indicated GPs. Luciferase activities were measured 72 hours after transduction. Two additional experiments yielded comparable results. Error bars indicate standard deviations.

Figure 3.

Subtle differences in the protease requirements of pseudotypes bearing glycoprotein (GP) from Ebola virus (EBOV) strain Mayinga from 1976 (EBOV-GP 1976) and EBOV variant Makona from 2014 (EBOV-GP 2014). A, 293T cells were preincubated with solvent or protease inhibitor (CA-074, 5 µM; CA-074Me, 0.4 µM; CatL, 1 µM; and AEBSF, 5 µg/mL) and thereafter transduced with infectivity-normalized pseudotypes and incubated for 72 hours in the presence of pseudotype and inhibitor. Then, luciferase activities were determined in cell extracts. Transduction in the absence of inhibitor was set as 100%. The average of 3 independent experiments is shown. Error bars indicate standard error of the mean (SEM). B, 293T cells were preincubated with solvent or protease inhibitor CatL III (CatL) at either 1 µM (white bars) or 10 µM (black bars), followed by transduction with infectivity-normalized pseudotypes. Luciferase activities were determined 72 hours after transduction. Transduction in the absence of inhibitor was set as 100%. The average of 3 independent experiments is shown. Error bars indicate SEM.

Figure 4.

Pseudoparticles bearing glycoprotein (GP) from Ebola virus (EBOV) strain Mayinga from 1976 (EBOV-GP 1976) and EBOV variant Makona from 2014 (EBOV-GP 2014) exhibit comparable stability. Pseudotypes normalized for comparable infectivity were incubated at the indicated temperatures for different periods, frozen at −80°C, and used for transduction of 293T target cells. Seventy-two hours after transduction, transduction efficiency was quantified by luciferase assay. The results are representative of 3 independent experiments performed with triplicate samples. Error bars indicate standard deviations. Abbreviation: VSV-G, glycoprotein of vesicular stomatitis virus.

Figure 5.

Pseudoparticles bearing glycoprotein (GP) of Ebola virus (EBOV) strain Mayinga from 1976 (EBOV-GP 1976) and EBOV variant Makona from 2014 (EBOV-GP 2014) are susceptible to inhibition by interferon-induced transmembrane (IFITM) proteins. 293T cells engineered to express IFITM proteins 1, 2, or 3 or no IFITM protein (control) were transduced with pseudoparticles bearing the indicated GPs. Luciferase activities were assessed 72 hours after transduction; transduction efficiency in cells expressing no IFITM protein (control) was set as 100%. The average of 2 separate experiments performed with triplicate samples is shown. Error bars indicate standard deviations.

Figure 6.

Host cell entry driven by pseudotypes bearing glycoprotein (GP) of ebolavirus (EBOV) strain Mayinga from 1976 (EBOV-GP 1976) and EBOV variant Makona from 2014 (EBOV-GP 2014) is inhibited by a neutralizing antibody. Pseudotypes carrying the indicated GPs were normalized for comparable infectivity and incubated with the indicated dilutions of monoclonal antibody KZ52 for 1 hour at 37°C. Thereafter, the complexes were used for transduction of 293T cells, and luciferase activities in cell lysates were measured 72 hours after transduction. Transduction in the absence of antibody was set as 100%. The results of a single experiment performed with triplicate samples are shown and are representative of 3 independent experiments. Error bars indicate standard deviations.