A Polymorphism within the Internal Fusion Loop of the Ebola Virus Glycoprotein Modulates Host Cell Entry

Abstract

The large scale of the Ebola virus disease (EVD) outbreak in West Africa in 2013-2016 raised the question whether the host cell interactions of the responsible Ebola virus (EBOV) strain differed from those of other ebolaviruses. We previously reported that the glycoprotein (GP) of the virus circulating in West Africa in 2014 (EBOV2014) exhibited reduced ability to mediate entry into two nonhuman primate (NHP)-derived cell lines relative to the GP of EBOV1976. Here, we investigated the molecular determinants underlying the differential entry efficiency. We found that EBOV2014-GP-driven entry into diverse NHP-derived cell lines, as well as human monocyte-derived macrophages and dendritic cells, was reduced compared to EBOV1976-GP, although entry into most human-and all bat-derived cell lines tested was comparable. Moreover, EBOV2014 replication in NHP but not human cells was diminished relative to EBOV1976, suggesting that reduced cell entry translated into reduced viral spread. Mutagenic analysis of EBOV2014-GP and EBOV1976-GP revealed that an amino acid polymorphism in the receptor-binding domain, A82V, modulated entry efficiency in a cell line-independent manner and did not account for the reduced EBOV2014-GP-driven entry into NHP cells. In contrast, polymorphism T544I, located in the internal fusion loop in the GP2 subunit, was found to be responsible for the entry phenotype. These results suggest that position 544 is an important determinant of EBOV infectivity for both NHP and certain human target cells. IMPORTANCEThe Ebola virus disease outbreak in West Africa in 2013 entailed more than 10,000 deaths. The scale of the outbreak and its dramatic impact on human health raised the question whether the responsible virus was particularly adept at infecting human cells. Our study shows that an amino acid exchange, A82V, that was acquired during the epidemic and that was not observed in previously circulating viruses, increases viral entry into diverse target cells. In contrast, the epidemic virus showed a reduced ability to enter cells of nonhuman primates compared to the virus circulating in 1976, and a single amino acid exchange in the internal fusion loop of the viral glycoprotein was found to account for this phenotype.

Spontaneous Mutation at Amino Acid 544 of the Ebola Virus Glycoprotein Potentiates Virus Entry and Selection in Tissue Culture

“…Our findings are in agreement with a recent report from Hoffman et al (7). In that report, T544I conferred enhanced entry of pseudotyped viruses on Vero E6 but not on Huh7 cells.…”

“…The bulk of our virus studies were performed on Vero E6 cells and are in agreement with recent reports that T544I enhanced GP 1,2 -mediated entry in these cells (5,6,7). Our studies further show that the T544I mutation is a rapid and spontaneous mutation, likely not "carried" with T544 virus from clinical samples.…”

“…A second study suggested that the I544/T544 polymorphism might be implicated in human-to-human transmission (6). A third report suggests that the I544 residue was a tissue culture adaptation, but that study was unable to determine whether this appearance was a mutation or an outgrowth of an I544 population circulating in patients that became dominant upon growth in culture (7).…”

Recently Identified Mutations in the Ebola Virus-Makona Genome Do Not Alter Pathogenicity in Animal Models

“…This observation may be supported by the recent finding that polymorphism at 544 in GP, located in the internal fusion loop on GP2, decreased entry into monkey and certain human target cells mediated by the EBOV-Makona GP compared to the EBOV-Mayinga GP (Hoffmann et al, 2017). By contrast, increased virulence of EBOV-Makona isolates was found when compared to EBOV-Kikwit, the isolate derived from the 1995 outbreak in the Democratic Republic of Congo (Wong et al, 2016).…”

Calu-3 cells are largely resistant to entry driven by filovirus glycoproteins and the entry defect can be rescued by directed expression of DC-SIGN or cathepsin L

“…Expression plasmids encoding for the envelope glycoproteins of EBOV, BDBV, TAFV, RESTV, SUDV, MARV, LLOV, NiV, MuV, LASV, LCMV, MACV and VSV have been described previously (Hoffmann et al, 2016;Wrensch et al, 2014;Krüger et al, 2015). The plasmids required for the trVLP system (Watt et al, 2014) and VP30-luciferase containing particles have also been described before (Hoffmann et al, 2017). For the generation of CatB and CatL encoding retroviral vectors, the respective open reading frames were amplified by PCR from cDNA prepared from A549 cells and inserted into the vector pQCXIP using NotI and BamHI restriction enzymes.…”