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

Abstract

Ebolaviruses have a surface glycoprotein (GP 1,2 ) that is required for virus attachment and entry into cells. Mutations affecting GP 1,2 functions can alter virus growth properties. We generated a recombinant vesicular stomatitis virus encoding Ebola virus Makona variant GP 1,2 (rVSV-MAK-GP) and observed emergence of a T544I mutation in the Makona GP 1,2 gene during tissue culture passage in certain cell lines. The T544I mutation emerged within two passages when VSV-MAK-GP was grown on Vero E6, Vero, and BS-C-1 cells but not when it was passaged on Huh7 and HepG2 cells. The mutation led to a marked increase in virus growth kinetics and conferred a robust growth advantage over wild-type rVSV-MAK-GP on Vero E6 cells. Analysis of complete viral genomes collected from patients in western Africa indicated that this mutation was not found in Ebola virus clinical samples. However, we observed the emergence of T544I during serial passage of various Ebola Makona isolates on Vero E6 cells. Three independent isolates showed emergence of T544I from undetectable levels in nonpassaged virus or virus passaged once to frequencies of greater than 60% within a single passage, consistent with it being a tissue culture adaptation. Intriguingly, T544I is not found in any Sudan, Bundibugyo, or Tai Forest ebolavirus sequences. Furthermore, T544I did not emerge when we serially passaged recombinant VSV encoding GP 1,2 from these ebolaviruses. This report provides experimental evidence that the spontaneous mutation T544I is a tissue culture adaptation in certain cell lines and that it may be unique for the species Zaire ebolavirus. IMPORTANCE The Ebola virus (Zaire) species is the most lethal species of all ebolaviruses in terms of mortality rate and number of deaths. Understanding how the Ebola virus surface glycoprotein functions to facilitate entry in cells is an area of intense research. Recently, three groups independently identified a polymorphism in the Ebola glycoprotein (I544) that enhanced virus entry, but they did not agree in their conclusions regarding its impact on pathogenesis. Our findings here address the origins of this polymorphism and provide experimental evidence showing that it is the result of a spontaneous mutation (T544I) specific to tissue culture conditions, suggesting that it has no role in pathogenesis. We further show that this mutation may be unique to the species Zaire ebolavirus, as it does not occur in Sudan, Bundibugyo, and Tai Forest ebolaviruses. Understanding the mechanism behind this mutation can provide insight into functional differences that exist in culture conditions and among ebolavirus glycoproteins.

Growth-Adaptive Mutations in the Ebola Virus Makona Glycoprotein Alter Different Steps in the Virus Entry Pathway

“…We analyzed over 1,500 EBOV sequences deposited in the NCBI database, including EBOV genomic sequences from clinical samples and more than a dozen sequences from laboratory-grown stocks. We have previously noted that the T544I mutation often arises in laboratory-passaged virus (25). Our analysis showed that, in addition to this mutation, the D552N mutation that spontaneously arose in our pseudotype was also found in five laboratory-passaged EBOV sequences (Table 3).…”

“…Emergence of VSV-MAK-GP clones containing single amino acid substitutions in the Makona glycoprotein during virus recovery. Analysis of recombinant VSVs (rVSVs) expressing EBOV glycoproteins has shown that growth in cell culture can lead to the selection of growth-adaptive GP mutations (19,25). Following recovery of a recombinant VSV encoding the glycoprotein (GP) from a clinically isolated Ebola Makona virus (VSV-MAK-GP), we assessed the extent of mutational pressure on the glycoprotein during early recovery and passage of the virus.…”

“…A striking aspect of our findings was the isolation of five different mutations that are within the fusion loop region of GP2, each providing a growth advantage. Of these five, the T544I mutation has been extensively discussed elsewhere (25). Two of the remaining four mutations in FL (D522E and D552N) occur spontaneously in TC-adapted EBOV.…”

“…Other tissue culture mutations in EBOV GP 1,2 that alter key aspects of GP function have been identified. These include mutation within the FL of EBOV GP that changes a threonine at 544 to an isoleucine (24,25). The T544I mutation occurs at a critical residue in the FL (18) and suggests that the amino acids present in the FL of clinical isolates of EBOV GP do not provide optimal entry into Vero cells.…”

A Rare Deletion in SARS-CoV-2 ORF6 Dramatically Alters the Predicted Three-Dimensional Structure of the Resultant Protein

“…Development of spontaneous mutation due to virus adaptation to both Vero and Vero E6 cells has been reported for viruses such as Ebolavirus, sometimes with altered pathogenesis. 26,27 Similar growth adaptative mutations have also been reported with Zika virus that led to attenuation of virulence in mice. 28 If the intent is to study the wild-type virus for basic or translational research studies, it may be useful to consider whether a more appropriate cell line could be used for SARS-CoV-2 propagation to ensure that the integrity of its native characteristics are maintained.…”