Epitopes of Naturally Acquired and Vaccine‐Induced Anti‐Ebola Virus Glycoprotein Antibodies in Single Amino Acid Resolution

ABSTRACT Ebola virus causes lethal hemorrhagic fever in humans, but currently there are no effective vaccines or antiviral compounds for this infectious disease. Passive transfer of monoclonal antibodies (MAbs) protects mice from lethal Ebola virus infection (J. A. Wilson, M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart, Science 287:1664-1666, 2000). However, the epitopes responsible for neutralization have been only partially characterized because some of the MAbs do not recognize the short synthetic peptides used for epitope mapping. To identify the amino acids recognized by neutralizing and protective antibodies, we generated a recombinant vesicular stomatitis virus (VSV) containing the Ebola virus glycoprotein-encoding gene instead of the VSV G protein-encoding gene and used it to select escape variants by growing it in the presence of a MAb (133/3.16 or 226/8.1) that neutralizes the infectivity of the virus. All three variants selected by MAb 133/3.16 contained a single amino acid substitution at amino acid position 549 in the GP2 subunit. By contrast, MAb 226/8.1 selected three different variants containing substitutions at positions 134, 194, and 199 in the GP1 subunit, suggesting that this antibody recognized a conformational epitope. Passive transfer of each of these MAbs completely protected mice from a lethal Ebola virus infection. These data indicate that neutralizing antibody cocktails for passive prophylaxis and therapy of Ebola hemorrhagic fever can reduce the possibility of the emergence of antigenic variants in infected individuals.

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Remdesivir targets a structurally analogous region of the Ebola virus and SARS-CoV-2 polymerases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2012294117 ◽

2020 ◽

Vol 117 (43) ◽

pp. 26946-26954 ◽

Cited By ~ 2

Author(s):

Michael K. Lo ◽

César G. Albariño ◽

Jason K. Perry ◽

Silvia Chang ◽

Egor P. Tchesnokov ◽

...

Keyword(s):

Amino Acid ◽

Homology Modeling ◽

Disease Control ◽

Broad Spectrum ◽

Active Site ◽

Ebola Virus ◽

Single Amino Acid ◽

Resistance Mutations ◽

Molecular Surveillance ◽

Low Level

Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention’s Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.

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Did a Single Amino Acid Change Make Ebola Virus More Virulent?

Cell ◽

10.1016/j.cell.2016.10.032 ◽

2016 ◽

Vol 167 (4) ◽

pp. 892-894 ◽

Cited By ~ 5

Author(s):

Trevor Bedford ◽

Harmit S. Malik

Keyword(s):

Amino Acid ◽

Amino Acid Change ◽

Ebola Virus ◽

Single Amino Acid ◽

Single Amino Acid Change

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Single amino acid change at position 255 in rabies virus glycoprotein decreases viral pathogenicity

The FASEB Journal ◽

10.1096/fj.201902577r ◽

2020 ◽

Vol 34 (7) ◽

pp. 9650-9663

Author(s):

Jun Luo ◽

Boyue Zhang ◽

Ziyu Lyu ◽

Yuting Wu ◽

Yue Zhang ◽

...

Keyword(s):

Amino Acid ◽

Rabies Virus ◽

Amino Acid Change ◽

Single Amino Acid ◽

Rabies Virus Glycoprotein ◽

Virus Glycoprotein ◽

Single Amino Acid Change

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Identification of two amino acid residues on Ebola virus glycoprotein 1 critical for cell entry

Virus Research ◽

10.1016/j.virusres.2006.06.002 ◽

2006 ◽

Vol 121 (2) ◽

pp. 205-214 ◽

Cited By ~ 28

Author(s):

Onesmo M. Mpanju ◽

Jonathan S. Towner ◽

Jason E. Dover ◽

Stuart T. Nichol ◽

Carolyn A. Wilson

Keyword(s):

Amino Acid ◽

Ebola Virus ◽

Cell Entry ◽

Amino Acid Residues ◽

Virus Glycoprotein

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Whole-Genome Expression Profiling Reveals That Inhibition of Host Innate Immune Response Pathways by Ebola Virus Can Be Reversed by a Single Amino Acid Change in the VP35 Protein

Journal of Virology ◽

10.1128/jvi.00215-08 ◽

2008 ◽

Vol 82 (11) ◽

pp. 5348-5358 ◽

Cited By ~ 76

Author(s):

Amy L. Hartman ◽

Ling Ling ◽

Stuart T. Nichol ◽

Martin L. Hibberd

Keyword(s):

Immune Response ◽

Amino Acid ◽

Host Cell ◽

Innate Immune Response ◽

Ebola Virus ◽

Innate Immune ◽

Single Amino Acid ◽

Cell Responses ◽

Single Amino Acid Change ◽

Whole Genome Expression

ABSTRACT Ebola hemorrhagic fever is a rapidly progressing acute febrile illness characterized by high virus replication, severe immunosuppression, and case fatalities of ca. 80%. Inhibition of phosphorylation of interferon regulatory factor 3 (IRF-3) by the Ebola VP35 protein may block the host innate immune response and play an important role in the severity of disease. We used two precisely defined reverse genetics-generated Ebola viruses to investigate global host cell responses resulting from the inhibition of IRF-3 phosphorylation. The two viruses encoded either wild-type (WT) VP35 protein (recEbo-VP35/WT) or VP35 with an arginine (R)-to-alanine (A) amino acid substitution at position 312 (recEbo-VP35/R312A) within a previously defined IRF-3 inhibitory domain. When sucrose-gradient purified virus was used for infection, host cell whole-genome expression profiling revealed striking differences in human liver cell responses to these viruses differing by a single amino acid. The inhibition of host innate immune responses by WT Ebola virus was so potent that little difference in interferon and antiviral gene expression could be discerned between cells infected with purified WT, inactivated virus, or mock-infected cells. However, infection with recEbo-VP35/R312A virus resulted in a strong innate immune response including increased expression of MDA-5, RIG-I, RANTES, MCP-1, ISG-15, ISG-54, ISG-56, ISG-60, STAT1, IRF-9, OAS, and Mx1. The clear gene expression differences were obscured if unpurified virus stocks were used to initiate infection, presumably due to soluble factors present in virus-infected cell supernatant preparations. Ebola virus VP35 protein clearly plays a pivotal role in the potent inhibition of the host innate immune responses, and the present study indicates that VP35 has a wider effect on host cell responses than previously shown. The ability to eliminate this inhibitory effect with a single amino acid change in VP35 demonstrates the critical role this protein must play in the severe aspects this highly fatal disease.

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Inhibition of IRF-3 Activation by VP35 Is Critical for the High Level of Virulence of Ebola Virus

Journal of Virology ◽

10.1128/jvi.02344-07 ◽

2008 ◽

Vol 82 (6) ◽

pp. 2699-2704 ◽

Cited By ~ 104

Author(s):

Amy L. Hartman ◽

Brian H. Bird ◽

Jonathan S. Towner ◽

Zoi-Anna Antoniadou ◽

Sherif R. Zaki ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Change ◽

Reverse Genetics ◽

Ebola Virus ◽

Single Amino Acid ◽

Wild Type Virus ◽

Hemorrhagic Disease ◽

Viral Virulence ◽

Single Amino Acid Change ◽

High Level

ABSTRACT Zaire ebolavirus causes a rapidly progressing hemorrhagic disease with high mortality. Identification of the viral virulence factors that contribute to the severity of disease induced by Ebola virus is critical for the design of therapeutics and vaccines against the disease. Given the rapidity of disease progression, virus interaction with the innate immune system early in the course of infection likely plays an important role in determining the outcome of the disease. The Ebola virus VP35 protein inhibits the activation of IRF-3, a critical transcription factor for the induction of early antiviral immunity. Previous studies revealed that a single amino acid change (R312A) in VP35 renders the protein unable to inhibit IRF-3 activation. A reverse-genetics-generated, mouse-adapted, recombinant Ebola virus that encodes the R312A mutation in VP35 was produced. We found that relative to the case for wild-type virus containing the authentic VP35 sequence, this single amino acid change in VP35 renders the virus completely attenuated in mice. Given that these viruses differ by only a single amino acid in the IRF-3 inhibitory domain of VP35, the level of alteration of virulence is remarkable and highlights the importance of VP35 for the pathogenesis of Ebola virus.

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A Single Residue in Ebola Virus Receptor NPC1 Influences Cellular Host Range in Reptiles

mSphere ◽

10.1128/msphere.00007-16 ◽

2016 ◽

Vol 1 (2) ◽

Cited By ~ 16

Author(s):

Esther Ndungo ◽

Andrew S. Herbert ◽

Matthijs Raaben ◽

Gregor Obernosterer ◽

Rohan Biswas ◽

...

Keyword(s):

Amino Acid ◽

Host Range ◽

Amino Acid Residue ◽

Ebola Virus ◽

Single Amino Acid ◽

Binding Interface ◽

Single Amino Acid Residue ◽

Ebov Infection ◽

Russell’S Viper ◽

Niemann Pick

ABSTRACT Identifying cellular factors that determine susceptibility to infection can help us understand how Ebola virus is transmitted. We asked if the EBOV receptor Niemann-Pick C1 (NPC1) could explain why reptiles are resistant to EBOV infection. We demonstrate that cells derived from the Russell’s viper are not susceptible to infection because EBOV cannot bind to viper NPC1. This resistance to infection can be mapped to a single amino acid residue in viper NPC1 that renders it unable to bind to EBOV GP. The newly solved structure of EBOV GP bound to NPC1 confirms our findings, revealing that this residue dips into the GP receptor-binding pocket and is therefore critical to the binding interface. Consequently, this otherwise well-conserved residue in vertebrate species influences the ability of reptilian NPC1 proteins to bind to EBOV GP, thereby affecting viral host range in reptilian cells. Filoviruses are the causative agents of an increasing number of disease outbreaks in human populations, including the current unprecedented Ebola virus disease (EVD) outbreak in western Africa. One obstacle to controlling these epidemics is our poor understanding of the host range of filoviruses and their natural reservoirs. Here, we investigated the role of the intracellular filovirus receptor, Niemann-Pick C1 (NPC1) as a molecular determinant of Ebola virus (EBOV) host range at the cellular level. Whereas human cells can be infected by EBOV, a cell line derived from a Russell’s viper (Daboia russellii) (VH-2) is resistant to infection in an NPC1-dependent manner. We found that VH-2 cells are resistant to EBOV infection because the Russell’s viper NPC1 ortholog bound poorly to the EBOV spike glycoprotein (GP). Analysis of panels of viper-human NPC1 chimeras and point mutants allowed us to identify a single amino acid residue in NPC1, at position 503, that bidirectionally influenced both its binding to EBOV GP and its viral receptor activity in cells. Significantly, this single residue change perturbed neither NPC1’s endosomal localization nor its housekeeping role in cellular cholesterol trafficking. Together with other recent work, these findings identify sequences in NPC1 that are important for viral receptor activity by virtue of their direct interaction with EBOV GP and suggest that they may influence filovirus host range in nature. Broader surveys of NPC1 orthologs from vertebrates may delineate additional sequence polymorphisms in this gene that control susceptibility to filovirus infection. IMPORTANCE Identifying cellular factors that determine susceptibility to infection can help us understand how Ebola virus is transmitted. We asked if the EBOV receptor Niemann-Pick C1 (NPC1) could explain why reptiles are resistant to EBOV infection. We demonstrate that cells derived from the Russell’s viper are not susceptible to infection because EBOV cannot bind to viper NPC1. This resistance to infection can be mapped to a single amino acid residue in viper NPC1 that renders it unable to bind to EBOV GP. The newly solved structure of EBOV GP bound to NPC1 confirms our findings, revealing that this residue dips into the GP receptor-binding pocket and is therefore critical to the binding interface. Consequently, this otherwise well-conserved residue in vertebrate species influences the ability of reptilian NPC1 proteins to bind to EBOV GP, thereby affecting viral host range in reptilian cells.

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