Filovirus receptor NPC1 contributes to species-specific patterns of ebolavirus susceptibility in bats

Biological factors that influence the host range and spillover of Ebola virus (EBOV) and other filoviruses remain enigmatic. While filoviruses infect diverse mammalian cell lines, we report that cells from African straw-colored fruit bats (Eidolon helvum) are refractory to EBOV infection. This could be explained by a single amino acid change in the filovirus receptor, NPC1, which greatly reduces the affinity of EBOV-NPC1 interaction. We found signatures of positive selection in bat NPC1 concentrated at the virus-receptor interface, with the strongest signal at the same residue that controls EBOV infection in Eidolon helvum cells. Our work identifies NPC1 as a genetic determinant of filovirus susceptibility in bats, and suggests that some NPC1 variations reflect host adaptations to reduce filovirus replication and virulence. A single viral mutation afforded escape from receptor control, revealing a pathway for compensatory viral evolution and a potential avenue for expansion of filovirus host range in nature.

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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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Analysis of Resistance of Ebola Virus Glycoprotein-Driven Entry Against MDL28170, An Inhibitor of Cysteine Cathepsins

Pathogens ◽

10.3390/pathogens8040192 ◽

2019 ◽

Vol 8 (4) ◽

pp. 192 ◽

Cited By ~ 3

Author(s):

Markus Hoffmann ◽

Svenja Victoria Kaufmann ◽

Carina Fischer ◽

Wiebke Maurer ◽

Anna-Sophie Moldenhauer ◽

...

Keyword(s):

Viral Entry ◽

Ebola Virus ◽

Mutational Analysis ◽

Cathepsin L ◽

Cysteine Proteases ◽

Host Cells ◽

Single Amino Acid ◽

Limited Information ◽

Cysteine Cathepsins ◽

Ebov Infection

Ebola virus (EBOV) infection can cause severe and frequently fatal disease in human patients. The EBOV glycoprotein (GP) mediates viral entry into host cells. For this, GP depends on priming by the pH-dependent endolysosomal cysteine proteases cathepsin B (CatB) and, to a lesser degree, cathepsin L (CatL), at least in most cell culture systems. However, there is limited information on whether and how EBOV-GP can acquire resistance to CatB/L inhibitors. Here, we addressed this question using replication-competent vesicular stomatitis virus bearing EBOV-GP. Five passages of this virus in the presence of the CatB/CatL inhibitor MDL28170 were sufficient to select resistant viral variants and sequencing revealed that all GP sequences contained a V37A mutation, which, in the context of native GP, is located in the base of the GP surface unit. In addition, some GP sequences harbored mutation S195R in the receptor-binding domain. Finally, mutational analysis demonstrated that V37A but not S195R conferred resistance against MDL28170 and other CatB/CatL inhibitors. Collectively, a single amino acid substitution in GP is sufficient to confer resistance against CatB/CatL inhibitors, suggesting that usage of CatB/CatL inhibitors for antiviral therapy may rapidly select for resistant viral variants.

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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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Species-Specific Variation in the B30.2(SPRY) Domain of TRIM5α Determines the Potency of Human Immunodeficiency Virus Restriction

Journal of Virology ◽

10.1128/jvi.79.5.3139-3145.2005 ◽

2005 ◽

Vol 79 (5) ◽

pp. 3139-3145 ◽

Cited By ~ 242

Author(s):

Matthew Stremlau ◽

Michel Perron ◽

Sohanya Welikala ◽

Joseph Sodroski

Keyword(s):

Human Immunodeficiency Virus ◽

Single Amino Acid ◽

Wild Type ◽

Old World ◽

Spry Domain ◽

Single Amino Acid Change ◽

Immunodeficiency Virus ◽

Specific Variation ◽

Species Specific ◽

Hiv 1

ABSTRACT Retroviruses encounter dominant postentry restrictions in cells of particular species. Human immunodeficiency virus type 1 (HIV-1) is blocked in the cells of Old World monkeys by TRIM5α, a tripartite motif (TRIM) protein composed of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. Rhesus monkey TRIM5α (TRIM5αrh) more potently blocks HIV-1 infection than human TRIM5α (TRIM5αhu). Here, by studying chimeric TRIM5α proteins, we demonstrate that the major determinant of anti-HIV-1 potency is the B30.2(SPRY) domain. Analysis of species-specific variation in TRIM5α has identified three variable regions (v1, v2, and v3) within the B30.2 domain. The TRIM5α proteins of Old World primates exhibit expansion, duplication, and residue variation specifically in the v1 region. Replacement of three amino acids in the N terminus of the TRIM5αhu B30.2 v1 region with the corresponding TRIM5αrh residues resulted in a TRIM5α molecule that restricted HIV-1 nearly as efficiently as wild-type TRIM5αrh. Surprisingly, a single-amino-acid change in this region of TRIM5αhu allowed potent restriction of simian immunodeficiency virus, a phenotype not observed for either wild-type TRIM5αhu or TRIM5αrh. Some of the chimeric TRIM5α proteins that are >98% identical to the human protein yet mediate a strong restriction of HIV-1 infection may have therapeutic utility. These observations implicate the v1 variable region of the B30.2(SPRY) domain in TRIM5αrh antiviral potency.

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The host-range tdCE phenotype of Chandipura virus is determined by mutations in the polymerase gene

Journal of General Virology ◽

10.1099/vir.0.059204-0 ◽

2014 ◽

Vol 95 (1) ◽

pp. 38-43

Author(s):

Emily L. Stock ◽

Anthony C. Marriott ◽

Andrew J. Easton

Keyword(s):

Host Range ◽

Mammalian Cells ◽

Reverse Genetics ◽

Single Amino Acid ◽

Permissive Temperature ◽

Dependent Manner ◽

Polymerase Gene ◽

Temperature Dependent ◽

Chandipura Virus ◽

Single Amino Acid Change

The emerging arbovirus Chandipura virus (CV) has been implicated in epidemics of acute encephalitis in India with high mortality rates. The isolation of temperature-dependent host-range (tdCE) mutants, which are impaired in growth at 39 °C in chick embryo (CE) cells but not in monkey cells, highlights a dependence on undetermined host factors. We have characterized three tdCE mutants, each containing one or more coding mutations in the RNA polymerase gene and two containing additional mutations in the attachment protein gene. Using reverse genetics, we showed that a single amino acid change in the virus polymerase of each mutant was responsible for the host-range specificity. In CE cells at the non-permissive temperature, the discrete cytoplasmic replication complexes seen in mammalian cells or at the permissive temperature in CE cells were absent with the tdCE mutants, consistent with the tdCE lesions causing disruption of the replication complexes in a host-dependent manner.

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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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Faculty Opinions recommendation of 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.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1103572.559645 ◽

2008 ◽

Author(s):

Catharine Bosio

Keyword(s):

Expression Profiling ◽

Amino Acid Change ◽

Ebola Virus ◽

Innate Immune ◽

Single Amino Acid ◽

Whole Genome ◽

Genome Expression ◽

Single Amino Acid Change ◽

Host Innate Immune Response ◽

Whole Genome Expression

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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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