Innate Immunity to Ebola virus
Ebola virus is a negative sense RNA virus and belongs to the family of Filoviridae. It can cause severe disease including hemorrhagic fever and multiorgan failure. Initial Ebola virus replication occurs in dendritic cells and macrophages, the sentinel cells of our immune system. However, infected...
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| Format: | Dissertation |
| Sprache: | Englisch |
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Philipps-Universität Marburg
2022
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| Zusammenfassung: | Ebola virus is a negative sense RNA virus and belongs to the family of Filoviridae. It can
cause severe disease including hemorrhagic fever and multiorgan failure. Initial Ebola
virus replication occurs in dendritic cells and macrophages, the sentinel cells of our
immune system. However, infected dendritic cells fail to orchestrate an effective
immune response. Virulence is partly associated to Ebola virus protein VP35, an
interferon-antagonist which effectively counteracts the activation of RNA receptors
from the RIG-I-like receptor pathway family. Furthermore, potential editing of viral RNA
could have pro-viral effects, avoiding interferon induction. ADAR1 is an RNA editing
enzyme, which modifies double-stranded RNA by adenosine-to-inosine editing,
essential for differentiation between self and foreign RNA. This crucial negative
regulator of the interferon response is expressed in two isoforms: the interferoninducible
p150 present in both the cytoplasm and nucleus, and the constitutively
expressed p110, which is restricted to the nucleus. Potential adenosine-to-inosine
editing of Ebola virus genomes was shown recently in different approaches and in
samples from Ebola virus disease survivors.
Therefore, the early immune response to Ebola virus seems to be crucial for disease
outcome. The aim of this thesis was to determine and analyze innate sensors and
regulators of the innate signaling pathways relevant for Ebola virus infections. The goal
was to research innate sensors of Ebola virus transcription and replication competent
virus-like particles as well as to analyze sensing of Ebola virus RNA in cell-based assays
and to investigate a link to altered innate sensing depending on the presence or absence
of ADAR1 isoforms.
The transcription and replication competent virus-like particle system allows life cycle
modeling of Ebola virus under biosafety level-1 conditions. Innate sensing was measured
by monitoring expression of the direct IRF3-target gene ISG54. Two different VP35
mutants were analyzed regarding their interferon antagonistic function as well as their
function in replication and transcription. Production of transcription and replication
competent virus-like particles including the VP35 mutants in HEK 293T cells, leads to a
strong interferon-β response compared to wildtype VP35, suggesting that mutant VP35
proteins lost their antagonistic activity compared to wildtype VP35. Nevertheless,
infection of cells with mutant VP35 virus-like particles does not lead to an immune
response. To further investigate the lack of innate response to particles infection, the
immunostimulatory potential of naked Ebola virus RNAs isolated from particles was
assessed in a quantitative assay using monocyte-derived dendritic cells and monocytederived
macrophages as a model for highly immunocompetent cells. Upon transfection
of viral nucleic acids into immunocompetent cells, high sensing inductions are observed,
suggesting that viral RNA components are sensed. To identify the particular innate
pathways that are triggered by Ebola RNA, THP-1 knock-out cell lines deficient for key
molecules of RNA and DNA sensing pathways were exposed to Ebola virus RNA. As
expected, THP-1 cells deficient in the key molecule of the RNA sensing pathway lose the
ability to trigger an immune response upon stimulation with Ebola virus RNA, suggesting
members of the RIG-I like receptor family as initial sensors. Indeed, in gene knock-down
experiments sensing of Ebola virus RNA was abrogated upon knock-down of RIG-I.
Furthermore, ADAR1 knock-out HEK 293T cells, as well as knock-out cells stably
expressing ADAR1p150, catalytically inactive ADAR1p150in, and the ADAR1p110 isoform were
generated. Ebola virus particles were produced in respective cells or wildtype cells,
followed by Ebola virus RNA extraction. Target cells were transfected with respective
Ebola virus RNA and innate sensing was measured by monitoring the expression of the
IRF-3 target gene ISG54 as well as by Western Blots for IRF-3 activation.
Here, it was shown that Ebola virus RNA extracted from particles produced in wildtype
cells induce an IRF-3-dependent response after transfection in primary myeloid cells.
Interestingly, Ebola virus RNA produced in ADAR1 knock-out cells induce a higher
immune response after transfection in A549 cells than RNA produced in wildtype cells.
This suggests ADAR1 as a negative regulator for sensing. In addition, the innate response
to particle-associated RNA stemming from cells overexpressing ADAR1p150 is strongly
diminished in comparison to RNA stemming from ADAR1 knock-out cells or cells
overexpressing the catalytically inactive form of p150 or the nuclear isoform p110. This
suggests that strong RNA editing activity by the active interferon-stimulated p150, but
not p110 influences the capacity for Ebola virus RNA sensing.
In conclusion, this work leads to a better understanding of Ebola virus-host interactions
and established ADAR1 as a pro-viral factor during Ebola virus infection and as a negative
regulator of innate sensing of Ebola virus RNA. A better understanding of the first
interactions between Ebola virus and innate regulators can help to advance therapeutic
strategies. |
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| Umfang: | 166 Seiten |
| DOI: | 10.17192/z2022.0376 |