title: Innate Immunity to Ebola virus
title: Angeborene Immunität gegen das Ebola-virus
titleType: AlternativeTitle
Creators: name: Wildemann, Johanna
Publisher: name: Philipps-Universität Marburg
Year: 2022
Subjects: VLPs
DI-RNA
angeborene Immunität
MAVS
RNA-editierung
ebola virus
innate immunity
ADAR1
RNA
Immunantwort
Ebola-virus
RIG-I
Interferon
interferon
sensing
RNA-editing
Medical sciences Medicine
Medizin

description: 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.