Charakterisierung der Interaktion der Coronaviren SARS-CoV sowie SARS-CoV-2 mit dem IRE1α-abhängigen Signalweg der Unfolded Protein Response
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Abstract
SARS-CoV and SARS-CoV-2 belong to the family of Coronaviridae and the species Betacoronavirus Pandemicum. Both viruses cause severe respiratory infections in humans. While SARS-CoV is the causative agent of the SARS-epidemic 2002/2003, SARS-CoV-2 caused a pandemic in 2020 lasting multiple years. Due to the pathogenicity of both viruses and their pandemic potential, a further characterisation of the interactions of both viruses with host cells is necessary to identify new potential targets for therapeutics.
The unfolded protein response is a cellular response to stress within the endoplasmic reticulum (ER), which is composed of three signaling pathways. In the present study, the most highly conserved signaling pathway of the UPR, the inositol-requiring enzyme 1 (IRE1α)-dependent signaling pathway, was analyzed. When this pathway is activated, IRE1α first oligomerizes and then performs different functions. The main function is to splice the X-box binding protein 1 (XBP1)-mRNA. The XBP1u (unspliced)-mRNA is spliced by IRE1α into XBP1s (spliced)-mRNA, from which the active transcription factor XBP1s is translated. XBP1s translocates into the nucleus to regulate the expression of various genes by binding to e.g. unfolded protein response element (UPRE) promoters. Furthermore, the UPR can also be chemically induced, e.g. by thapsigargin (Tg). Another function of IRE1α is the phosphorylation of c-Jun-N-terminal kinases (JNK). Activation of IRE1α can play a protective role and restore ER homeostasis, but strong activation can induce cell apoptosis via JNK. The UPR can therefore also play a decisive role in the context of viral infections.
In this study, I could show that infection of HEK293/ACE2 cells with both viruses leads to activation of the IRE1α-dependent signaling pathway (oligomerization of IRE1α, splicing of XBP1u-mRNA, upregulation of target genes), these effects were more pronounced after SARS CoV than after SARS-CoV-2 infection. Furthermore, I could show that JNK is phosphorylated in SARS-CoV- and SARS-CoV-2-infected cells. The use of the IRE1α activator IXA4, but also the inhibitor IRE1α, reduced the amount of phosphorylated JNK in infected cells. A clear dependency of JNK-phosphorylation could therefore not be shown, further analyses are therefore necessary.
Further studies investigated which viral proteins are involved in the activation of the IRE1α dependent signaling pathway and whether there are differences between SARS-CoV and SARS CoV-2. The viral proteins S, E, N, ORF7a and ORF7b were analyzed. Expression of the S and ORF7a proteins of both viruses led to complete activation of the IRE1α-dependent pathway, whereas the N proteins of both viruses had no activating effects. For the ORF7b proteins, the induction of IRE1α oligomerization, XBP1u-mRNA splicing and of one target gene could be observed. Activation of the UPRE-promoter was only detected in presence of SARS-CoV-2 HA ORF7b. While oligomerization of IRE1α was detected in presence of both E-proteins, only the expression of SARS-CoV-2 Flag-E led to enhanced splicing of XBP1u-mRNA and the upregulation of target genes. To further investigate the effects of both proteins, studies were performed using SARS-CoV Flag-E mutants which were cloned by in-vitro mutagenesis. Both E proteins only differ in four amino acids. The respective positions were mutated in the SARS-CoV Flag-E construct to the SARS-CoV-2 Flag-E phenotype. Here, only a deletion of Glycin at position 70 led to enhanced splicing of XBP1u. At the same time, the mutant showed higher expression levels, which might be responsible for the observed effect. To further investigate this, different concentrations of the E-proteins were tested in the XBP1 assay. Here, higher amounts of the SARS-CoV Flag-E protein did not induce an enhanced activation of XBP1, but identical expression levels of SARS-CoV- and SARS-CoV-2 Flag-E could not be achieved.
Since I could show that infection with both viruses led to activation of the IRE1α-dependent signaling pathway ad phosphorylation of JNK, a possible relevance of this activation for viral replication was investigated. Therefore, the IRE1α-inhibitor KIRA8 and the IRE1α-activator IXA4 as well as the JNK-inhibitor SP600125 were analyzed for their cytotoxicity and functionality and used in respective concentrations. An activation of IRE1α using IXA4 as well as an inhibition using KIRA8 and an inhibition of JNK using SP600125 led to a reduction of viral titers in HEK293/ACE 2 cells, whereas in Calu3-cells, only IXA4 led to a reduction of SARS-CoV titers. I could show that both cell lines react differently to ER-stress and differ in their interferon (IFN)-response, which could have an influence on the observed effects. In Calu3-cells, but not in HEK293/ACE-2 cells, I could show an upregulation of Erdj4 upon IFN-β treatment. Furthermore, IFN-λ was well as an upregulation of IFN or IFN-stimulated genes upon Tg treatment was only detected in Calu3-cells.
The data obtained in this study show that both viruses activate the IRE1α-dependent signaling pathway. Since both an inhibitor and activator of this pathway negatively impact viral replication, a balanced activation seems to be relevant for efficient replication of both viruses, highlighting the relevance as a potential therapeutic target.
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Issued: 2026-02-20
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FB20:Medizin
Language
de
Keywords
UPRSARS-CoVSARS-CoV-2IRE1ER-Stress
DFG-subjects
2.21-04 - Virologie
Funding
Funding Organisations:
Jürgen-Manchot-Stiftung
Project Information:Promotionsstipendium
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Pfeiffer, Sebastian: Charakterisierung der Interaktion der Coronaviren SARS-CoV sowie SARS-CoV-2 mit dem IRE1α-abhängigen Signalweg der Unfolded Protein Response. : 2026-02-20. DOI: https://doi.org/10.17192/openumr/649.
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Except where otherwised noted, this item's license is described as Attribution-NonCommercial-NoDerivatives 4.0 International