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Analysis of the Cell Entry of Enveloped Viruses and Identification of Potential Intervention Strategies

dc.contributor.advisorHahn, Alexander Dr.
dc.contributor.authorHörnich, Bojan
dc.titleAnalysis of the Cell Entry of Enveloped Viruses and Identification of Potential Intervention Strategiesde
dc.contributor.refereeHahn, Alexander Dr.
dc.description.abstractengViruses are a continuous threat for the human population. They are causing tremendous damage to human health and economy. Not only zoonotic viruses, which jump from animals to humans, but also the spread of highly adapted human pathogens, results in an increase of epidemics. To be prepared for future challenges it is necessary to gain fundamental knowledge of viruses and utilize this knowledge to invent applicable counteractions. Antiviral intervention is possible at several key points of the viral replication cycle. Preventing already the entry of the virus into the host cell holds great potential for effective treatment strategies, as it completely prevents virus induced damage to the cell. We therefore investigated fundamental basics of the entry and fusion mechanism as well as potential intervention strategies of two distinct viruses, one zoonotic and recently emerged, the other one evolutionary ancient and highly adapted. First, we analyzed the viral surface glycoprotein Spike (S) of the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and compared it with the Spike of the severe acute respiratory syndrome coronavirus (SARS-CoV). Both viruses require the viral surface protein Spike, the cell surface receptor ACE2 and an activating protease to enter the cell. We were able to show that SARS-CoV-2 Spike mediated cell-cell fusion was less dependent on the protease TMPRSS2 and more on the receptor ACE2; Vice versa the SARS-CoV Spike-mediated cell-cell fusion was more dependent on TMPRSS2 and less on ACE2 expression. We could show that this observation was based on the fact that the SARS-CoV-2 Spike can be activated, in addition to members of the transmembrane protease serine subtype (TMPRSS)-family and cathepsins, by metalloproteases. The TMPRSS2-independent activation of the Spike for cell-cell fusion and the ability of forming syncytia was related to the multibasic cleavage motif present in the SARS-CoV-2 Spike. Furthermore, we identified a KRmotif in the SARS-CoV-2 Spike subunit 2 (S2) as the key site for TMPRSS2 proteolytic activation. While mutation of this site abolished any TMPRSS2-related activation, the SARS-CoV-2 Spike was still proteolytically activated by metalloproteases for cell-cell fusion and cathepsins for particle entry. In addition, we were able to identify the overthe-counter medication Ambroxol as inhibitor of SARS-CoV-2 replication in Calu-3 cells. In contrast to the entry of SARS-CoV-2, the high degree of complexity of the entry of Kaposi’s sarcoma herpesvirus (KSHV) does not allow for a simple intervention. We therefore analyzed the antiviral activity of the host cell broad-spectrum restriction factors Interferon (IFN)-inducible transmembrane proteins (IFITMs) on the gamma-2-herpesviruses KSHV and the closely related primate virus rhesus monkey rhadinovirus (RRV). We were able to show that the entry of KSHV and RRV is inhibited by IFITM1 in a cell-dependent manner. While knockout of IFITMs in cells of epithelial and fibroblast origin enhanced KSHV and RRV infection, IFITM-knockout in endothelial cells remained without effect. We could furthermore show that in epithelial and fibroblast cells, IFITM1 overexpression inhibited the KSHV and RRV infection more effectively than IFITM2 and IFITM3. By showing that all IFITMs inhibit gamma-2- herpesvirus glycoprotein-mediated cell-cell fusion and that KSHV partially evades this restriction, likely by avoiding IFITM-positive compartments, we could contribute to the understanding of the IFITM mechanism against the entry of enveloped viruses. Taken together this thesis investigated fundamental basics of SARS-CoV-2 entry and fusion as well as the impact of IFITMs on gamma-2-herpesviruses, leading to future perspectives for intervention strategies against the entry process of enveloped
dc.contributor.coRefereePöhlmann, Stefan Prof. Dr.
dc.subject.engSARS-CoV-2 entryde
dc.affiliation.instituteBiologische Fakultät für Biologie und Psychologiede
dc.subject.gokfullBiologie (PPN619462639)de

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