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dc.contributor.advisorKracht, Michael
dc.contributor.advisorHerker, Eva
dc.contributor.advisorEwers, Christa
dc.contributor.advisorWeber, Friedemann
dc.contributor.authorAlbert, Benadict Vincent
dc.date.accessioned2023-11-29T16:47:16Z
dc.date.available2023-11-29T16:47:16Z
dc.date.issued2023
dc.identifier.urihttps://jlupub.ub.uni-giessen.de//handle/jlupub/18721
dc.identifier.urihttp://dx.doi.org/10.22029/jlupub-18085
dc.description.abstractHuman coronaviruses frequently cause respiratory tract infections ranging from mild upper respiratory illness to severe acute respiratory distress syndrome and death. For example, the global spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has triggered a public health and economic crisis. Although the development of mRNA vaccines and antiviral drugs has helped reduce the extent and severity of SARS-CoV-2 infections, vaccines and drugs targeting viral proteins have limitations in terms of potential drug resistance or side effects and a limited number of viral targets. CoV replicate in infected host cells using a variety of cellular factors for their replication. Therefore, it is of great importance to identify the set of human genes required and manipulated by coronaviruses for propagation and to elucidate their function in the viral replication cycle. In this dissertation, the complex relationship between human coronaviruses and the essential host factors responsible for viral replication and cell survival was systematically investigated using modern genome-wide sequencing techniques and loss-of-function approaches. Focusing on human coronavirus 229E (HCoV-229E) and Middle East respiratory syndrome coronavirus (MERS-CoV), CRISPR-Cas9-based genetic tools were used to identify specific host factors and investigate their functional relevance. In addition, the antiviral effects of thapsigargin, a small molecule activator of an ER stress response and effective inhibitor of coronavirus replication, were investigated using genetic screening methods. Initially, we investigated the hypothesis that host cell genes particularly strongly induced by CoV play a critical role in coronavirus replication and their disruption could represent a potential target for antiviral intervention. From a genome-wide transcriptome analysis of Huh7 cells infected with HCoV-229E, 11 genes induced by HCoV-229E were selected and characterized in more detail. Here, subsequent knockouts of ANKRD1, EDEM1, KLF6, and FICD did not significantly affect viral replication and the host cell transcriptome. To identify essential host factors in an expanded, genome-wide approach, an unbiased, CRISPR-Cas9-based knockout screening system was employed, which led to the identification of ANPEP, the cellular receptor for HCoV-229E, as the strongest hit. Similarly, the cellular surface proteins DPP4 and TMPRSS2 were identified as the strongest hits in additional screens of MERS-CoV-infected cells. Validation experiments on the functional role of host factors in viral release and cell viability after infection revealed that the absence of host factors SFTA2, HDAC4, and ZDHHC3 (which emerged as significant hits from HCoV-229E selection) each slightly impaired viral release during HCoV-229E infection, providing new insights into the complex mechanisms employed by CoV to ensure successful replication. In the final part of this work, genetic screens identified over 1000 host factors as significant hits in thapsigargin-treated and / or HCoV-229E-infected cells. For functional follow-up, the intersection of this group of genes with significantly deregulated proteins from comparable studies of the nascent proteome was determined and 40 thapsigargin-specific factors were identified. Individual knockouts of ARFGAP3, EMC6, and CLU each increased the cell viability of thapsigargin-treated infected cells, and the absence of SEC24A reduced the long-term antiviral effect of thapsigargin on HCoV-229E replication. In summary, this work provides valuable insights into specific host factors involved in antiviral effects and CoV-induced cell death and points to potential targets of antiviral agents. Based on these findings, the molecular mechanisms involved in the underlying host-virus interactions and their applicability to suppress future emerging CoV infections can be further investigated.de_DE
dc.language.isoende_DE
dc.rightsAttribution 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/*
dc.subject.ddcddc:570de_DE
dc.subject.ddcddc:610de_DE
dc.titleIdentification and validation of the human genes that support human coronavirus infection using functional genomics approachesde_DE
dc.typedoctoralThesisde_DE
dcterms.dateAccepted2023-11-13
local.affiliationFB 11 - Medizinde_DE
thesis.levelthesis.doctoralde_DE


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