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  • Book
    Andreas Sebastian Puschnik.
    As humans we are under constant threat from viruses. They have evolved to infect our cells, hijack cellular components and machineries, and interfere with our innate immune system in order to propagate and spread. For example, they bind to cell surface molecules to enter our cells, utilize intracellular factors to make genome copies and assemble new virus particles as well as disable viral nucleic acid sensing. The identification of host factors, which facilitate infection, can provide important insights in the molecular interactions of the virus with its host, and may ultimately lead to the discovery of new antiviral targets. However, the diploid nature of the human genome has been a challenge for high-throughput interrogation using knockout genetics. The work in this thesis describes two genetic screening approaches, haploid and CRISPR-Cas screens, which have overcome this obstacle and yielded important insights into the biology of important human viruses. We describe how these techniques resulted in the identification of host components used by dengue virus and other mosquito-borne flaviviruses (including Zika, yellow fever and West Nile virus) for infection. We further show how these insights can be translated in the development of a novel, specific antiviral compound. Furthermore, we reveal a comprehensive set of host factors for hepatitis C virus, a more distantly related member in the Flaviviridae family, and show remarkable divergence in the molecular interactions with its host compared to the mosquito-borne flaviviruses. Next, we demonstrate how haploid screening led to the discovery of cell components important for Monkeypox virus, a DNA virus related to the etiological agent of smallpox. Lastly, we utilize our screening approach to study entry of the adeno-associated virus, a virus with gene therapy application, which resulted in the discovery of its universal entry receptor. Overall, this thesis demonstrates the robustness and versatility of knockout screens in the study of virus-host interactions. Genetic screens are powerful tools for the identification of critical cell components in viral infections, enabling development of therapeutic applications for current and future epidemics.