Vorschau

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Abstract

Zoonotic viruses pose a global threat to both human and veterinary health, as recently demonstrated by the emergence of SARS-CoV-2, the causative agent of the COVID-19 pandemic. In addition to airborne viruses, highly pathogenic arthropod-borne viruses have emerged globally in recent decades, essentially due to human activities, global warming, habitat destruction, and globalization. Ideally, the prevention of the emergence and spread of emerging pathogens will require approaches that target the first steps of infection and that block the release of the viral genome into the cytosol, prerequisites for productive infection. This PhD thesis work was dedicated to elucidating the entry mechanisms of two unrelated zoonotic emerging enveloped viruses, SARS-CoV-2 and Toscana virus (TOSV), at the cellular and molecular levels. TOSV is a sand fly-borne neurotropic pathogen of the family Phenuiviridae in the order Bunyavirales. TOSV is widely distributed in Mediterranean countries, where it is one of the most common causes of human meningitis during the summer. However, TOSV remains a neglected pathogen and little is known about its cell life cycle. Here, I developed sensitive, quantitative, and accurate assays involving flow cytometry, fluorimetry, and microscopy to decipher each step of the TOSV entry program, including virus binding, internalization, intracellular trafficking, and membrane fusion. Using fluorescently labeled TOSV particles, I showed that TOSV traffics along the endosomal machinery in induced pluripotent stem cell-derived human neurons and cell lines, first entering Rab5a+ early endosomes and then Rab7a+ and LAMP1+ late endosomal compartments. TOSV entry required intact late endosomes, from which acid-activated membrane fusion occurred. The pH threshold for fusion was optimal and faster at pH 5.5, but fusion also happened with prolonged pre-exposure of viral particles to the slightly acidic pH present in early endosomes. Unexpectedly for a class-II fusion virus like TOSV, the virus and other bunyaviruses remained infectious when exposed to low pH in the absence of a target membrane. In parallel, I studied the mechanism of entry of SARS-CoV-2 into various cell lines representing the tissues targeted during infection. I found that authentic SARS-CoV-2 entered the cytosol from or near the plasma membrane in a rapid, pH-independent manner when host cells expressed the trypsin-like protease TMPRSS2. In contrast, in cells lacking TMPRSS2 expression, SARS-CoV-2 entry was slower and relied on both endosome maturation and acid-dependent endolysosomal cathepsins. Pre-activation of viral particles by proteases bypassed the need for acidification and cathepsin L activity. In addition, I established a microscopy-based cell-cell fusion assay and found that proteolytic processing of S was necessary and sufficient to induce fusion, whereas acidification was not required. In conclusion, my results expand our knowledge of the entry of emerging zoonotic viruses. TOSV makes atypical use of endosomal acidity to find its way out of the endocytic machinery, whereas SARS-CoV-2 uses different cellular proteases for membrane fusion and penetration independent of acidification. While the TOSV fusion process itself is triggered by low pH, SARS-CoV-2 requires acidification only for the activity of cathepsins that activate the viral particles. Overall, our study highlights the diversity of strategies developed by viruses to subvert cellular machinery and enter host cells and may provide a basis for the development of antiviral strategies.