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Abstract

Viruses initiate infection of host cells by entering through a variety of different pathways. Their entry is concluded by the release of the viral genome into the cytoplasm, where the cellular machinery gets repurposed for virus replication. Prerequisite for genome release is the uncoating of the viral particles, a process which requires the destabilization of interactions established during virus assembly. Ebola viruses (EBOVs) are highly pathogenic, enveloped RNA viruses of remarkable filamentous morphology. Their shape is dictated by the viral matrix protein VP40, which forms a tubular scaffold underneath the viral envelope and confers stability to the particles during EBOV transmission. EBOVs enter host cells via the endocytic pathway and release their genome into the cytoplasm after fusion of their envelope with the endosomal membrane. The first line of defence against a viral infection is blocking viral entry, and EBOV entry has accordingly been well investigated with respect to receptor engagement and potential membrane fusion triggers. However, key mechanisms governing the final step of virus entry are still unknown, including the central question of how these unusually shaped virions undergo uncoating. Whether and how the VP40 matrix disassembles to enable membrane fusion; whether uncoating involves additional triggers; and finally, how and where the viral genome gets released from the viral particles and nucleocapsids remains to be elucidated. In this thesis, I investigate EBOV uncoating during entry into host cells and shed light on the fate of the most abundant and versatile viral protein, VP40. As a main tool, I use in situ cryo-electron tomography and provide structural insights into EBOV uncoating both in vitro and in infected host cells at molecular resolution. I discover that at low endosomal pH, the VP40 matrix detaches from the viral envelope and disassembles. This is caused by the disruption of electrostatic interactions between membrane lipids and anionic amino acids exposed on the surface of VP40 dimers, which I show are the structural units of the VP40 matrix. The strong effect of low pH on the integrity of the VP40 matrix is a consequence of acidification of the viral lumen, which I further investigate to uncover its mechanism. I show that protons diffuse passively across the viral envelope independently of a dedicated ion channel, which might be relevant for other late-penetrating viruses lacking viroporins. Finally, I provide the first high-resolution images of Ebola virions in endolysosomal compartments of infected cells. These images confirm the disassembly of the VP40 matrix in virions located in acidified compartments while clearly showing that their nucleocapsids remain intact. Together, these findings reveal that VP40 matrix disassembly is an essential step during EBOV uncoating, which precedes membrane fusion and genome release from the nucleocapsids. Overall, this thesis extends the current understanding of virus uncoating and indicates that pH-driven structural remodeling of viral matrix proteins may act as a switch coupling matrix uncoating to membrane fusion during host cell entry of enveloped viruses.