Structural and functional organization of Ebola virus induced cytoplasmic inclusion bodies

• Applicant: Dr. Melina Vallbracht
• Subject Area: Virology ; Structural Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 469065579

A hallmark of infections by the filovirus Ebola Virus (EBOV) and many other non-segmented negative-sense RNA viruses is the formation of membraneless viral inclusion bodies (IBs) in the cytoplasm of the infected cell. These unique and highly specialized structures are induced by viral proteins and serve as an important platform for efficient viral genome replication and transcription. As such IBs represent potential targets for the development of novel antiviral strategies. However, although IBs have been shown to play a key role in the life cycle of EBOV, there are substantial gaps in the knowledge of how these important structures are organized, both structurally and functionally. Therefore, this research propopsal aims to elucidate the previously inaccessible, three-dimensional molecular architecture and internal structural organization of ebolavirus induced cytoplasmic IBs. By applying cutting edge on-lamella cryo-correlative light (cryo-CLEM) and electron tomography (cryo-ET) techniques we will investigate IBs in their native environment inside the cell. With this research proposal we aim to address three unsolved hypotheses and questions regarding EBOV IBs. 1) Ebolavirus IBs are the venue for a variety of different processes, including viral RNA replication, transcription and nucleocapsid assembly. To orchestrate and effectively facilitate these functions within a confined space, we hypothesize that EBOV IBs contain a high degree of organization and feature sub-compartments. Using fluorescently labelled viral proteins that accumulate in IBs as well as a new, cryo-CLEM compatible method to label nascent RNA in living cells, we will uncover the spatiotemporal organization of IBs inside the host cell. These studies aim at understanding how IBs are functionally organized and will clarify if IBs undergo further compartmentalization and whether functionally different classes of IBs exist.2) A current model on EBOV replication proposes that only condensed nucleocapsids are released from IBs and transported to the budding sites. However, the assembly cascade and in particular the assembly intermediates of nucleocapsids in infected cells remain elusive. Using on-lamella cryo-ET and subvolume averaging we will investigate the different nucleocapsid assembly states inside infected cells to provide a structural perspective of the nucleocapsid maturation process. Special focus will be put on the role of VP24 and VP40 in nucleocapsid condensation. 3) While it is well established that nucleocapsids are rapidly released from IBs and move within the cytosol in an actin-dependent and directed, polar fashion, it remains unknown how and in which orientation nucleocapsids are released from IBs. Using in cellulo cryo-ET we want to address this question and clarify whether only condensed nucleocapsids are transported. Finally we want to elucidate the role of the cytoskeleton network in IB-egress.

• DFG Programme: WBP Position