Labelling of Ebola Virus Inclusion Body Constituents by Proximity Ligation

Inclusion body (IB) formation is one major characteristic of an Ebola virus (EBOV) infection and is essential for viral RNA synthesis since IBs are the site of replication and transcription. IBs are most likely liquid organelles formed by liquid-liquid phase separation. Standard approaches and techniques to identify protein-protein interactions, which could be leveraged to identify IB constituents, can only identify highaffinity binding partners, so they might miss recruited constituents because, in liquid organelles, low-affinity interactions occur. Therefore, this research project aimed to label EBOV IB constituents for subsequent mass spectrometry (MS) analysis by proximity ligation in transfected cells to identify such low-affinity interacting host factors. This knowledge might lead to a better understanding of which host proteins interact with viral proteins that are important for IB formation and, therefore, which host protein might contribute to the formation of IBs and, ultimately, result in new therapeutic approaches. For performing an enzyme-catalysed proximity ligation inside IBs to detect host proteins that interact with viral proteins, the two promiscuous biotinylation enzymes, TurboID or miniTurboID, were fused to EBOV viral protein 30 (VP30), which is located in IBs, since VP30 is more likely to accept such a fusion without losing its functionality. The generated VP30-TurboID and VP30-miniTurboID were characterised, and their biological functionality was tested and compared. VP30-TurboID, which showed a minimal reduction in functionality, was then used for testing and optimisation of biotinylation, which served as preparation for extracting and purifying these proteins found in near proximity of VP30-TurboID. As a last step, the samples containing purified biotin-labelled proteins were analysed via MS to identify EBOV IB constituents. Over 500 host proteins were identified among three replicates, with six overlapping in every replicate. Additionally, VP30-TurboID was cloned into the EBOV full-length genome plasmid to rescue respective recombinant EBOV, which can be used to identify EBOV IB constituents in infected cells.