Functional interactomes of the Ebola virus polymerase identified by proximity proteomics in the context of viral replication

TurboID is a biotin ligase optimized for biotilylation of exposed lysine residues on proximal proteins. The split TurboID is composed by sN- and sC-TurboID, leading to  be active when both of which interacts. The sN- and sC-TurboID was inserted to Ebola virus L protein and VP35 protein, and further a minigenome system using these proteins for viral genome replication and transcription was established. When the polymerase complex is active, the split TurboID can also be active. During the process of minigenome replication, the proximal labelled host factors were identified by proteomic analysis. 64 hits were selected for further validation to identify as viral replication coordinator by using siRNA screening. As the result, two functional polymerase interactors, eRF3a/GSP1 and UPF1, which are key components of the cellular nonsense-mediated mRNA decay pathway, were identified. It was shown that manipulation of these factors was associated with viral replication efficiency. Use of a compound targeting the GSP1 was shown to be effective for Ebola viral replication inhibitor.