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Ebola virus inclusion bodies are liquid organelles whose formation is facilitated by nucleoprotein oligomerization

Viral RNA synthesis of several non-segmented, negative-sense RNA viruses (NNSVs) takes place in inclusion bodies (IBs)that show properties of liquid organelles, which are formed by liquid–liquid phase separation of scaffold proteins. It isbelieved that this is driven by intrinsically disordered regions (IDRs) and/or multiple copies of interaction domains, whichfor NNSVs are usually located in their nucleo–and phosphoproteins. In contrast to other NNSVs, the Ebola virus (EBOV)nucleoprotein NP alone is sufficient to form IBs without the need for a phosphoprotein, and to facilitate the recruitmentof other viral proteins into these structures. While it has been proposed that also EBOV IBs are liquid organelles, this hasso far not been formally demonstrated. Here we used a combination of live cell microscopy,fluorescence recovery afterphotobleaching assays, and mutagenesis approaches together with reverse genetics-based generation of recombinantviruses to study the formation of EBOV IBs. Our results demonstrate that EBOV IBs are indeed liquid organelles, and thatoligomerization but not IDRs of the EBOV nucleoprotein plays a key role in their formation. Additionally, VP35 (oftenconsidered the phosphoprotein-equivalent of EBOV) is not essential for IB formation, but alters their liquid behaviour.Thesefindings define the molecular mechanism for the formation of EBOV IBs, which play a central role in the lifecycle of this deadly virus.

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