Characterization of Ebola Virus Inclusion Bodies Using reverse genetics
2022
Viral RNA synthesis of non-segmented negative-sense RNA viruses (NNSVs) often takes place in inclusion bodies (IBs), which for some of these viruses have been shown to have properties of liquid organelles. Such liquid organelles are formed by liquid-liquid phase separation of scaffold proteins, and often additional polymers such as RNA. It is believed that this is driven by intrinsically disordered regions (IDRs) and/or multiple copies of interaction domains (MCIDs), which for NNSVs are located in the respective phosphoproteins and nucleoproteins. For Ebola virus (EBOV), its nucleoprotein NP has been shown to be sufficient to form IBs. Using a combination of live cell microscopy, fluorescence recovery after photobleaching assays, and mutagenesis approaches together with reverse genetics-based generation of recombinant viruses we have studied the formation of EBOV IBs. Our results demonstrate that also EBOV IBs are, indeed, liquid organelles, but that the largest IDR of NP does not play a major role in their formation, whereas an NP:NP interaction domain is essential for this. Further, our data indicate that while the phosphoprotein-equivalent of EBOV is not required for IB formation, it modulates their biophysical properties, as does RNA synthesis. Given the central role of IBs in the EBOV life cycle, this insight into their formation and characteristics has the potential to provide new possibilities for countermeasure development.
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