Biology of ebolavirus inclusion bodies

• Applicant: Privatdozent Dr. Thomas Hoenen
• Subject Area: Virology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 554609800

Ebola virus (EBOV) inclusion bodies (IBs) are virus-induced structures in the cytoplasm of infected cells where viral RNA synthesis takes place, and they are known to incorporate host factors that are important for this process. However, many basic principles underlying IB formation and function were unclear at the onset of this research grant. In the first funding phase of this research grant we could show that EBOV IBs are liquid organelles, i.e. compartments that are not defined by delineating membranes, but rather are held together by liquid-liquid phase separation (LLPS). This is consistent with findings from a growing number of other non-segmented negative-sense RNA viruses (NNSVs). Interestingly, while for most of these viruses both the nucleoprotein and phosphoprotein are required for IB formation, in case of EBOV its nucleoprotein NP alone was sufficient for IB formation. Given that one of the main driving forces of LLPS are promiscuous low affinity interactions between intrinsically disordered regions (IDR), we initially hypothesized that the unusually high abundance of IDRs in EBOV NP (for most other NNSV, IDRs are predominantly found in the phosphoprotein and not in the nucleoprotein) might be responsible for this difference. Surprisingly, however, our work from the first funding phase showed that this is not the case, and that the IDRs in NP are most likely not required for IB formation. Given that other viral proteins are also not required for IB formation, this then suggests that host proteins (and particularly their IDRs), might be supporting the formation of EBOV IBs. This led us to assess the presence of host factors in IBs using proximity ligation. Among the host proteins that were consistently enriched in IBs, there are four proteins that are predicted to contain large IDRs. We now hypothesize that one or more of these proteins play(s) an important role in the biology of EBOV IBs, and propose to study their recruitment and their contributions to the formation and function of EBOV IB in the second funding phase of this grant. Further, based on a serendipitous finding that suggests an important function of NP phosphorylation for IB formation and/or function, and earlier observations of IBs during mitosis which showed dramatic formation/dissolution events, we will also assess the role of host kinases, and particularly cyclin-dependent kinases. These investigations will allow us to further decipher the general principles underlying IB formation, and to also investigate the basis for recruitment of host factors into IBs.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Jens Bernhard Bosse