Role of lipid droplets in the formation of coronavirus-induced replicative organelles

• Applicant: Dr. Christin Müller-Ruttloff
• Subject Area: Virology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 530813989

Viral RNA synthesis by coronaviruses and numerous other plus-strand RNA viruses (+ssRNA viruses) occurs at virus-induced membrane structures in the cytoplasm of infected cells. These are called replicative organelles (ROs) and serve as scaffolds for the assembly of protein-RNA complexes of viral replicative enzymes and their cofactors and substrates. The formation of coronaviral ROs is poorly understood to date. However, there is increasing evidence that membrane-enclosed lipid droplets (LDs) are instrumental in their formation. LDs are dynamic cellular organelles with key functions in (i) membrane biogenesis, (ii) lipid transport, (iii) energy storage, and (iv) prevention of lipotoxicity. In our preliminary work, coronavirus-induced ROs were observed in close proximity to LDs. Moreover, increased accumulation of LDs that colocalized with virus-induced ROs was detected in coronavirus-infected lung fibroblasts, and pharmacological inhibition of LD formation resulted in a reduction in the number of viral ROs. These data support the hypothesis that LDs are involved in the formation of coronaviral ROs by providing lipids and other essential factors. Similar mechanisms and observations have previously been described for other +ssRNA viruses (poliovirus, dengue virus), increasingly supporting the idea that (some of the) mechanisms involved in the structural reorganization of +ssRNA virus-infected cells are conserved across virus families. To corroborate this hypothesis, the project proposal aims to evaluate a potential, functionally relevant LD involvement in the replication of different corona- and other nidoviruses. This will include (i) studies to assess a possible enrichment of LDs in corona- and/or nidovirus-infected cells. Furthermore, (ii) LD-associated host factors will be identified by mass spectrometric analysis of purified LDs and their potential (essential or supporting) function in coronavirus replication will be assessed by further analysis. In this context, the localization of the identified factors will also be analyzed by CLEM analysis, for example, to provide evidence for functions as binding partners between LDs and ROs. Finally, (iii) a potential direct association of LDs with ROs via membrane contact sites will be investigated and characterized in detail by cryoelectron tomography. This work will provide important new insights into the molecular mechanisms involved in the formation of ROs essential for coronavirus replication. A better understanding of these LD-RO interactions will contribute significantly to the identification of new conserved cellular and viral targets that may may be used in the development of novel therapeutic strategies against current and emerging coronaviruses.

• DFG Programme: Research Grants