DFG-Funding KR1143/13-1 "Regulation of virus-host membrane contact sites during replication organelle formation and endomembrane remodeling in coronavirus-infected cells"
Since 2026, our research work has been supported by the DFG-Funding KR1143/13-1 "Regulation of virus-host membrane contact sites during replication organelle formation and endomembrane remodeling in coronavirus-infected cells".
Short Summary:
Coronaviruses (CoV) rely on host cell membranes for most steps of their replication cycle. CoVs induce so-called replication organelles (ROs) that mainly originate from the endoplasmic reticulum. This process interferes with intrinsic cellular surveillance and signaling mechanisms and is incompletely understood.
The present proposal will focus on mechanisms involving membrane contact sites (MCSs). MCSs are dynamic junctions that orchestrate key processes such as signaling, membrane dynamics, and organelle biogenesis through specialized MCS tethering complexes to physically bridge two organelles.
Published and unpublished data obtained by the applicants strongly support the idea that, in CoV-infected cells, lipid-driven MCS are formed between nsp3/nsp4 (i.e., the two CoV proteins that induce RO formation) and different organelles.
Additional data imply a role of certain phosphoinositides in CoV replication, a class of modified lipids suggested to coordinate factors essential for MCS formation. Specifically, we found that pharmacological and genetic inhibition of non-canonical phosphoinositide kinases reduces the formation and growth of ROs. Collectively, these findings indicate that several layers of MCS regulation contribute to RO biogenesis.
Against this background, the two applicants will combine their complementary expertise in CoV biology and reverse genetics, molecular imaging, CRISPR-Cas9 technology and proteomics / bioinformatics to evaluate their main hypothesis according to which CoVs regulate specific phosphoinositide lipid signals as well as critical protein interactions and pathways involved in MCS formation and endomembrane remodeling.
Initially, they will combine unbiased multi-level proteomics approaches to identify MCS tethers connecting ROs and cellular organelles in a minimal nsp3/nsp4-driven RO formation system and in cells infected with genetically engineered CoV mutants.
Subsequently, they will systematically validate key factors of MCS formation by proximity-based molecular imaging and functional assays in infected cells.
In addition, the subcellular localization of selected phosphoinositides and the role of the involved (non)canonical lipid kinases in the context of MCS and RO formation will be systematically investigated.
Bioinformatic meta-analyses of the large data sets generated during this work will be employed to define canonical, non-canonical and conserved MCS landscapes in cells infected with genetically diverged CoVs with the overarching goal to gain a better understanding of virus-induced MCSs.
In the long term, this project will not only provide important new insights into how CoV remodel cell architecture to their advantage, but may also help to identify new hubs suitable for future antiviral interventions.