In situ studies of viral replication organelles using cryo-electron tomography

Viruses can induce significant functional and morphological changes in host cells. For single-stranded positive-sense RNA (ss(+)RNA) viruses, these changes often involve extensive remodeling of host cell membranes to form viral replication organelles. These specialized organelles create a protective environment for viral RNA replication while enabling selective exchange with the cytoplasm. Despite their importance, the molecular mechanisms underlying formation and function of replication organelles remain poorly understood for most ss(+)RNA viruses. This study aimed to investigate macromolecular complexes associated with viral replication organelles using in situ cryo-electron tomography, focusing on murine norovirus (MNV) and Hepatitis C virus (HCV) as model systems. In human hepatoma Huh7.5 cells, a fluorescently labeled HCV-derived subgenomic replicon induced the formation of fluorescent foci, which corresponded to regions containing doublemembrane vesicles (DMVs) in reconstructed 3D tomograms. These DMVs likely represented HCV replication organelles. However, no distinct electron densities indicative of DMV-associated macromolecular complexes were detected. In murine microglial BV2 cells infected with MNV, 3D tomograms revealed a high abundance of singlemembrane vesicles (SMVs), potentially representing MNV replication organelles. These SMVs were often filled with filamentous electron densities but lacked distinct features indicative of macromolecular complexes. MNV particles, appearing either "empty" or "filled," frequently aligned along the cytoplasmic side of SMVs. However, subtomogram averaging of viral particles did not reveal any direct connections with SMVs, leaving the significance of their spatial association unresolved. In conclusion, no electron densities indicative of macromolecular complexes could be observed in association with either HCV DMVs or MNV SMVs. / © 2025 The Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY-NC-SA 4.0). To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-sa/4.0/deed.en