Using correlative cryo-SXT and cryo-SIM imaging to study the morphology of membranous rearrangements of Hepatitis C Virus & West Nile Virus-infected cells

Recent advancements in virology have revealed a common feature among positive-strand viruses: the alteration of cellular membranes to form replication complexes (Paul, D. et al. 2013). Despite variations in origin and structure, these membranous compartments, classified as double-membrane vesicles (DMVs) or invaginated vesicles (IVs), are observed across diverse viruses, including hepatitis C virus (HCV) and SARS coronavirus (Castro, V. et al. 2023; Romero-Brey, I. et al. 2012; Perez-Berna, A. et al. 2021). Notably, West Nile virus (WNV) generates IVs, suggesting a conserved strategy across distant viruses (Romero-Brey, I. et al. 2012).

In this study we have performed cryo correlative Light-soft X-ray Tomography (CLXT) in the water window photon energy range to investigate in whole, unstained cells, the morphology of the membranous rearrangements induced by WNV infection.

Our CLXT results on human epithelial cells (HuH-7) infected with a fluorescent virus reporter allowed us localizing the membranous replication complexes associated with WNV. The morphology of the membranous WNV factory entails a WN-induced membrane structures and endoplasmic reticulum form a continuous intracellular membrane network which is delimited by a cytosketal cage. We discovered also an enlargement and reshaping of the mitochondria caused by WNV infection compared to non-infected cells, characterized by an increase in area but not in volume. This difference is statistically significant, with a P value of 0.0029.

In summary, our research aims to advance our understanding of viral pathogenesis by elucidating the 3D spatial organization of membranous replication complexes and their interactions with host organelles in close-to-native conditions.