Multimodal imaging of membranous rearrangements of West Nile Virus-infected cells

This project is the continuation of our previous proposal (2023087667, see report, and 2024098732 which beamtime has not been allocated yet), where we investigated WNV. WNV induces significant morphological changes in host cells during infection, such as convoluted membranes and vesicle packages1. Membrane rearrangements driven by viral replication are closely associated with lipid metabolism 2. Specific lipid content in membranes has been linked to achieving proper membrane fluidity, plasticity, and topology (facilitating membrane curvature) as well as promoting the recruitment of viral and cellular factors to the replication complex3. Flaviviruses exploit host-cell machinery to create a tailored lipid microenvironment for assembling their replication complex, where cholesterol plays an essential role4. Using a correlative approach combining cryo-SXT and cryo-3DSIM, we successfully identified and obtained volumetric data of several WNV infection features. We now aim to extend this approach by incorporating nanoIR and deep-UV-fluorescence to not only identify viral replication complexes but also characterize their composition and other induced features

In our previous work, we have observed notable mitochondrial alterations, specifically observing the elongation of mitochondria characterized by an increase in surface area without a corresponding increase in volume. This phenomenon suggests a significant structural adaptation within the mitochondria during infection. Additionally, we successfully identified the presence of convoluted membranes enclosed in a cytoskeletal cage induced by the WNV as illustrated in Fig1. The convoluted membranes are highlighted in red, while the surrounding cytoskeletal cage, segmented in orange, is situated in the perinuclear area, underscoring the complex interactions between viral components and host cell structures.

In addition, specific mitochondrial alterations, such as inhibition of mitochondrial fission and subsequent elongation, have been associated with West Nile virus infection3. Note that morphological membranous rearrangements induced by SARS-CoV-28 and HCV9 replicon in whole, hydrated cells, have been reported using cryo-SXT in the past.