Structural Changes In Cells Imaged by Soft X-ray Cryo-Tomography During Hepatitis C Virus Infection

Abstract

Chronic hepatitis C virus (HCV) infection causes severe liver disease in millions of humans worldwide. Pathogenesis of HCV infection is strongly driven by a deficient immune response of the host, although intersection of different aspects of the virus life cycle with cellular homeostasis is emerging as an important player in the pathogenesis and progression of the disease. Cryo soft X-ray tomography (cryo-SXT) was performed to investigate the ultrastructural alterations induced by the interference of HCV replication with cellular homeostasis. Native, whole cell, three-dimensional (3D) maps were obtained in HCV replicon-harboring cells and in a surrogate model of HCV infection. Tomograms from HCV-replicating cells show blind-ended endoplasmic reticulum tubules with pseudospherical extrusions and marked alterations of mitochondrial morphology that correlated spatially with the presence of endoplasmic reticulum alterations, suggesting a short-range influence of the viral machinery on mitochondrial homeostasis. Both mitochondrial and endoplasmic reticulum alterations could be reverted by a combination of sofosbuvir/daclatasvir, which are clinically approved direct-acting antivirals for the treatment of chronic HCV infection. In addition to providing structural insight into cellular aspects of HCV pathogenesis, our study illustrates how cryo-SXT is a powerful 3D wide-field imaging tool for the assessment and understanding of complex cellular processes in a setting of near-native whole hydrated cells. Our results also constitute a proof of concept for the use of cryo-SXT as a platform that enables determining the potential impact of candidate compounds on the ultrastructure of the cell that may assist drug development at a preclinical level.

Characterization of transfer function, resolution and depth of field of a soft X-ray microscope applied to tomography enhancement by Wiener deconvolution

“…Most work performed on SXT microscopes addresses samples a few microns thick [4,[12][13][14][15][16][17][18]. Depending on the ratio between the sample thickness and the DOF, standard reconstruction algorithms introduce different artifacts which can be better estimated once the experimental DOF is known [19].…”

Encapsulation, Visualization and Expression of Genes with Biomimetically Mineralized Zeolitic Imidazolate Framework‐8 (ZIF‐8)

“…Cellular uptake of plGFP@ZIF‐8 was investigated by transfecting the biocomposites into mammalian cells. Internalization of plGFP@ZIF‐8 particles were visualized at 24 h using correlative cryo‐epifluorescence microscopy and soft X‐ray cryo‐tomography (cryo‐SXT), the only modality that allows imaging of whole cell samples in their native state without any chemical treatment or sectioning . Gradual protein expression as a green fluorescence in the cells could be recorded at 96 h posttreatment.…”

Quantitative three-dimensional nondestructive imaging of whole anaerobic ammonium-oxidizing bacteria

“…Thus, unlike other imaging methods, synchrotron soft X-ray nano-CT provides a unique opportunity for imaging intact anammox cells in the in-situ state. The synchrotron soft X-ray imaging technique was successfully applied to the 3D mapping of eukaryotic cells, such as the 3D reconstruction of saccharomyces organelles (26), imaging of the structure of hepatitis C virus (HCV) replicon-harboring cells (28), and research of superparamagnetic iron oxide nanoparticle reactions with a breast cancer cell (29). However, to date, the synchrotron soft X-ray nano-CT technique has not been applied to anammox cells; therefore, this topic requires investigation.…”