Abstract
Viruses are obligate parasites that depend on a host cell for replication and survival. Consequently, to fully understand the viral processes involved in infection and replication, it is fundamental to study them in the cellular context. Often, viral infections induce significant changes in the subcellular organization of the host cell due to the formation of viral factories, alteration of cell cytoskeleton and/or budding of newly formed particles. Accurate 3D mapping of organelle reorganization in infected cells can thus provide valuable information for both basic virus research and antiviral drug development. Among the available techniques for 3D cell imaging, cryo–soft X-ray tomography stands out for its large depth of view (allowing for 10 µm thick biological samples to be imaged without further thinning), its resolution (about 50 nm for tomographies, sufficient to detect viral particles), the minimal requirements for sample manipulation (can be used on frozen, unfixed and unstained whole cells) and the potential to be combined with other techniques (i.e., correlative fluorescence microscopy). In this review we describe the fundamentals of cryo–soft X-ray tomography, its sample requirements, its advantages and its limitations. To highlight the potential of this technique, examples of virus research performed at BL09-MISTRAL beamline in ALBA synchrotron are also presented.
Highlights
Viruses occur universally, presumably infecting all cellular life from the early stages of life development on the planet [1]
Viral factories (VFs) with rather specific morphologies, such as the tubular membrane structures that Bunyaviridae produce around the Golgi [18] or the giant volcano-shaped viral factories of the Mimivirus, which can grow to a size similar to that of the nucleus of the infected host cell [19]
These can exit the host cell by exocytosis, losing the outermost membrane bilayer, yielding the extracellular enveloped virus (EEV). Such a complex maturation process, involving multiple sets of temporal interactions between cellular membranes and organelles with viral components, takes place in extended regions of the cytoplasm of the infected cell. This makes it difficult to study with highresolution imaging techniques, mainly because the information is retrieved from a small percentage of the volume of the whole cell, preventing a quantitative analysis of the viral life cycle at the cellular level
Summary
Presumably infecting all cellular life from the early stages of life development on the planet [1]. Other viruses induce the formation of VFs with rather specific morphologies, such as the tubular membrane structures that Bunyaviridae produce around the Golgi [18] or the giant volcano-shaped viral factories of the Mimivirus, which can grow to a size similar to that of the nucleus of the infected host cell [19]. Up-to-date cell-scale imaging techniques are fundamental to characterize these virus-induced alterations in the host cell substructures and, to understand the multiple steps involved in the viral life cycle In this context, cryo–soft X-ray tomography (cryo-SXT) stands out as a powerful tool for current virology research, providing 3D information on cellular infection processes and helping to understand drug action at the cellular level [22]. Several examples of cryo-SXT research on virus-infected cells are shown
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