viruses Review Imaging of Virus-Infected Cells with Soft X-ray Tomography Damià Garriga 1 , Francisco Javier Chichón 2 ,Bárbara M. Calisto 1 , Diego S. Ferrero 3 , Pablo Gastaminza 2 , Eva Pereiro 1 and Ana Joaquina Pérez-Berna 1, *   Citation: Garriga, D.; Chichón, F.J.; Calisto, B.M.; Ferrero, D.S.; Gastaminza, P.; Pereiro, E.; Pérez-Berna, A.J. Imaging of Virus-Infected Cells with Soft X-ray Tomography. Viruses 2021, 13, 2109. https://doi.org/10.3390/v13112109 Academic Editor: Fasséli Coulibaly Received: 2 August 2021 Accepted: 14 October 2021 Published: 20 October 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 ALBA Synchrotron Light Source, 08290 Cerdanyola del Vallès, Spain; dgarriga@cells.es (D.G.); bcalisto@cells.es (B.M.C.); epereiro@cells.es (E.P.) 2 Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain; fjchichon@cnb.csic.es (F.J.C.); pgastaminza@cnb.csic.es (P.G.) 3 Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Parc Científic de Barcelona, 08028 Barcelona, Spain; dfecri@ibmb.csic.es * Correspondence: anperez@cells.es; Tel.: +34-93-592-4371 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 sam- ples to be imaged without further thinning), its resolution (about 50 nm for tomographies, suffi- cient 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. Keywords: cryo–soft X-ray tomography (cryo-SXT); hepatitis C virus (HCV); vaccinia virus (VACV); Zika virus (ZIKV); direct-acting antiviral (DAAs) 1. Introduction Viruses occur universally, presumably infecting all cellular life from the early stages of life development on the planet [1]. For humans, viruses represent a major burden, causing dramatic losses of human life worldwide, and constitute a challenge for society, public health and the economy, as exemplified by the current SARS-CoV-2 pandemic. Despite their vast diversity, viruses share some common features, the fundamental one being that all viruses are obligate parasites, lacking metabolic mechanisms of their own to generate energy or to synthesize proteins. Due to their unavoidable interaction with the cellular environment, any proliferative virus infection of a host cell leads, to a larger or smaller degree, to alterations in the cell metabolism and/or its substructures, to build a favorable environment for viral multiplication [2,3]. In eukaryotic viruses, one of the most relevant of such alterations is the formation of viral factories or viral replication compartments. These are complex, dynamic, intracellular compartments or inclusions that arise from extensive rearrangement of host cell cytoskeletal and membrane compartments and are used by the virus as platforms for genome replication and morphogenesis. Viral factories (VFs) increase the efficiency of viral replication by concentrating the required cellular and viral materials while shielding the virus from host defenses [4–7]. Viruses 2021, 13, 2109. https://doi.org/10.3390/v13112109 https://www.mdpi.com/journal/viruses