Multiscale Electron Microscopy for the Study of Viral Replication Organelles.

During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human pathogens like coronaviruses, enteroviruses, or flaviviruses. From their discovery to date, progress in our understanding of viral ROs has closely followed new developments in electron microscopy (EM). This review gives a chronological account of this progress and an introduction to the different EM techniques that enabled it. With an ample repertoire of imaging modalities, EM is nowadays a versatile technique that provides structural and functional information at a wide range of scales. Together with well-established approaches like electron tomography or labeling methods, we examine more recent developments, such as volume scanning electron microscopy (SEM) and in situ cryotomography, which are only beginning to be applied to the study of viral ROs. We also highlight the first cryotomography analyses of viral ROs, which have led to the discovery of macromolecular complexes that may serve as RO channels that control the export of newly-made viral RNA. These studies are key first steps towards elucidating the macromolecular complexity of viral ROs.

Coronavirus infection induces progressive restructuring of the endoplasmic reticulum involving the formation and degradation of double membrane vesicles.

Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection.

Return of the Neurotropic Enteroviruses: Co-Opting Cellular Pathways for Infection.

Enteroviruses are among the most common human infectious agents. While infections are often mild, the severe neuropathogenesis associated with recent outbreaks of emerging non-polio enteroviruses, such as EV-A71 and EV-D68, highlights their continuing threat to public health. In recent years, our understanding of how non-polio enteroviruses co-opt cellular pathways has greatly increased, revealing intricate host-virus relationships. In this review, we focus on newly identified mechanisms by which enteroviruses hijack the cellular machinery to promote their replication and spread, and address their potential for the development of host-directed therapeutics. Specifically, we discuss newly identified cellular receptors and their contribution to neurotropism and spread, host factors required for viral entry and replication, and recent insights into lipid acquisition and replication organelle biogenesis. The comprehensive knowledge of common cellular pathways required by enteroviruses could expose vulnerabilities amenable for host-directed therapeutics against a broad spectrum of enteroviruses. Since this will likely include newly arising strains, it will better prepare us for future epidemics. Moreover, identifying host proteins specific to neurovirulent strains may allow us to better understand factors contributing to the neurotropism of these viruses.

Ultrastructural modifications induced by SARS-CoV-2 in Vero cells: a kinetic analysis of viral factory formation, viral particle morphogenesis and virion release.

Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early infection events were characterized by the presence of clusters of single-membrane vesicles and stacks of membrane containing nuclear pores called annulate lamellae (AL). A large network of host cell-derived organelles transformed into virus factories was subsequently observed in the cells. As previously described for other RNA viruses, these replication factories consisted of double-membrane vesicles (DMVs) located close to the nucleus. Viruses released at the cell surface by exocytosis harbored the typical crown of spike proteins, but viral particles without spikes were also observed in intracellular compartments, possibly reflecting incorrect assembly or a cell degradation process.

Integrative Imaging Reveals SARS-CoV-2-Induced Reshaping of Subcellular Morphologies.

Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.

Identification of putative viroplasms within banana cells infected by banana streak MY virus.

The badnavirus replication cycle is poorly understood and most knowledge is based on extrapolations from model viruses such as Cauliflower mosaic virus (CaMV). However, in contrast to CaMV, badnaviruses are thought not to produce viroplasms and therefore it has been a mystery as to where virion assembly occurs. In this study, ultrathin sections of a banana leaf infected with a badnavirus, banana streak MY virus (BSMYV), were examined by transmission electron microscopy. Electron-dense inclusion bodies (EDIBs) were sporadically distributed in parenchymatous tissues of the leaf, most commonly in the palisade and spongy mesophyll cells. These EDIBs had a characteristic structure, comprising an electron-dense core, a single, encircling lacuna and an outer ring of electron-dense material. However, much less frequently, EDIBs with two or three lacunae were observed. In the outer ring, densely packed virions were visible with a shape and size consistent with that expected for badnaviruses. Immunogold labelling was done with primary antibodies that detected the N-terminus of the capsid protein and strong labelling of the outer ring but not the central core or lacuna was observed. It is concluded that the EDIBs that were observed are equivalent in function to the viroplasms of CaMV, although obviously different in composition as there is not a paralogue of the transactivation/viroplasm protein in the badnavirus genome. It is postulated that production of a viroplasm could be a conserved characteristic of all members of the Caulimoviridae.