Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress

Abstract

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events – e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir

“…To date, little is known about the SARS-CoV-2 egress. SARS-CoV-2 may use the multivesicular body (MVB)-like structure for packaging virions that will be released to the extracellular space via exocytosis [ 28 , 40 , 41 , 42 , 43 , 44 ]. Alternatively, SARS-CoV-2 may release from infected cells via unconventional egress by lysosomal exocytosis instead of the conventional biosynthetic secretory pathway [ 42 , 45 ], by which it will likely be released as microvesicles [ 28 , 43 ].…”

Imaging of Virus-Infected Cells with Soft X-ray Tomography

“…In this study, the morphology of >500 mitochondria (obtained from ~10 whole-cell volumes) was characterized for each sample type (control cells, non-treated and treated) [22]. Other examples of quantitative cryo-SXT studies include the measurement of the average volumes of the nuclei at different times of HSV-1 infection [45], the determination of the number of mitochondria in SARS-CoV-2-infected cells [43] and the statistical characterization of the 3D configuration of centrioles in human primary CD4 T cells [67].…”

“…Cryo-SXT can provide understanding of the spatial organization of the different cellular organelles during the infection, but to achieve a better comprehension of the complexity of virus-host interactions, it is recommended to combine cryo-SXT data with other imaging techniques that can provide the precise localization or the higher resolution structure of the particular viral or host factors being studied. Correlative workflows combining cryo-SXT with techniques such as cryo-ET, visible light fluorescence microscopy, hard X-ray fluorescence tomography or numerical simulations have already proven their potential in several multi-length scale structural studies [24,[43][44][45][46][75][76][77]. A particularly powerful approach is a correlative cryo-imaging pipeline, in which the same sample is imaged with super-resolution 3D structured illumination fluorescence microscopy (cryo-SIM) and with cryo-SXT, allowing for a comprehensive view of both cellular ultrastructure and the related molecular organization over extended cellular volumes [78,79].…”

“…Several virus-related studies have been performed with different soft X-ray microscopes worldwide (e.g., [43][44][45][46][47][48]). In this section, three such applications performed at the MISTRAL beamline are described, to illustrate the potential of cryo-SXT for virology research.…”

Current Understanding of the Innate Control of Toll-like Receptors in Response to SARS-CoV-2 Infection

“…Two major ORFs, ORF1a and ORF1b, encode pp1a and pp1b polyproteins that are proteolytically cleaved into 16 nonstructural proteins (nsps) [15,31]. The nsps compose the viral replication and transcription complex (RTC), and nsp12, the RNA-dependent RNA polymerase, synthesizes viral RNAs, including genomic RNA and subgenomic (sg) RNA, in double-membrane vesicles (DMVs) in the perinuclear region [29,[32][33][34]. The other ORFs encode structural proteins S, E, M, and N, as well as accessory proteins [31].…”