Multiscale Electron Microscopy for the Study of Viral Replication Organelles

Abstract

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.

Cryo-electron microscopy of nodavirus RNA replication organelles illuminates positive-strand RNA virus genome replication

“…In the first three dimensional (3D) electron microscope (EM) tomography of a (+)RNA virus RC, nodavirus RCs were revealed as clustered, ∼50 nm diameter vesicular invaginations of the OMM, each connected to the cytosol via an ∼10 nm diameter necked pore ( Figure 2 a) [ 19 • ]. While these classical 3D EM studies resolved the membrane topology of nodavirus RCs, visualizing the RNA and protein components required cryo-electron microscopy (cryo-EM), free from the limitations and artefacts of classical EM chemical fixation and heavy metal staining [ 20 , 21 , 22 ]. An early two-dimensional cryo-EM study of FHV RCs began to characterize heterogeneity in RC sizes and, building on prior indications of filamentous density inside chemically fixed RC spherules [ 18 , 23 ], showed swirls of density within RC vesicles [ 24 ].…”

Viral-Host Dependency Factors as Therapeutic Targets to Overcome Antiviral Drug-Resistance: A Focus on Innate Immune Modulation

“…Advanced imaging methodologies, including improved highresolution light microscopy, confocal microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) or cryo-EM, among others, take advantage of new optical imaging tools to perform spatio-temporal assessment of the viral replication cycle, for a better understanding of viral structures and interactions within the cell between host proteins and viral counterparts. These techniques have been extensively reviewed elsewhere (56)(57)(58)(59)(60)(61).…”

Reinventing positive-strand RNA virus reverse genetics

“…The processes of positive-strand RNA virus genome replication and transcription occur within the cytosol of infected cells, in association with membrane-bound replication organelles (ROs), which have two general morphologies ( Nguyen-Dinh and Herker, 2021 ; Unchwaniwala et al, 2021 ; Wolff and Barcena, 2021 ). Some viruses, such as flaviviruses and nodaviruses, form “spherule”-like ROs, sac-like invaginations into larger membranes that feature a neck, such that the RNA-containing spherule interior is contiguous with the cytosol.…”