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Drug repurposing is a valuable source of new antivirals because many compounds used to treat a variety of pathologies can also inhibit viral infections. In this work, we have tested the antiviral capacity of four repurposed drugs to treat Bunyamwera virus (BUNV) infection in cell cultures. BUNV is the prototype of the Bunyavirales order, a large group of RNA viruses that includes important pathogens for humans, animals and plants. Mock- and BUNV-infected Vero and HEK293T cells were treated with non-toxic concentrations of digoxin, cyclosporin A, sunitinib and chloroquine. The four drugs inhibited BUNV infection with varying potency in Vero cells, and all except sunitinib also in HEK293T cells, with digoxin rendering the lowest half maximal inhibitory concentration (IC50). Since digoxin rendered the best results, we selected this drug for a more detailed study. Digoxin is an inhibitor of the Na+/K+ ATPase, a plasma membrane enzyme responsible for the energy-dependent exchange of cytoplasmic Na+ for extracellular K+ in mammalian cells and involved in many signalling pathways. Digoxin was shown to act at an early time point after viral entry reducing the expression of the viral proteins Gc and N. Effects on the cell cycle caused by BUNV and digoxin were also analysed. In Vero cells, digoxin favoured the transition from G1 phase of the cell cycle to S phase, an effect that might contribute to the anti-BUNV effect of digoxin in this cell type. Transmission electron microscopy showed that digoxin impedes the assembly of the characteristic spherules that harbour the BUNV replication complexes and the morphogenesis of new viral particles. Both BUNV and digoxin induce similar changes in the morphology of mitochondria that become more electron-dense and have swollen cristae. The alterations of this essential organelle might be one of the factors responsible for digoxin-induced inhibition of viral infection. Digoxin did not inhibit BUNV infection in BHK-21 cells that have a digoxin-resistant Na+/K+ ATPase, which suggests that the effects of the blockade of this enzyme is a key factor of the antiviral activity of digoxin in BUNV-infected Vero cells.

The SARS-CoV-2 pandemic made evident that we count with few coronavirus-fighting drugs. Here we aimed to identify a cost-effective antiviral with broad spectrum activity and high safety and tolerability profiles. We began elaborating a list of 116 drugs previously used to treat other pathologies or characterized in pre-clinical studies with potential to treat coronavirus infections. We next employed molecular modelling tools to rank the 44 most promising inhibitors and tested their efficacy as antivirals against a panel of alpha and beta coronavirus, e.g., the HCoV-229E and SARS-CoV-2 viruses. Four drugs, OSW-1, U18666A, hydroxypropyl-beta-cyclodextrin (HbetaCD) and phytol, showed antiviral activity against both HCoV-229E (in MRC5 cells) and SARS-CoV-2 (in Vero E6 cells). The mechanism of action of these compounds was studied by transmission electron microscopy (TEM) and by testing their capacity to inhibit the entry of SARS-CoV-2 pseudoviruses in ACE2-expressing HEK-293T cells. The entry was inhibited by HbetaCD and U18666A, yet only HbetaCD could inhibit SARS-CoV-2 replication in the pulmonary cells Calu-3. With these results and given that cyclodextrins are widely used for drug encapsulation and can be safely administered to humans, we further tested 6 native and modified cyclodextrins, which confirmed β-cyclodextrins as the most potent inhibitors of SARS-CoV-2 replication in Calu-3 cells. All accumulated data points to beta-cyclodextrins as promising candidates to be used in the therapeutic treatments for SARS-CoV-2 and possibly other respiratory viruses., This work has been funded by grant RTI2018-094445-B100 (MCIU/AEI/FEDER, UE) from the Ministry of Science and Innovation of Spain (C.R.), by Palex Medical S.A., Sika S.A.U. and 7 more companies, and by Ms. Raquel Casaus Alvarez, Mr. Miguel Pardo Gil, Mr. Jacques Noguès and a total of 2,916 citizens through the Precipita crowdfunding platform of Fecyt (Fundación Española para la Ciencia y la Tecnología). NI-U is supported by the Spanish Ministry of Science and Innovation (grant PID2020-117145RB-I00), EU HORIZON-HLTH-2021-CORONA-01 (grant 101046118) and by institutional funding of Grifols, Pharma Mar, HIPRA, Amassence and Palobiofarma. This work used the computational resources of the Centro de Supercomputación de Galicia (CESGA) supported by the Partnership for Advanced Computing in Europe (PRACE) COVID-19 Fast Track Call for Proposals – Allocation Decision – Proposal COVID19-85.

Macrophages are a heterogeneous population of innate immune cells that support tissue homeostasis through their involvement in tissue development and repair, and pathogen defense. Emerging data reveal that metabolism may control macrophage polarization and function and, conversely, phenotypic polarization may drive metabolic reprogramming. Here, using biochemical analysis, correlative cryogenic fluorescence microscopy and cryo-focused ion-beam scanning electron microscopy, we demonstrate that growth hormone (GH) functions as a metabolic modulator to reprogram inflammatory GM-CSF-primed monocyte-derived macrophages (GM-MØ). We found that exogenous treatment of GM-MØ with recombinant human GH suppressed glycolysis, lactate production and non-mitochondrial respiration, and enhanced mitochondrial oxidative phosphorylation. Likewise, GH treatment augmented mitochondrial volume and altered mitochondrial dynamics, including the remodeling of the inner membrane to increase the density of cristae. Our data demonstrate that GH likely serves a modulatory role in the metabolism of inflammatory macrophages and suggest that metabolic reprogramming of macrophages should be considered a new target to intervene in multiple inflammatory diseases.

Correction to: https://doi.org/10.1038/s41423-021-00794-6; http://hdl.handle.net/10261/257710, In the version of this correspondence initially published, one of the SARS-CoV-2 variants used in Fig. 1B, which was originally described in the article as the SARS-CoV-2 variant ‘B.1.1.248.2 Gamma’, is actually the ‘P.2 Zeta’ SARS-CoV-2 variant of interest. The GISAID accession ID EPI_ISL_1831696 provided is correct, but it belongs to the Zeta variant. The results and conclusions are not affected by this unintentional inaccuracy.

The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation elongation factor 1 alpha) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work., This research was funded by Pharma Mar, which commercializes Aplidin/Plitidepsin. The authors also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com) and the CBIG consortium supported by Grifols. N.I–U. is supported by grant PID2020-117145RB-I00 from the Spanish Ministry of Science and Innovation. C.R. is supported by grant RTI2018-094445-B-I00 (MCI/AEI/FEDER, UE) from the Spanish Ministry of Science and Innovation.

The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation factor 1 alpha 1) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin completely blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work., This research was funded by Pharma Mar, which commercializes Aplidin/Plitidepsin. The authors also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com). N.I-U. is supported by grant PID2020-117145RB-I00 from the Spanish Ministry of Science and Innovation. C.R. is supported by grant RTI2018-094445-B-I00 (MCI/AEI/FEDER, UE) from the Spanish Ministry of Science and Innovation

Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-β-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles from endosomes into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection.

The function of the mammalian orthoreovirus (reovirus) σNS nonstructural protein is enigmatic. σNS is an RNA-binding protein that forms oligomers and enhances the stability of bound RNAs, but the mechanisms by which it contributes to reovirus replication are unknown. To determine the function of σNS-RNA binding in reovirus replication, we engineered σNS mutants deficient in RNA-binding capacity. We found that alanine substitutions of positively charged residues in a predicted RNA-binding domain decrease RNA-dependent oligomerization. To define steps in reovirus replication facilitated by the RNA-binding property of σNS, we established a complementation system in which wild-type or mutant forms of σNS could be tested for the capacity to overcome inhibition of σNS expression. Mutations in σNS that disrupt RNA binding also diminish viral replication and σNS distribution to viral factories. Moreover, viral mRNAs only incorporate into viral factories or factory-like structures (formed following expression of nonstructural protein μNS) when σNS is present and capable of binding RNA. Collectively, these findings indicate that σNS requires positively charged residues in a putative RNA-binding domain to recruit viral mRNAs to sites of viral replication and establish a function for σNS in reovirus replication. IMPORTANCE Viral replication requires the formation of neoorganelles in infected cells to concentrate essential viral and host components. However, for many viruses, it is unclear how these components coalesce into neoorganelles to form factories for viral replication. We discovered that two mammalian reovirus nonstructural proteins act in concert to form functioning viral factories. Reovirus μNS proteins assemble into exclusive factory scaffolds that require reovirus σNS proteins for efficient viral mRNA incorporation. Our results demonstrate a role for σNS in RNA recruitment to reovirus factories and, more broadly, show how a cytoplasmic non-membrane-enclosed factory is formed by an RNA virus. Understanding the mechanisms of viral factory formation will help identify new targets for antiviral therapeutics that disrupt assembly of these structures and inform the use of nonpathogenic viruses for biotechnological applications.

Highlights • Emerging or reemerging viruses are a major public health concern. • New, efficient, broad-spectrum antivirals are urgently needed. • Drugs that inhibit cellular pathways needed for viral replication could work for several viruses. • Drug repurposing can lead to less costly and faster approval of new treatments for viral infections. • Several drugs in the market have been proved to be potential candidates for repurposing as broad spectrum antivirals., Emerging viruses are a major threat to human health. Recent outbreaks have emphasized the urgent need for new antiviral treatments. For several pathogenic viruses, considerable efforts have focused on vaccine development. However, during epidemics infected individuals need to be treated urgently. High-throughput screening of clinically tested compounds provides a rapid means to identify undiscovered, antiviral functions for well-characterized therapeutics. Repurposed drugs can bypass part of the early cost and time needed for validation and authorization. In this review we describe recent efforts to find broad spectrum antivirals through drug repurposing. We have chosen several candidates and propose strategies to understand their mechanism of action and to determine how resistance to antivirals develops in infected cells.

Mammalian orthoreoviruses (reoviruses) are nonenveloped, double-stranded RNA viruses that replicate and assemble in cytoplasmic membranous organelles called viral inclusions (VIs). To define the cellular compartments involved in nonlytic reovirus egress, we imaged viral egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs). Electron and confocal microscopy showed that reovirus mature virions are recruited from VIs to modified lysosomes termed sorting organelles (SOs). Later in infection, membranous carriers (MCs) emerge from SOs and transport new virions to the plasma membrane for nonlytic egress. Transmission electron microscopy (TEM) combined with electron tomography (ET) and three-dimensional (3D) reconstruction revealed that these compartments are connected and form the exit pathway. Connections are established by channels through which mature virions are transported from VIs to MCs. In the last step, MCs travel across the cytoplasm and fuse with the plasma membrane, which facilitates reovirus egress. This bio-protocol describes the combination of imaging approaches (TEM, ET, and 3D reconstruction) to analyze reovirus egress zones. The spatial information present in the 3D reconstructions, along with the higher resolution relative to 2D projections, allowed us to identify components of a new nonlytic viral egress pathway., This work was supported in part by Public Health Service awards AI032539 (T.S.D. and C.R.).

Antigen-presenting cells (APCs) may be resistant to SARS-CoV-2 infection but still contribute to viral pathogenesis. Lectins such as sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169) mediate the attachment of viruses to APCs. Here, we show that APCs effectively capture SARS-CoV-2 within compartments via recognition of Siglec-1. This receptor interacts with sialylated gangliosides on membranes of SARS-CoV-2 variants, as previously shown for retroviruses or filoviruses [1]. Blockage of Siglec-1 on monocyte-derived dendritic cells (MDDCs) decreased SARS-CoV-2 viral transfer or trans-infection to bystander target cells. However, monocyte-derived macrophages (MDMs) capturing SARS-CoV-2 via Siglec-1 did not transmit infectious particles. The presence of pulmonary APCs co-expressing Siglec-1 and SARS-CoV-2 corroborated these findings in vivo., The research of the CBIG consortium (constituted by IRTA-CReSA, BSC, & IrsiCaixa) is supported by Grifols Pharmaceutical. The authors also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com). N.I.-U. is supported by the grant PID2020-117145RB-I00 from the Spanish Ministry of Science and Innovation. J.M.-P. is supported by the grant PID2019-109870RB-I00 from the Spanish Ministry of Science and Innovation and in part also by Grifols. The C.R. laboratory is funded by RTI2018-094445-B100 (MCIU/AEI/FEDER, UE). The NHP study was primarily supported by a YNPRC Coronavirus Pilot Research Project Program grant to M.Pa. under award P51 OD11132, Emergent Venture Fast grant program to M.Pa. under awards #2206 and #2144, and William and Lula Pitts Foundation (to M.Pa.). X.M.-T. is supported by the Spanish Ministry of Science and Innovation and the European Regional Development Fund under agreement BES-2017-082900.

Drug repurposing is an important source of new antivirals because many compounds used to treat a variety of pathologies also hamper viral infections. Habitually, silver nanoparticles (AgNPs) have been used to treat bacterial and fungal infections and their antiviral properties have been also reported. In this work, we have studied the antiviral capacity of AgNPs in cells infected with Bunyamwera virus (BUNV), the prototype of the Bunyavirales order. This group of viruses contains important pathogens for humans, animals and plants. Incubation of BUNV-infected Vero cells with non-toxic concentrations of AgNPs, reduced the production of extracellular infectious viruses in up to three orders of magnitude. With a combination of imaging techniques, we have visualized the intracellular distribution of AgNPs in mock- and BUNV-infected cells and studied their effects on intracellular organelles. In mock-infected cells and at short times post-incubation, AgNPs were detected inside nuclei and mitochondria by transmission electron microscopy (TEM). At long times post-treatment, they accumulated inside lysosome-like organelles. Cell compartments did not exhibit any appreciable ultrastructural alterations after incubation with AgNPs. In BUNV-infected cells, AgNPs attached to extracellular virions, that showed a disrupted morphology. Inside cells, they were detected inside the nucleus, in mitochondria and around characteristic Golgi-associated, single-membrane spherules. These membranous structures are the replication organelles (ROs) of bunyaviruses and contain active viral replication complexes (VRCs). Compared to normal spherules that are round, compact and have an electron-dense core, spherules in AgNPs-treated cells were deformed and their core was electron-lucent. Interestingly, in BUNV-infected cells treated with the typical antiviral ribavirin (RBV), spherules with VRCs exhibit also an anomalous morphology and an electron-lucent core. Both AgNPs and RBV might interfere with BUNV-induced dismantling of cell nucleoli and with the intercellular propagation of large groups of virions, a mechanism of BUNV transmission observed for the first time in cultured cells. Our results point to silver nanoparticles as good candidates for antiviral therapy, either alone or in combination with other antiviral drugs, such as RBV-related compounds.

© 2020 Fernández de Castro et al., Mammalian orthoreoviruses (reoviruses) are nonenveloped viruses that replicate in cytoplasmic membranous organelles called viral inclusions (VIs) where progeny virions are assembled. To better understand cellular routes of nonlytic reovirus exit, we imaged sites of virus egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs) and observed one or two distinct egress zones per cell at the basal surface. Transmission electron microscopy and 3D electron tomography (ET) of the egress zones revealed clusters of virions within membrane-bound structures, which we term membranous carriers (MCs), approaching and fusing with the plasma membrane. These virion-containing MCs emerged from larger, LAMP-1–positive membranous organelles that are morphologically compatible with lysosomes. We call these structures sorting organelles (SOs). Reovirus infection induces an increase in the number and size of lysosomes and modifies the pH of these organelles from ∼4.5–5 to ∼6.1 after recruitment to VIs and before incorporation of virions. ET of VI–SO–MC interfaces demonstrated that these compartments are connected by membrane-fusion points, through which mature virions are transported. Collectively, our results show that reovirus uses a previously undescribed, membrane-engaged, nonlytic egress mechanism and highlights a potential new target for therapeutic intervention., This work was supported in part by Public Health Service awards AI032539 (T.S. Dermody and C. Risco), AI122563 (J.J. Knowlton), and GM007347 (J.J. Knowlton).

Transmission electron microscopy (TEM) has been crucial to study viral infections. As a result of recent advances in light and electron microscopy, we are starting to be aware of the variety of structures that viruses assemble inside cells. Viruses often remodel cellular compartments to build their replication factories. Remarkably, viruses are also able to induce new membranes and new organelles. Here we revise the most relevant imaging technologies to study the biogenesis of viral replication organelles. Live cell microscopy, correlative light and electron microscopy, cryo-TEM, and three-dimensional imaging methods are unveiling how viruses manipulate cell organization. In particular, methods for molecular mapping in situ in two and three dimensions are revealing how macromolecular complexes build functional replication complexes inside infected cells. The combination of all these imaging approaches is uncovering the viral life cycle events with a detail never seen before.

Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Reoviruses are common pathogens of mammals that have been linked to celiac disease and show promise for oncolytic applications. These viruses form nonenveloped, double-shelled virions that contain ten segments of double-stranded RNA. Replication organelles in reovirus-infected cells are nucleated by viral nonstructural proteins µNS and σNS. Both proteins partition the endoplasmic reticulum to form the matrix of these structures. The resultant membranous webs likely serve to anchor viral RNA⁻protein complexes for the replication of the reovirus genome and the assembly of progeny virions. Ongoing studies of reovirus replication organelles will advance our knowledge about the strategies used by viruses to commandeer host biosynthetic pathways and may expose new targets for therapeutic intervention against diverse families of pathogenic viruses., This work was supported in part by Public Health Service awards F30 AI122563, R01 AI032539, T32 AI060525, and T32 GM007347 as well as by the Vanderbilt Lamb Center for Pediatric Research and the Heinz Endowments.

Bacteria and viruses pose serious challenges for humans because they evolve continuously. Despite ongoing efforts, antiviral drugs to treat many of the most troubling viruses have not been approved yet. The recent launch of new antimicrobials is generating hope as more and more pathogens around the world become resistant to available drugs. But extra effort is still needed. One of the current strategies for antiviral and antibiotic drug development is the search for host cellular pathways used by many different pathogens. For example, many viruses and bacteria alter lipid synthesis and transport to build their own organelles inside infected cells. The characterization of these interactions will be fundamental to identify new targets for antiviral and antibiotic drug development. This review discusses how viruses and bacteria subvert cell machineries for lipid synthesis and transport and summarises the most promising compounds that interfere with these pathways., This work has been funded by Contract BES‐2016‐078317 for predoctoral fellows (to A. F.‐ O.) and Research Grant BIO2015‐68758‐R (to C. R.) from the Ministry of Science, Innovation and Universities of Spain.

Many viruses build specialized structures known as viral factories, a protected environment in which viral genome replication and morphogenesis take place. Recent findings show that viruses manipulate lipid flows to assemble these replication platforms. Viruses are thus able to create new membranes by interfering with lipid metabolism, targeting and transport; they make use of specific lipid transfer proteins (LTP) at membrane contact sites, and frequently recruit endoplasmic reticulum (ER), ER export sites, and mitochondria. Some factories, such as those built by plant and certain animal viruses, are motile membranous structures involved in intracellular or intercellular transport of the replicated viral genome. The identification of lipids and LTP subverted by viruses might lead to better understand and fight viral infections.

Plus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs). Tomato bushy stunt virus (TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast ( Saccharomyces cerevisiae ) vps23 Δ bro1 Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant-negative versions of Arabidopsis homologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression in Nicotiana benthamiana leaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast and in vitro . Electron microscopic imaging of vps23 Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed in N. benthamiana cells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ∼600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a “measuring string” during VRC assembly and spherule formation. IMPORTANCE Plant positive-strand RNA viruses, similarly to animal positive-strand RNA viruses, replicate in membrane-bound viral replicase complexes in the cytoplasm of infected cells. Identification of cellular and viral factors affecting the formation of the membrane-bound viral replication complex is a major frontier in current virology research. In this study, we dissected the functions of co-opted cellular ESCRT-I (endosomal sorting complexes required for transport I) and ESCRT-III proteins and the viral RNA in tombusvirus replicase complex formation using in vitro , yeast-based, and plant-based approaches. Electron microscopic imaging revealed the lack of tombusvirus-induced spherule-like structures in ESCRT-I or ESCRT-III deletion yeasts replicating TBSV RNA, demonstrating the requirement for these co-opted cellular factors in tombusvirus replicase formation. The work could be of broad interest in virology and beyond.

Transmission electron microscopy (TEM) has been instrumental for studying viral infections. In particular, methods for labeling macromolecules at the ultrastructural level, by integrating biochemistry, molecular biology, and morphology, have allowed to study the functions of viral macromolecular complexes within the cellular context. Here, we describe a strategy for imaging influenza virus ribonucleoproteins in infected cells with two complementary labeling methods, metal-tagging transmission electron microscopy or METTEM, a highly sensitive technique based on the use of a metal-binding protein as a clonable tag, and immunogold labeling on thawed cryosections, a very specific labeling method that allows to study the distribution of different proteins simultaneously. The combination of both labeling methods offers new possibilities for TEM analysis of viral components in cells.

Like most viruses that replicate in the cytoplasm, mammalian reoviruses assemble membranous neo-organelles called inclusions that serve as sites of viral genome replication and particle morphogenesis. Viral inclusion formation is essential for viral infection, but how these organelles form is not well understood. We investigated the biogenesis of reovirus inclusions. Correlative light and electron microscopy showed that endoplasmic reticulum (ER) membranes are in contact with nascent inclusions, which form by collections of membranous tubules and vesicles as revealed by electron tomography. ER markers and newly synthesized viral RNA are detected in inclusion internal membranes. Live-cell imaging showed that early in infection, the ER is transformed into thin cisternae that fragment into small tubules and vesicles. We discovered that ER tubulation and vesiculation are mediated by the reovirus σNS and μNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles., IMPORTANCE Viruses modify cellular structures to build replication organelles. These organelles serve as sites of viral genome replication and particle morphogenesis and are essential for viral infection. However, how these organelles are constructed is not well understood. We found that the replication organelles of mammalian reoviruses are formed by collections of membranous tubules and vesicles derived from extensive remodeling of the peripheral endoplasmic reticulum (ER). We also observed that ER tubulation and vesiculation are triggered by the reovirus σNS and μNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles and provide functions for two enigmatic reovirus replication proteins. Most importantly, this research uncovers a new mechanism by which viruses form factories for particle assembly.

Transmission electron microscopy (TEM) has been instrumental for studying viral infections. In particular, methods for labeling macromolecules at the ultrastructural level, by integrating biochemistry, molecular biology, and morphology, have allowed to study the functions of viral macromolecular complexes within the cellular context. Here, we describe a strategy for imaging influenza virus ribonucleoproteins in infected cells with two complementary labeling methods, metal-tagging transmission electron microscopy or METTEM, a highly sensitive technique based on the use of a metal-binding protein as a clonable tag, and immunogold labeling on thawed cryosections, a very specific labeling method that allows to study the distribution of different proteins simultaneously. The combination of both labeling methods offers new possibilities for TEM analysis of viral components in cells.

ABSTRACT RNA viruses replicate in the cytoplasm in close association with host cell membranes. Both viral and cellular factors generate organelle-like structures termed viral factories, viral inclusions or viroplasms. Biochemical, light and electron microscopy analyses, including 3D models, have improved our understanding of the architecture and function of RNA virus replication factories. In these structures, the virus compartmentalizes genome replication and transcription, thus enhancing replication efficiency and protection from host defenses. Recent studies with diverse RNA viruses have elucidated the ultrastructure of replication organelles and shown how these structures act in close coordination with virion assembly. This review focuses on a general description of RNA virus factories and summarizes recent progress in the characterization of those assembled by bunyaviruses and reoviruses. We describe how these viruses modify intracellular membranes; we highlight similarities with the structures induced by viruses of other families, and discuss how these structures might be formed.

Viruses are molecular machines sustained through a life cycle that requires replication within host cells. Throughout the infectious cycle, viral and cellular components interact to advance the multistep process required to produce progeny virions. Despite progress made in understanding the virus–host protein interactome, much remains to be discovered about the cellular factors that function during infection, especially those operating at terminal steps in replication. In an RNA interference screen, we identified the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC; also called CCT for chaperonin containing TCP-1) as a cellular factor required for late events in the replication of mammalian reovirus. We discovered that TRiC functions in reovirus replication through a mechanism that involves folding the viral σ3 major outer-capsid protein into a form capable of assembling onto virus particles. TRiC also complexes with homologous capsid proteins of closely related viruses. Our data define a critical function for TRiC in the viral assembly process and raise the possibility that this mechanism is conserved in related non-enveloped viruses. These results also provide insight into TRiC protein substrates and establish a rationale for the development of small-molecule inhibitors of TRiC as potential antiviral therapeutics.

Xenophagy has been studied in epithelial cells infected with Salmonella enterica serovar Typhimurium (S. Typhimurium). Distinct autophagy receptors target this pathogen to degradation after interacting with ubiquitin on the surface of cytosolic bacteria, and the phagophore- and autophagosome-associated protein MAP1LC3/LC3. Glycans exposed in damaged phagosomal membranes and diacylglycerol accumulation in the phagosomal membrane also trigger S. Typhimurium xenophagy. How these responses control intraphagosomal and cytosolic bacteria remains poorly understood. Here, we examined S. Typhimurium interaction with autophagy in fibroblasts, in which the pathogen displays limited growth and does not escape into the cytosol. Live-cell imaging microscopy revealed that S. Typhimurium recruits late endosomal or lysosomal compartments that evolve into a membranous aggregate connected to the phagosome. Active dynamics and integrity of the phagosomal membrane are requisite to induce such aggregates. This membranous structure increases over time to become an aggresome that engages autophagy machinery at late infection times (> 6 h postentry). The newly formed autophagosome harbors LC3 and the autophagy receptor SQSTM1/p62 but is devoid of ubiquitin and the receptor CALCOCO2/NDP52. Live-cell imaging showed that this autophagosome captures and digests within the same vacuole the aggresome and some apposed intraphagosomal bacteria. Other phagosomes move away from the aggresome and avoid destruction. Thus, host endomembrane accumulation resulting from activity of intracellular S. Typhimurium stimulates a novel type of aggrephagy that acts independently of ubiquitin and CALCOCO2, and destroys only a few bacteria. Such selective degradation might allow the pathogen to reduce its progeny and, as a consequence, to establish persistent infections.

Three-dimensional (3D) imaging technologies are beginning to have significant impact in the field of virology, as they are helping us understand how viruses take control of cells. In this article we review several methodologies for 3D imaging of cells and show how these technologies are contributing to the study of viral infections and the characterization of specialized structures formed in virus-infected cells. We include 3D reconstruction by transmission electron microscopy (TEM) using serial sections, electron tomography, and focused ion beam scanning electron microscopy (FIB-SEM). We summarize from these methods selected contributions to our understanding of viral entry, replication, morphogenesis, egress and propagation, and changes in the spatial architecture of virus-infected cells. In combination with live-cell imaging, correlative microscopy, and new techniques for molecular mapping in situ, the availability of these methods for 3D imaging is expected to provide deeper insights into understanding the structural and dynamic aspects of viral infection.

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions., Author Summary The actin network, which is a central node in cellular pathways, is frequently targeted by various pathogens to modulate cellular responses. In this paper, the authors show that TBSV interacts with cofilin actin depolymerization factor leading to inhibition of the dynamic function of the actin network in infected cells. This allows TBSV to utilize the existing actin filaments to efficiently recruit host proteins and lipids for viral replication and to build viral replication compartments for robust viral replication. Altogether, subversion of the actin network by TBSV is a key step for the virus to gain access to cellular resources required for virus replication.

Positive-strand RNA viruses, which can be devastating pathogens in humans, animals and plants, replicate their genomes on intracellular membranes. Here we describe the three-dimensional ultrastructural organization of a tombusvirus replicase in yeast, a valuable model for exploring virus-host interactions. We visualized the intracellular distribution of a viral replicase protein using metal-tagging transmission electron microscopy, a highly sensitive nanotechnology whose full potential remains to be developed. These three-dimensional images show how viral replicase molecules are organized when they are incorporated into the active domains of the intracellular replication compartment. Our approach provides a means to study protein activation mechanisms in cells and to identify targets for new antiviral compounds.