Antiviral Research 200 (2022) 105270 Contents lists available at ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral Unraveling the antiviral activity of plitidepsin against SARS-CoV-2 by subcellular and morphological analysis Martin Sachse a, Raquel Tenorio a, 1, Isabel Fernández de Castro a, 1, Jordana Muñoz-Basagoiti b, 2, Daniel Perez-Zsolt b, 2, Dàlia Raïch-Regué b, 2, Jordi Rodon c, 2, Alejandro Losada h, Pablo Avilés h, Carmen Cuevas h, Roger Paredes b, Joaquim Segalés d, e, Bonaventura Clotet b, f, g, Júlia Vergara-Alert c, Nuria Izquierdo-Useros b, f, *, Cristina Risco a, ** a Centro Nacional de Biotecnología, CSIC, 28049, Madrid, Spain b IrsiCaixa AIDS Research Institute, 08916, Badalona, Spain c Unitat mixta d’investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193, Catalonia, Spain d IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193, Catalonia, Spain e Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona (UAB), Campus de la UAB, Bellaterra, 08193, Catalonia, Spain f Germans Trias i Pujol Research Institute (IGTP), Can Ruti Campus, 08916, Badalona, Spain g University of Vic–Central University of Catalonia (UVic-UCC), 08500, Vic, Spain h PharmaMar S.A, 28770, (Colmenar Viejo), Madrid, Spain A R T I C L E I N F O A B S T R A C T Keywords: The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, Plitidepsin efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of SARS-CoV-2 marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host Antiviral protein eEF1A (eukaryotic translation elongation factor 1 alpha) and affects viral infection at an early, post-entry Covid-19 step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action Electron microscopy 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 pli­ tidepsin, 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. 1. Introduction coronavirus 2 (SARS-CoV-2) urgently needs effective antiviral treat­ ments with a significant clinical benefit for patients. An increasing Infection caused by the severe acute respiratory syndrome number of randomized clinical trials are starting to identify potent * Corresponding author. IrsiCaixa AIDS Research Institute, 08916, Badalona, Spain. ** Corresponding author. Centro Nacional de Biotecnología, CSIC, 28049 Madrid, Spain. E-mail addresses: nizquierdo@irsicaixa.es (N. Izquierdo-Useros), crisco@cnb.csic.es (C. Risco). 1 Equal contribution. 2 Equal contribution. https://doi.org/10.1016/j.antiviral.2022.105270 Received 1 December 2021; Received in revised form 10 February 2022; Accepted 23 February 2022 Available online 26 February 2022 0166-3542/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). M. Sachse et al. Antiviral Research 200 (2022) 105270 antivirals targeting the virus. Indeed, remdesivir, molnupiravir and proteins (Baggen et al., 2021; Wolff et al., 2020a). Translation of viral nirmatrelvir have recently shown clinical benefits when administered proteins is facilitated by mRNA export via molecular pores located in the early upon infection (Beigel et al., 2020; Garibaldi et al., 2021; Grein DMV that enable viral protein production in the cytoplasm (Wolff et al., et al., 2020; Imran et al., 2021; Owen et al., 2021). However, corona­ 2020b). Yet, how plitidepsin exerts its intracellular antiviral activity and viruses have a reduced number of molecular druggable targets, and as influences the formation of viral replication DMV remains unknown. new variants arise, these targets evolve and could eventually develop Here we aimed to explore the antiviral effect of plitidepsin at the antiviral resistance. Monoclonal antibodies such as cellular level to understand its impact on SARS-CoV-2 replication and casirivimab-imdevimab have shown activity in clinical trials before the DMV formation. Using transmission electron microscopy (TEM), we surge of omicron variant, but pre-clinical evidence suggest now that this recapitulated the infectious SARS-CoV-2 cycle in Vero E6 cells and combination lacks neutralization activity against omicron (Agarwal observed a lack of viral DMVs in plitidepsin-treated cells. Complemen­ et al., 2020). Although Sotrovimab may retain activity, higher concen­ tary immunohistochemistry analyses using nucleocapsid and dsRNA trations are needed for neutralization of omicron (Agarwal et al., 2020). immunogold labeling unambiguously confirmed the lack of viral repli­ An interesting approach to overcome these limitations relies on the cation in plitidepsin treated cells. use of compounds against highly conserved cellular host factors required to complete the replication cycle of distinct types of viruses, which offer 2. Material & methods a common targeted solution to diverse viral threats. Furthermore, tar­ geting host factors could offer a pan-antiviral strategy to combat not Biosafety Approval. The biologic biosafety committee of the only viruses known at present, but also future pandemics to come Research Institute Germans Trias i Pujol approved the execution of (Baggen et al., 2021). Presently, there are only a limited number of SARS-CoV-2 experiments at the BSL3 laboratory of the Center for Bio­ approved drugs involved in targeting host factors at post-entry steps imaging and Comparative Medicine, CMCiB (CSB-20-015-M3). (Baggen et al., 2021). This approach is especially relevant for Materials. Plitidepsin was synthesized at PharmaMar, S.A. (Colme­ pan-antiviral solutions given that viruses may use alternative pathways nar Viejo, Madrid, Spain). to enter a cell, but will most likely converge at intracellular processes Cell culture, viral isolation and titration. Vero E6 cells (ATCC CRL- involving genome replication and protein production. One of these 1586) were cultured in Dulbecco’s modified Eagle medium (Invitrogen) compounds is plitidepsin, which has shown a potent preclinical efficacy supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/mL by targeting the host protein eEF1A (Losada et al., 2016; Rodon et al., penicillin, 100 μg/mL streptomycin (all from Invitrogen). 2021; White et al., 2021). In 2018, the Therapeutic Goods Administra­ SARS-CoV-2 D614G was isolated from a nasopharyngeal swab tion (TGA; Australian Government) approved the combination of pliti­ collected in March 2020 in Spain in Vero E6 cells as previously described depsin with dexamethasone for the treatment of patients with in detail (Rodon et al., 2021). The virus was propagated for two passages relapsed/refractory multiple myeloma (https://www.tga.gov.au/au and a virus stock was prepared collecting the supernatant from Vero E6 spar-plitidepsin). The results of randomized phase I studies of plitidep­ cell and sequenced as described elsewhere (Rodon et al., 2021). sin in adults hospitalized with Covid-19 have been recently published Genomic sequence was deposited at GISAID repository (http://gisaid. (Varona et al., 2022) and plitidepsin has been successfully used as a org) with accession ID EPI_ISL_510689. Viral stock was titrated in rescue treatment for prolonged viral SARS-CoV-2 replication in a patient 10-fold serial dilutions on Vero E6 cells to calculate the TCID50 per mL. with chronic lymphocytic leukemia (Guisado-Vasco et al., 2022). The following SARS-CoV-2 variants with deposited genomic Currently, plitidepsin is being evaluated in a phase 3, multicenter, sequence at the GISAID repository were also tested: Delta or B.1.617.2 randomized, controlled trial to determine the efficacy and safety of two (originally detected in India; EPI_ISL_3342900) isolated in Spain in dose levels of plitidepsin versus control in adult patients requiring hos­ February 2021, and Omicron or B.1.1.529 isolated in Spain in December pitalization for management of moderate coronavirus infectious disease 2021 (originally detected in South Africa; EPI_ISL_8151031). Genomic 2019 (COVID-19) (ClinicalTrials.gov Identifier: NCT04784559). eEF1A sequencing was performed from viral supernatant by using standard is necessary to transport aminoacyl-tRNAs to the A site of the ribosome ARTIC v3 or v4 based protocols followed by Illumina sequencing (dx. during protein translation, but is also implicated in other activities doi.org/10.17504/protocols.io.bhjgj4jw). Viral stocks were obtained as (Mateyak and Kinzy, 2010) such as inhibition of apoptosis (Sun et al., previously described and titrated in 1/3 serial dilutions on Vero E6 cells 2014), proteasome degradation (Hotokezaka et al., 2002), and actin to achieve a 50% of cytopathic effect three days post infection as bundling and cytoskeleton reorganization (Edmonds et al., 1998) among described in the antiviral activity section. other non-canonical functions. Also, eEF1A is implicated in the repli­ Antiviral Activity. Increasing concentrations of plitidepsin were cation of distinct viruses, including coronaviruses (Zhang et al., 2014), added to Vero E6 cells and SARS-CoV-2 viruses were added subsequently and was identified as a potential SARS-CoV-2 interacting protein in one as described in (Rodon et al., 2021). Assay was set up to achieve a 50% of the first screenings performed to identify novel targets (Gordon et al., of cytopathic effect three days post infection. Cytopathic or cytotoxic 2020). effects of viruses or drugs were measured using the CellTiter-Glo lumi­ Upon SARS-CoV-2 infection, plitidepsin inhibits nucleocapsid viral nescent cell viability assay (Promega). Luminescence was measured in a protein expression and viral induced cytopathic effect in vitro (Rodon Fluoroskan Ascent FL luminometer (ThermoFisher Scientific). Response et al., 2021; White et al., 2021). In addition, it also reduces genomic and curves were adjusted to a non-linear fit regression model, calculated subgenomic RNA expression (White et al., 2021). In contrast to with a four-parameter logistic curve with variable slope to calculate the Remdesivir, the in vitro inhibitory activity of plitidepsin is enhanced IC50. Cells not exposed to the virus were used as negative controls of when the drug is added several hours post-infection (White et al., 2021). infection, and were set as 100% of viability to normalize data and Current models of SARS-CoV-2 replication propose that upon viral calculate the percentage of cytopathic effect. Analyses were generated fusion, non-structural viral proteins form a replication–transcription with the GraphPad Prism v9 Software (GraphPad Software, San Diego, complex that is continuous with the ER and has a double membrane CA, USA). vesicle (DMV) morphology that shelters the viral genome replication Cellular infection. Vero E6 cells were infected with SARS-CoV-2 at a (Baggen et al., 2021; Wolff et al., 2020a). A negative RNA strand is used multiplicity of infection MOI of 0.02 for 24 and 48 hours (h) in the as a template for the generation of positive strands that are translated presence or absence of 0.2 and 0.05 μM of plitidepsin added at the time and incorporated into nascent viruses (Baggen et al., 2021; Wolff et al., of infection. These two concentrations are close to the IC90 and IC50 of 2020a). Discontinued transcription of positive RNA strands produce plitidepsin, respectively, determined by the SARS-CoV-2 induced cyto­ negative subgenomic RNAs, which are then used as templates for posi­ pathic effect on Vero E6 cells (Rodon et al., 2021). As negative controls, tive subgenomic RNA generation that codify for structural and accessory Vero E6 cells were treated with or without plitidepsin for the same time 2 M. Sachse et al. Antiviral Research 200 (2022) 105270 but without virus. At each studied time point, cell monolayers were TEM operated at 100 kV with a Gatan ES1000W camera. At least 50 cells chemically fixed using two different protocols depending on the type of per condition were studied by TEM. analysis (morphology or immunohistochemistry). Morphology Analysis. Cell monolayers were fixed with 4% para­ 3. Results formaldehyde and 1% glutaraldehyde in phosphate buffered saline (PBS) for 2h at room temperature (RT). Cells were removed from the To understand how plitidepsin exerts its mechanism of action, we plates in the fixative, pelleted by centrifugation and washed three times studied how plitidepsin interferes with viral replication of three SARS- with PBS. Post-fixation of cell pellets was done on ice with 1% CoV-2 variants (D614G, Delta and Omicron) and observed a dose osmiumtetroxide +0.8% potassium ferrocyanide in water. Afterwards response reduction of the viral-induced cytopathic effect three days the pellets were dehydrated on ice with increasing concentrations of post-infection on Vero E6 cells (Fig. 1). acetone and processed for embedding in the epoxy resin EML-812 (Taab We then followed SARS-CoV-2 D614G variant replication cycle using Laboratories), as previously described (Tenorio et al., 2018). Sections of electron microscopy. cells in epoxy resins have high contrast and optimal morphological de­ We found no replication organelles nor viral particles in cells infec­ tails. Infiltration with epoxy resin was performed at RT. All samples ted for 24h at an MOI of 0.02, although compared to control cells, the were polymerized at 60 ◦ C for 48h. Ultrathin sections (50–70 nm) were viral infection induced some stress in cells, which showed mitochondria cut with a Leica UC6 microtome and placed on uncoated 300 mesh with swollen cristae and Golgi with swollen cisternae (not shown). copper grids. Sections were contrasted with 4% uranyl acetate and In Vero E6 cells infected for 48h at an MOI of 0.02 with the D614G Reynold’s lead citrate. Images were taken with a Tecnai G2 TEM oper­ variant, clusters of characteristic DMVs, which are the replication or­ ated at 120 kV with a Ceta camera or with a Jeol 1400 operated at 120 ganelles of SARS-CoV-2 (Ogando et al., 2020), were present in the kV with a Gatan Rio camera. At least 50 cells per condition were studied cytoplasm and occupied a significant area of the cytosol (Fig. 2A and B). by TEM. DMVs often had an electron lucent content with fibrillar material Immunohistochemistry. Cells were fixed with 4% paraformaldehyde (Fig. 2B). Intracellular viral particles and extracellular virions attached and 0.1% glutaraldehyde in PBS for 1h at RT and removed from the to the plasma membrane were abundant (Fig. 2A and C). On average, in plates in the fixative, pelleted by centrifugation and washed three times ultrathin sections of infected cells we found 30 intracellular viral par­ with PBS. Cell pellets were incubated 30 min with 2% uranyl acetate in ticles and 73 extracellular particles attached to the plasma membrane water at RT, gradually dehydrated on ice with ethanol and infiltrated at per cell. Single or very few viral particles were present inside small RT with LR-White acrylic resin as previously described (de Castro Martin vesicles with an electron lucent lumen while larger vacuoles with et al., 2017). Sections of cells in acrylic resins have low contrast but an numerous viral particles were often observed (Fig. 2C). These vacuoles optimal preservation of protein epitopes, which is fundamental for that contained internal membranes and/or amorphous electron dense immunolabeling studies. After polymerization at 60 ◦ C for 48h, the material might represent late endosomes/lysosomes. We seldom samples were sectioned using a Leica UC6 ultramicrotome. Ultrathin observed budding of viral particles into the endoplasmic reticulum (ER) sections were collected on copper grids with a carbon-coated Formvar (Fig. 2C). film. For immunogold labeling, unspecific binding on sections was When 0.2 μM of plitidepsin was added to Vero E6 cells, the drug had blocked by incubation with Tris-buffered gelatin (TBG) for 5 min. N a profound impact on viral replication 48h later (Fig. 2D–F). We did not protein was labeled with the rabbit anti-SARS-CoV-2 nucleocapsid observe any DMVs in the cytoplasm, nor viral particles inside cells or at antibody (GXT 135357 GeneTex) diluted 1/50 in TBG for 1h. Double the plasma membrane (Fig. 2D). We found clusters of potential single stranded RNA (dsRNA), an intermediate of viral replication, was labeled membrane vesicles present in the cytoplasm, which are formed in early with the mouse monoclonal anti-dsRNA J2 antibody (10010200, English stages of SARS-CoV-2 infection (Eymieux et al., 2021a), that could and Scientific Consulting Kft, SCICONS) diluted 1/30 in TBG for 1h. however also reflect parts of a swollen Golgi (Fig. 2E). Lipid droplets After three washes with PBS and incubation with TBG for 5 min, grids (LDs) and glycogen granules were abundant (Fig. 2F), something also were labeled with a secondary antibody, corresponding to the species of observed in mock-infected cells treated with 0.2 μM of plitidepsin the first antibody, conjugated with 10-nm colloidal gold particles (BB (Fig. S1). This effect was observed in cells incubated with a lower con­ international) in TBG for 30 min. Sections were washed three times with centration of plitidepsin, that is 0.05 μM, only less pronounced (Fig. S2). PBS and five times with milliQ water and stained with saturated uranyl We then studied in detail the effects of 0.05 or 0.2 μM of plitidepsin acetate for 20 min. After washing three times with milliQ water, grids on the assembly of SARS-CoV-2 replication organelles or DMVs. Of note, were allowed to dry at RT. Images were taken with a Jeol 1400 TEM these two concentrations are close to the IC50 and IC90 estimated for operated at 80 kV with a Gatan One view camera or with a Jeol 1011 SARS-CoV-2 induced cytopathic effect on Vero E6 cells (Rodon et al., Fig. 1. Viral induced cytopathic effect on Vero E6 cells exposed to a fixed concentration of different SARS-CoV-2 VOC (D614G, Delta and Omicron var­ iants) in the presence of increasing concentrations of plitidepsin. Non-linear fit to a variable response curve showing mean values and S.E.M from two representative experiments with four replicates are shown (red lines), excluding data from drug con­ centrations with associated toxicity. The IC50 value for each VOC is indicated. Cytotoxic effect on Vero E6 cells exposed to increasing concentrations of plitidepsin in the absence of virus is also shown (grey lines) in relative light units (RLUs). 3 M. Sachse et al. Antiviral Research 200 (2022) 105270 Fig. 2. Transmission electron microscopy of Vero E6 cells infected with SARS-CoV-2 and effects of plitidepsin. (A) to (C) ultrathin sections of cells infected at an MOI of 0.02 and 48 h post-inoculation. (A) Cell containing a viral replication organelle (asterisk) made of a collection of double-membrane vesicles (DMVs) near the nucleus (N). Numerous viral particles are seen on the cell surface (arrows and inset in A). (B) DMVs have a round shape and fibrillar content. (C) Individual intracellular viral particles (arrows) are seen inside single-membrane vesicles and a large group of viruses inside a vacuole (V). Budding of viral particles in RER membranes (arrowhead and inset). (D) to (F) Ultrathin sections of Vero E6 cells infected with SARS-CoV-2 and treated with 0.2 μM plitidepsin. Low (D) and high (E and F) magnification views show that these cells contain lipid droplets (LD), large glycogen deposits (GD) and an altered Golgi complex (G) but no viral structures. M, mitochondrion. Scale bars, 1 μm in A and D; 200 nm in B, C, E and F. 2021). When plitidepsin was added to Vero E6 cells at the same time of μM) (Fig. 4G–I). In SARS-CoV-2 infected cells without plitidepsin, the viral infection (MOI of 0.02 for 48h), both concentrations had a deep labeling for dsRNA was restricted to the lumen of large electron lucent impact on the biogenesis of DMVs (Fig. 3). Cells infected without pliti­ vesicles, which represent the DMVs (Fig. 5A–C). Due to the low contrast depsin contained numerous, typical DMVs with fibrillar content sur­ of ultrathin sections of cells embedded in the LR-White acrylic resin, the rounded by viral particles inside single-membrane vesicles (Fig. 3A–C) fibrillar content in the lumen of DMVs is not visible in these samples but but when the drug was added no DMVs were found (Fig. 3D–I). Already the labeling of dsRNA marked them unequivocally as DMVs. After at the low concentration, no DMVs were found in the cytosol (Fig. 3D–F). application of plitidepsin at 0.05 or 0.2 μM, no signal for dsRNA was Single membrane vesicles were present but they did not contain fibrillar detected by immunogold labelling (Fig. 5D–I). Overall, immunode­ content in their lumen as opposed to infected cells, and this could reflect tection of viral proteins and genetic material confirmed that the a part of a swollen Golgi or ER (Fig. 3F and I). These morphological morphological analysis performed corresponded to viral replication analyses on plitidepsin-treated cells failed to identify any structures structures and that plitidepsin completely blocked the assembly of the implicated in SARS-CoV-2 replication that were unambiguously detec­ viral replication organelles or DMVs and the accumulation or viral ted in untreated infected cells (Fig. 2A–C and 3A-C). nucleocapsid protein and dsRNA. Taken together these results provide The absence of structures implied in viral replication was confirmed evidence that plitidepsin inhibits intracellular viral replication and ab­ by immunodetection of two key products of SARS-CoV-2: the nucleo­ rogates the formation of DMVs. capsid and double stranded RNA (dsRNA). The nucleocapsid partici­ pates in replication and translation of viral RNA and maintains the 4. Discussion structure of ribonucleoprotein complex, whereas the dsRNA is synthe­ sized by the viral polymerase in the host cell, once viral replication has The TEM analysis of SARS-CoV-2-infected Vero E6 cells performed started in DMVs. For both targets, no signal by immunogold labeling was herein reveals the formation of DMV structures characteristic of the viral detected in non-infected cells, either non treated with plitidepsin or replication organelle, and the accumulation of intracellular viral parti­ incubated with low or high concentration of plitidepsin (Figs. S3 and cles in small vesicles and large vacuoles as well as extracellular virions at S4). However, virus-infected cells showed a strong labeling for the the plasma membrane. These structures have been described before as nucleocapsid in the cytosol and in viral particles that accumulated intra- characteristic features of SARS-CoV-2 infection in cell culture (Eymieux as well as extracellularly (Fig. 4A–C). Coherent with the morphological et al., 2021a, 2021b; Ogando et al., 2020). In this study, infected cells observations, after application of plitidepsin at 0.05 μM, no signal was displayed a clear specific labelling for both the nucleocapsid viral pro­ detected by antibody labelling for nucleocapsid (Fig. 4D–F). The absence tein and the dsRNA viral replication intermediate. Specific labelling of of labelling was also found for the high concentration of plitidepsin (0.2 two key viral products is highly relevant in a context in which TEM 4 M. Sachse et al. Antiviral Research 200 (2022) 105270 Fig. 3. Transmission electron microscopy of Vero E6 cells infected with SARS-CoV-2 in the absence or presence of two doses of plitidepsin. (A) to (C) Cells infected in the absence of plitidepsin. Low (A) and high (B and C) magnification of ultrathin sections of cells infected 48 h at an MOI of 0.02. (A) Groups of characteristic double membrane vesicles (DMVs; asterisks) occupy large areas of infected cells. (B) Typical DMVs exhibit electron dense membranes and sometimes a fibrillar content. (C) Single membrane vesicles with viruses (arrows) are often found in the close vicinity of DMVs. (D) to (F) Low (D) and high (E) and (F) magnification of Vero E6 cells infected with SARS-CoV-2 48 h at an MOI of 0.02 and treated with 0.05 μM plitidepsin. Cells contain lipid droplets (arrows in D), swollen Golgi stacks (G), mitochondria with swollen cristae (M) and swollen endoplasmic reticulum (ER) cisternae. No viral structures are seen. (G) to (I) Cells infected with SARS-CoV-2 48 h at an MOI of 0.02 and treated with 0.2 μM plitidepsin. Cells contain lipid droplets (arrows in G), swollen ER and altered mitochondria (I). N, nucleus; P, plasma membrane. Scale bars, 1 μm in A, D and G; 200 nm in B, C, E, F, H and I. analysis of SARS-CoV-2-infected samples has led to the misidentification et al., 2014). In addition, the exposure of plitidepsin to cells at the of viral particles and to ambiguous results (Dittmayer et al., 2020; Miller moment of SARS-CoV-2 infection reduced the viral replication and and Goldsmith, 2020). Together, the morphological analysis along with transcription, which induced a reduction in the accumulation of the immunogold detection of two complementary SARS-CoV-2 products genomic RNA and sub-genomic N RNA viral levels, respectively (White performed herein provided conclusive evidence that the subcellular et al., 2021). Due to the known high sensitivity of coronavirus to structures detected in infected cells represented viral replication or­ translation inhibitors (Bojkova et al., 2020; van den Worm et al., 2011), ganelles and virus progeny. In the present set of experiments, plitidepsin especially at an early stage of infection, a plitidepsin-mediated inhibi­ abrogated the formation of DMVs and the detection of nucleocapsid and tion of SARS-CoV-2 protein translation process could represent one dsRNA viral products in SARS-CoV-2-infected Vero E6 cells. Since pli­ possible mechanism, which could lead to the abrogation of DMV for­ tidepsin interferes with the host factor eEF1A implicated in RNA mation. The results gathered from the present experiments demonstrate translation (Losada et al., 2016), it is worthwhile to know the impact of that a concentration as low as 50 nM of plitidepsin, completely abol­ this mechanism on the formation of DMVs. ished the formation of DMVs, and the synthesis of N protein and viral The antiviral effect of plitidepsin against SARS-CoV-2 is mediated dsRNA in SARS-CoV-2 infected Vero E6 cells both at 24 and 48 h throughout inhibition of the host protein eEF1A. siRNA silencing of post-inoculation. The plitidepsin-mediated anti-SARS-CoV-2 activity at eEFA1A in host cells induces a significant reduction in the nucleocapsid such a low concentration may be the result of a highly specific inhibition protein levels, as well as a reduction in the viral RNA, which demon­ of genomic RNA viral translation. This specific antiviral effect is strates a direct involvement of eEF1A in the viral replication (Zhang explained by the lack of plitidepsin-mediated protein synthesis 5 M. Sachse et al. Antiviral Research 200 (2022) 105270 Fig. 4. Immunogold detection of SARS-CoV-2 nucleocapsid protein in infected cells in the absence or presence of two doses of plitidepsin. Ultrathin sections of cells were incubated with anti-N primary antibody followed by a secondary antibody conjugated with 10 nm colloidal gold particles and visualized by TEM. (A) to (C) Ultrathin sections of cells infected for 48 h with SARS-CoV-2 at an MOI of 0.02. (A) Low magnification image of an infected cell with characteristic DMVs (asterisks) near the nucleus (N). (B) High magnification image of a group of intracellular viruses (arrowheads) inside a vacuole. Viral particles and areas of cytosol are labeled. (C) Labeled extracellular virions (arrows) on the cell surface. (D) to (F) Cells infected 48 h with SARS-CoV-2 at an MOI of 0.02 and treated with 0.05 μM plitidepsin. Low (D) and high (E) and (F) magnification images of cells show no labeling. (G) to (I) Cells infected 48 h with SARS-CoV-2 at an MOI of 0.02 and treated with 0.2 μM plitidepsin. Low (G) and high (H) and (I) magnification images of cells show no labeling. P, plasma membrane. Scale bars, 0.5 μm in A, D and G; 200 nm in B, C, E, F, H and I. inhibition in eukaryotic cells at high concentrations (e.g., 450 nM or by exerting a direct action against a viral protein. In contrast to those higher), far above those used herein (Losada et al., 2016). Inhibition of monoclonal antibodies that have lost activity with the surge of omicron the viral protein translation may affect several essential proteins, such as variant (Agarwal et al., 2020), the mechanism of action of plitidepsin non-structural proteins, whose predicted transmembrane domains are remains active against omicron as we have demonstrated herein, and in key for DMV formation during the replication of distinct coronaviruses agreement with a recent publication (Varona et al., 2022). Plitidipsin is (Angelini et al., 2013; Oudshoorn et al., 2017; Wolff et al., 2020a). It is therefore a one of a kind compound that exerts a potent antiviral activity tempting to speculate that the lack of translation of these viral proteins against different SARS-CoV-2 variants by targeting a cellular host factor. could directly abrogate the formation of these replication–transcription In the present study, TEM morphological analysis coupled to complexes necessary for DMV formation. This possible mechanism is immunogold labeling of SARS-CoV-2 products has demonstrated to be a supported by prior observations where plitidepsin treatment decreased valuable tool to better understand the antiviral effects, as previously nucleocapsid content and reduced subgenomic RNA detection (Rodon reported for other viruses (García-Serradilla and Risco, 2021; Sachse et al., 2021; White et al., 2021). An alternative mechanism has been et al., 2019). Remarkably, the plitidepsin-induced complete blockade of recently proposed with eEF1A binding to membranes and thereby the assembly of viral structures detected here by electron microscopy is recruiting a subset of proteins related to DMV formation (Carriles et al., rather unique. In the presence of non-toxic, high concentrations of other 2021). It is worth highlighting that the activity of plitidepsin against antiviral drugs, SARS-CoV-2 often assembles small amounts of DMVs SARS-CoV-2 is mediated by targeting the host factor eEF1A rather than and viral particles (Izquierdo-Useros, Cerón and Risco, unpublished 6 M. Sachse et al. Antiviral Research 200 (2022) 105270 Fig. 5. Immunogold labelling of SARS-CoV-2 dsRNA in infected cells in the absence or presence of two doses of plitidepsin. Ultrathin sections of cells were incubated with anti-dsRNA primary antibody followed by a secondary antibody conjugated with 10 nm colloidal gold particles and visualized by TEM. (A) to (C) Ultrathin sections of cells infected 48 h with SARS-CoV-2 at an MOI of 0.02. (A) Low magnification image of an infected cell with characteristic DMVs (asterisks) near the nucleus (N). (B) and (C) High magnification images of labeled DMVs (arrowheads). (D) to (F) Cells infected 48 h with SARS-CoV-2 at an MOI of 0.02 and treated with 0.05 μM plitidepsin. Low (D) and high (E) and (F) magnification images of cells show no labeling. (G) to (I) Cells infected 48 h with SARS-CoV-2 at an MOI of 0.02 and treated with 0.2 μM plitidepsin. Low (G) and high (H) and (I) magnification images of cells show no labeling. N, nucleus; EA, empty areas; M, mitochondrion. Scale bars, 1 μm in A, D and G; 200 nm in B, C, E, F, H and I. results). The generated insights warrant new experiments that aim to Data availability better understand the mechanism of plitidepsin-induced antiviral ac­ tivity. This knowledge will be crucial to identify the mechanism of ac­ Data related to this work is available from corresponding authors tion for promising compounds that interfere with host factors whose upon reasonable request. implication in key biological processes can be applied as a pan-antiviral strategies. Declaration of competing interest Financial support P.A., J.V-A and N.I–U. are inventors in a patent application related to Aplidin/Plitidepsin (EP20382821.5). Unrelated to the submitted work, This research was funded by Pharma Mar, which commercializes N.I–U. reports institutional grants from Grifols, Dentaid, Hipra and Aplidin/Plitidepsin. The authors also acknowledge the crowdfunding Palobiofarma. The authors declare that no other competing financial initiative #Yomecorono (https://www.yomecorono.com) and the CBIG interests exist. A.L., P.A. and C.C. are PharmaMar S.A. employees or consortium supported by Grifols. N.I–U. is supported by grant PID2020- PharmaMar S.A. shareholders or both. 117145RB-I00 from the Spanish Ministry of Science and Innovation. C. R. is supported by grant RTI2018-094445-B-I00 (MCI/AEI/FEDER, UE) Acknowledgements from the Spanish Ministry of Science and Innovation. We acknowledge J. Díaz Pedroza from the CMCiB for his constant help at the BSL3 facility, I. Blanco, E. Martro, and V. Saludes from the 7 M. Sachse et al. Antiviral Research 200 (2022) 105270 Microbiology department of HUGTIP for providing key samples and Wang, H.-Y., Trenker, R., Young, J.M., Cavero, D.A., Hiatt, J., Roth, T.L., Rathore, U., Subramanian, A., Noack, J., Hubert, M., Stroud, R.M., Frankel, A.D., Rosenberg, O. outstanding help to sequence all VOC, and C. Andres, A. Antón and T. S., Verba, K.A., Agard, D.A., Ott, M., Emerman, M., Jura, N., von Zastrow, M., Pumarola from Respiratory Virus Unit, Microbiology Department, Vall Verdin, E., Ashworth, A., Schwartz, O., d’Enfert, C., Mukherjee, S., Jacobson, M., d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Hospital Uni­ Malik, H.S., Fujimori, D.G., Ideker, T., Craik, C.S., Floor, S.N., Fraser, J.S., Gross, J. versitari for providing key samples. We are grateful to M. Noguera and D., Sali, A., Roth, B.L., Ruggero, D., Taunton, J., Kortemme, T., Beltrao, P., Vignuzzi, M., García-Sastre, A., Shokat, K.M., Shoichet, B.K., Krogan, N.J., 2020. M. 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