key: cord-0880979-jry9wppw authors: Sachse, Martin; Tenorio, Raquel; de Castro, Isabel Fernández; Muñoz-Basagoiti, Jordana; Perez-Zsolt, Daniel; Raïch-Regué, Dàlia; Rodon, Jordi; Losada, Alejandro; Avilés, Pablo; Cuevas, Carmen; Paredes, Roger; Segalés, Joaquim; Clotet, Bonaventura; Vergara-Alert, Júlia; Izquierdo-Useros, Nuria; Risco, Cristina title: Unraveling the antiviral activity of plitidepsin by subcellular and morphological analysis date: 2021-12-20 journal: bioRxiv DOI: 10.1101/2021.12.16.472880 sha: 29692db7e8725a60ad6273c481ef19a01444e3ea doc_id: 880979 cord_uid: jry9wppw 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. Infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently 62 needs effective antiviral treatments with a significant clinical benefit for hospitalized patients. So 63 far, randomized clinical trials have failed to identify potent antivirals targeting the virus, with the 64 only exception of remdesivir and molnupiravir, which have recently shown clinical benefits when 65 administered early upon infection (Beigel et al., 2020; Garibaldi et al., 2021; Grein et al., 2020; 66 Imran et al., 2021) . As it happens with all viruses, coronaviruses have a reduced number of 67 molecular druggable targets, and as new variants arise, these targets evolve and could eventually 68 develop antiviral resistance. An interesting approach to overcome these limitations relies on the 69 use of compounds against highly conserved cellular host factors required to complete the 70 replication cycle of distinct types of viruses, which offer a common targeted solution to diverse 71 viral threats. Furthermore, targeting host factors could offer a pan-antiviral strategy to combat not 72 only viruses known at present, but also future pandemics to come (Baggen et al., 2021) . Presently, there are only a limited number of approved drugs involved in targeting host factors at 74 post-entry steps (Baggen et al., 2021) . This approach is especially relevant for pan-antiviral 75 solutions given that viruses may use alternative pathways to enter a cell, but will most likely 76 converge at intracellular processes involving genome replication and protein production. One of 77 these compounds is plitidiepsin, which has shown a potent preclinical efficacy against SARS-78 CoV-2 by targeting the host protein eEF1A (Losada et al., 2016; Rodon et al., 2021; White et al., (ClinicalTrials.gov Identifier: NCT04784559). eEF1A2 is necessary to transport aminoacyl-86 tRNAs to the A site of the ribosome during protein translation, but is also implicated in other 87 activities (Mateyak and Kinzy, 2010) such as inhibition of apoptosis (Sun et al., 2014) , proteasome 88 degradation (Hotokezaka et al., 2002) , and actin bundling and cytoskeleton reorganization 89 (Edmonds et al., 1998) among other non-canonical functions. Also, eEF1A2 is implicated in the 90 replication of distinct viruses, including coronaviruses , and was identified as 91 4 a potential SARS-CoV-2 interacting protein in one of the first screenings performed to identify 92 novel targets (Gordon et al., 2020) . Upon SARS-CoV-2 infection, plitidepsin inhibits nucleocapsid viral protein expression and viral 95 induced cytopathic effect in vitro (Rodon et al., 2021; White et al., 2021) . In addition, it also 96 reduces genomic and subgenomic RNA expression (White et al., 2021) . Current models of SARS- CoV-2 replication propose that upon viral fusion, non-structural viral proteins form a replication-98 transcription complex that is continuous with the ER and has a double membrane vesicle (DMV) 99 morphology that shelters the viral genome replication (Baggen et al., 2021; Wolff et al., 2020a) . A negative RNA strand is used as a template for the generation of positive strands that are 101 translated and incorporated into nascent viruses (Baggen et al., 2021; Wolff et al., 2020a) . Discontinued transcription of positive RNA strands produce negative subgenomic RNAs, which 103 are then used as templates for positive subgenomic RNA generation that codify for structural and 104 accessory proteins (Baggen et al., 2021; Wolff et al., 2020a) . Translation of viral proteins is 105 facilitated by mRNA export via molecular pores located in the DMV that enable viral protein 106 production in the cytoplasm (Wolff et al., 2020b ). Yet, how plitidepsin exerts its intracellular 107 antiviral activity and influences the formation of viral replication DMV remains unknown. Here we aimed to explore the antiviral effect of plitidepsin at the cellular level to understand its 110 impact on SARS-CoV-2 replication and DMV formation. Using transmission electron microscopy 111 (TEM), we recapitulated the infectious SARS-CoV-2 cycle in Vero E6 cells and observed a lack Materials. Plitidepsin was synthesized at PharmaMar, S.A. (Colmenar Viejo, Madrid, Spain). Cell culture, viral isolation and titration. Vero E6 cells (ATCC CRL-1586) were cultured in 126 Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum 127 (FBS; Invitrogen), 100 U/mL penicillin, 100 μg/mL streptomycin (all from Invitrogen). SARS- CoV-2 D614G was isolated from a nasopharyngeal swab collected in March 2020 in Spain in Vero 129 E6 cells as previously described in detail (Rodon et al., 2021) . The virus was propagated for two 130 passages and a virus stock was prepared collecting the supernatant from Vero E6 cell and 131 sequenced as described elsewhere (Rodon et al., 2021) . Genomic sequence was deposited at 132 GISAID repository (http:// gisaid.org) with accession ID EPI_ISL_510689. Viral stock was titrated 133 in 10-fold serial dilutions on Vero E6 cells to calculate the TCID50 per mL. To understand how plitidepsin exerts its mechanism of action, we first followed SARS-CoV-2 184 replication cycle using electron microscopy. We found no replication organelles nor viral particles We then studied in detail the effects of 0.05 or 0.2 µM of plitidepsin on the assembly of SARS- CoV-2 replication organelles or DMVs. Of note, these two concentrations are close to the IC50 and 210 IC90 estimated for SARS-CoV-2 induced cytopathic effect on Vero E6 cells (Rodon et al., 2021) . (Figure 3D-F) . The absence of labelling was also found for the high concentration of plitidepsin 232 (0.2 µM) (Figure 3G-I) . In SARS-CoV-2 infected cells without plitidepsin, the labeling for Since plitidepsin interferes with the host factor eEF1A implicated in RNA translation (Losada et 262 al., 2016) , it is worthwhile to know the impact of this mechanism on the formation of DMVs. The antiviral effect of plitidepsin against SARS-CoV-2 is mediated throughout inhibition of the 265 host protein eEF1A. siRNA silencing of eEFA1A in host cells induces a significant reduction in 266 the nucleocapsid protein levels, as well as a reduction in the viral RNA, which demonstrates a 267 direct involvement of eEF1A in the viral replication . In addition, the exposure formation. This possible mechanism is supported by prior observations where plitidepsin treatment 287 decreased nucleocapsid content and reduced subgenomic RNA detection (Rodon et al., 2021; 288 White et al., 2021 ). An alternative mechanism has been recently proposed with eEF1A binding to 289 membranes and thereby recruiting a subset of proteins related to DMV formation (Carriles et al., In the present study, TEM morphological analysis coupled to immunogold labeling of SARS-CoV- This knowledge will be crucial to identify the mechanism of action for promising compounds that 301 interfere with host factors whose implication in key biological processes can be applied as a pan-302 antiviral strategies. A 368 SARS-CoV-2 protein interaction map reveals targets for drug repurposing Compassionate Use of Remdesivir for Patients with 379 Severe Covid-19 Interaction of the Eukaryotic Elongation Factor 1A 383 with Newly Synthesized Polypeptides Discovery, Development, and Patent 388 Trends on Molnupiravir: A Prospective Oral Treatment for COVID-19 Factor eEF1A2 is a Novel Anticancer Target for the Marine Natural Product Plitidepsin eEF1A: Thinking Outside the Ribosome Caution in Identifying Coronaviruses by Electron 397 SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid 402 14 adaptation and cytopathology Expression and Cleavage of Middle East Respiratory Syndrome 406 Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles 407 That Mimic Those Associated with Coronaviral RNA Replication 412 Identification of Plitidepsin as Potent Inhibitor of SARS-CoV-2-Induced Cytopathic 413 The viral replication 416 organelles within cells studied by electron microscopy Up-regulation of eEF1A2 419 promotes proliferation and inhibits apoptosis in prostate cancer Reovirus NS and NS Proteins Remodel the Endoplasmic 423 Reticulum to Build Replication Neo-Organelles Development and RNA-Synthesizing Activity of 426 Coronavirus Replication Structures in the Absence of Protein Synthesis Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by 433 targeting the host protein eEF1A Double-Membrane Vesicles as 436 Platforms for Viral Replication A molecular pore spans the 439 double membrane of the coronavirus replication organelle EF1A interacting with nucleocapsid 441 protein of transmissible gastroenteritis coronavirus and plays a role in virus replication We acknowledge J. Pedroza from the CMCiB for his constant help at the BSL3 facility.