key: cord-0936069-tmle1amw
authors: Kumar, Ram; Khandelwal, Nitin; Chander, Yogesh; Riyesh, Thachamvally; Gulati, Baldev R.; Pal, Yash; Tripathi, Bhupendra N.; Barua, Sanjay; Kumar, Naveen
title: Emetine as an antiviral agent suppresses SARS-CoV-2 replication by inhibitinginteraction of viral mRNAwith eIF4E: An in vitro study
date: 2020-11-30
journal: bioRxiv
DOI: 10.1101/2020.11.29.401984
sha: dacff366e79443a9688ba83da739c2712ede38c3
doc_id: 936069
cord_uid: tmle1amw

Emetine is a FDA-approved drug for the treatment of amebiasis. In the recent times we had also demonstrated the antiviral efficacy of emetine against some RNA and DNA viruses. Following emergence of the COVID-19, we further evaluated thein vitro antiviral activity of emetine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The therapeutic index of emetine was determined to be 10910.4, at a cytotoxic concentration 50 (CC50) of 1603.8 nM and effective concentration 50 (EC50) of 0.147 nM.Besides, we also demonstrated the protective efficacy of emetine against lethal challenge with infectious bronchitis virus (IBV; a chicken coronavirus) in the embryonated chicken egg infection model. Emetine treatment was shown to decrease viral RNA and protein synthesis without affecting other steps of viral life cycle such as attachment, entry and budding.In a chromatin immunoprecipitation (CHIP) assay, emetine was shown to disrupt the binding of SARS-CoV-2 RNA with eIF4E (eukaryotic translation initiation factor 4E, a cellular cap-binding protein required for initiation ofprotein translation). Further, SARS-CoV-2 was shown to exploit ERK/MNK1/eIF4E signalling pathwayfor its effective replication in the target cells. To conclude, emetine targets SARS-CoV-2 protein synthesis which is mediated via inhibiting the interaction of SARS-CoV-2 RNA with eIF4E. This is a novel mechanistic insight on the antiviral efficacy of emetine. In vitro antiviral efficacy against SARS-CoV-2 and its ability to protect chicken embryos against IBV suggests that emetine could be repurposed to treat COVID-19.

The ongoing pandemic of COVID-19 has become a health emergency of international concern. Therapeutic agents that can be used against COVID-19 or other coronavirus diseases are unavailable.Emetine dihydrochloride hydrate or emetine isa natural alkaloid found in three plant families:Alangiaceae, Icacinaceae, and Rubiaceae.

Emetine is a FDA-approved drug for the treatment of amebiasis (Bleasel and Peterson, 2020) .

Emetine has beenshownto disrupt protein synthesis in mammalian, yeast and plant cells (Grollman, 1966 (Grollman, , 1968 Gupta and Siminovitch, 1976; Han et al., 2014; Jimenez et al., 1977; Smirnova et al., 2003) . Recent studies by our and other groups have suggested in vitroantiviral efficacy of emetine against some RNA and DNA viruses (Chaves Valadao et al., 2015; Choy et al., 2020; Deng et al., 2007; Khandelwal et al., 2017; Mukhopadhyay et al., 2016; Ramabhadran and Thach, 1980; Yang et al., 2018) .Emetine can directly inhibit viral polymerase (Chaves Valadao et al., 2015; Khandelwal et al., 2017; Yang et al., 2018) , although the major antiviral activity of emetine is believed to be mediated via targeting certain cellular factors (Khandelwal et al., 2017) . While most studies are based on simply measuring the reduction in virus yield in the cell culture system, some mechanistic insights of emetine action have also been provided. For instance, in a study on human cytomegalovirus (HCMV), emetine was shown to target themouse double minute 2 homolog (MDM2)-p53 interaction, which is required for efficient HCMV replication (Mukhopadhyay et al., 2016) . However, the 5 precise natures of the cellular factors targeted by emetine and their roles in viral life cycle remain unknown. In this study, weprovide some novel mechanistic insights on the antiviral efficacy of emetine againstSARS-CoV-2, besides evaluating its protective efficacy against lethal challenge with infectious bronchitis virus (IBV, a chicken coronavirus) in embryonated chicken eggs.

African green monkey kidney (Vero)cells, available at the National Centre for Veterinary Type Cultures(NCVTC), Hisar were grown in Minimum Essential Medium 

The cytotoxicity (Kumar et al., 2008) 

Vero cells, in triplicates were pre-incubated with 500 nM emetine or equivalent volume of PBS for 1 h and then infected with SARS-CoV-2 at MOI of 5 for 2 h at 4°C.

The cells were then washed 5 times with PBS and the cell lysates were prepared by rapid freeze-thaw method. The viral titres were determined by plaque assay. 

The effect of emetine on SARS-CoV-2 entry was assessed using a previously described assay (Khandelwal et al., 2014) . Briefly, Vero cell monolayers (in triplicates)

were pre-chilled to 4 o C and infected with SARS-CoV-2 at MOI of 5 in the emetine-free medium for 1 h at 4 o C to permit attachment. This was followed by washing and addition of fresh MEM containing the drug or its vehicle-control. Entry was allowed to proceed at 37 o C for 1 h after which the cells were washed with PBS and incubated with MEMwithout any drug. The progeny virus particles released in the infected cell culture supernatant at 16 h were titrated by plaque assay.

The levels of SARS-CoV-2 RNA in the infected cells were quantified by quantitative analyzed to determine relative fold change in SARS-CoV-2 RNA copy numbers (E gene) as described previously (Kumar et al., 2016) . The primers used for amplification 9 of SARS-CoV-2 E gene were as follows: [forward primer: 5'-ACCGACGACGACTACTAGCG-3'and reverse primer: 5'-AGCTCTTCAACGGTAATAGTACCG-3'.

Confluent monolayers of Vero cells, in triplicates, were infected with SARS-CoV-2

for 1 h at MOI of 5, followed by washing with PBS and addition of fresh MEM. At 10 hpi, cells were washed 5 times with chilled PBS followed by addition of fresh MEM containing 500nM emetine or PBS. Virus release at various time points (post-emetine addition) was quantified by plaque assay.

Confluent monolayers of Vero cells were infected with SARS-CoV-2 at MOI of 5for 1h, followed by washing with PBS and addition of fresh MEM. Cell lysates were prepared at 12 hpi in RIPA buffer (Kumar et al., 2011) and subjected to Western blot analysis to probe the viral/cellularproteins.

CHIP assay was carried out to evaluate the interaction of viral mRNA with a cellular cap-binding protein (eIF4E) as per the previously described method (Bier et al., 2011; Carey et al., 2009 ) with some modifications. Briefly, Vero cells, in triplicates were infected with SARS-CoV-2 at MOI of 5. At 10hpi, when the RNA levels were expected to be at its peak, the cells were treated with 1% formaldehyde for 10 min to covalently cross-link interacting proteins and nucleic acid. Thereafter, the cross-linking reaction 

Emetine at concentrations of ~500 nMhad no effects onthe viability ofVero cells ( Fig.   2a ), though at higher concentrations (≥2000 nM), it was found to be quite toxic to the cells (Fig. 2a) . The CC 50 was determined to be 1603.8 nM. To analysethe effect administration also resulted in normal development of embryos as compared to vehiclecontrol-treated group wherein stunted growth and defective feather development was observed (Fig. 3c) .At an EC 50 of 2.3 ng/egg, the therapeutic index (LD 50 /EC 50 ) of emetine was determined to be 159.1. Taken together, we conclude that emetine prevents the chicken embryos against lethal challenge with IBV.

In order to evaluate which specific step ( Typically, the receptor-virus interactions are specific and the binding is independent of energy or temperature. The attachmentassay was carried out at 4 o C which allowed only attachment but restricted other post-attachment steps of the viral life cycle which are energy and temperature dependent. As shown in (Fig. 4b) , we did not observe any significant difference in the viral tittersattached in the presence of emetine or vehicle-control. In order to determine whetherthe pre-attached virus was able to enter 1 3 the cells in the presence of emetine, a standard entry assay was also performed.

Emetine was not shown to affect viral entry as the viral titres were comparable between emetine-treated and vehicle-control-treated cells (Fig. 4c) . To determine the effect of emetine on virus release (budding), it was applied at the time when the virus was presumed to be completing its life cycle viz; ~10 hpi. As shown in Fig. 4d ,no significant difference in the viral titres was observed between emetine-treated and control-treated cells, suggesting that emetine does not affect SARS-CoV-2 release from the infected cells.

In order to determinethe effect of emetine on the synthesis of viral genome, we first evaluated the kinetics of the viral RNA synthesis in the infected cells (cell pellet). As shown in Fig. 5a , the amount of viral RNA in the infected cell pellet was comparable at 1 hpi, 2 hpi, 4 hpi and 6 hpi. The detectable amounts of viral RNA at these early time points represent the virus particles which entered in the target cells following infection (input). A sharp rise in the viral RNA was observed at 8 hpi which peaked at 10 hpi before declining sharply at 12 hpi (Fig. 5a) .

To evaluate the effect of emetine on synthesis of SARS-CoV-2 RNA, emetine was applied when early steps of the virus life cycle (attachment/entry) were expected to occur (~4hpi) and the cells were harvested when the levels of viral RNA were expected to be maximum (~10 hpi, Fig. 5a ). As shown in Fig. 5b , a highly significantreduction in the relative copy numbers of viral RNAas well as viral mRNA was observed in emetine-1 4 treated cells whichsuggestedthat emetine could affect viral genome synthesis in infected cells.

Emetine-induced decreasedsynthesis of viral genome could be due to the reduced synthesis of viral proteins (viral polymerase and other accessory proteins required for virus replication).Nevertheless, Western blot analysis of cells infected withSARS-CoV-2 ( Fig. 5c, upper panel) showed decreased levels of viral proteins in the presence of emetine. However, there were similar levels of cellular housekeeping proteinGAPDH in emetine-treated and untreated cells (Fig. 5c, lowerpanel) which suggested that emetine does not lead to a general shut off /inhibitionof cellular protein synthesis(at least at the concentration we employed).

We further explored the possible inhibitory mechanism of emetine in suppressingthe synthesis of SARS-CoV-2 proteins.In coronaviruses, viral mRNA translation takes place in a cap-dependent manner wherein the eIF4E plays a central role in the initiation of translation (Cencic et al., 2011; Gordon et al., 2020; Kumar et al., 2018; Müller et al., 2018) . Upon activation (phosphorylation) by upstream kinase(s), elF4E binds to 5′ cap of mRNA to initiate translation (Kumar et al., 2018) . We first evaluated the impact of the inhibition of eIF4E activity on SARS-CoV-2 replication. As shown in Fig. 6a ,at a noncytotoxic concentration [(0.5 µg/ml, determined previously by our group (Khandelwal et al., 2020)], addition of 4EGI-1 resulted in a highly significant reduction invirus yield (Fig.   6a )whichsuggested that eIF4E is essential for SARS-CoV-2 replication.

1 5

Next we evaluated if emetine inhibits interaction of the viral RNAwith eIF4E (capbinding protein). At 10 hpi when SARS-CoV-2 RNA was expected to be at its peak level, cells were covalently cross-linked and evaluated for viral RNA and eIF4E interaction in a CHIP assay. As shown in Fig. 6b , α -eIF4E (reactive antibody) but not α -ERK (nonreactive antibody) or beads control immunoprecipitated SARS-CoV-2 RNA. The levels of viral RNA immunoprecipitated by α -eIF4E were >99.9% lower in emetine-treated cells as compared to the PBS-treated cells (Fig. 6b) which confirmed that emetine inhibits eIF4E/SARS-CoV-2 mRNA interaction. In qRT-PCR, the levels of SARS-CoV-2 RNA in α -ERK-treated (but notα-eIF4E-treated cell aliquots) were undetectable (Ct values undetermined) which suggested that α -eIF4E specifically interacted with SARS-CoV-2 RNA (Fig. 6b) . 2020).Addition of a non-cytotoxic concentration (2.5µg/ml) of Apigenin resulted in reduced production ofSARS-CoV-2 in Vero cells (Fig. 7) which further confirmed the virus supportive role of eIF4E in SARS-CoV-2 replication.

We along with some other groups have demonstrated thein vitro antiviral efficacy of emetine against some RNA and DNA viruses (Chaves Valadao et al., 2015; Choy et al., 2020; Deng et al., 2007; Khandelwal et al., 2017; Mukhopadhyay et al., 2016; Ramabhadran and Thach, 1980) . While the development of entirely new antiviral drugs may take several months or even years, repurposing the existing approved drugs could save time and a lot of investments. Following the emergence of COVID-19, we evaluated thein vitro antiviral activity of emetine against SARS-CoV-2. In this study, we have provided some novel mechanistic insights on the antiviral efficacy of emetine against SARS-CoV-2, besides analysingthe antiviral potential of emetine againsta lethal challenge with IBV for the first time.

of emetine is also known to inhibit the replication of certainviruses. While most of the studies on the inhibitory effect of emetine on virus replication are simply based on measuring the virus yields in cell culture (Choy et al., 2020; Ramabhadran and Thach, 1980; Yang et al., 2018) ,somemechanistic insights have also been provided. Emetine can directly inhibit viral polymerase(Chaves Valadao et al., 2015; Khandelwal et al., 2017; Yang et al., 2018) , although the major antiviral activity of emetine is believed to be mediated via targeting certain cellular factors (Khandelwal et al., 2017) . Depending (Yang et al., 2018) . In human cytomegalovirus (HCMV) infection, emetine was shown to inhibit HCMV replication after entry but before initiation of DNA synthesis (Mukhopadhyay et al., 2016) . Likewise, in rabies virus, emetine was also shown to block the axonal transport of the virus particles (MacGibeny et al., 2018) .

Similarly, in vaccinia virus, it was shown to interfere with the virus assembly (Deng et al., 2007) . Most of these studies provide insights on the inhibitory effect of emetine on the specific step(s) of viral life cycle. However, the precise molecular mechanism of emetine action remains unknown.

Viruses are well known to exploit several cellular factors to effectively transcribe and translate their genome (Kumar et al., 2020) . Like several other viruses, coronaviruses also synthesizetheir protein in cap-dependent manner wherein eIF4E plays a critical role in the initiation of translation (Nakagawa et al., 2016) . Since emetine was found to decrease synthesis of viral proteins, we hypothesize ifinhibitory effect of emetine is mediated via disrupting viral mRNA and eIF4E interaction. As in the case of other coronaviruses (Nakagawa et al., 2016) , inhibiting the cellular eIF4E activityresulted in decreasedSARS-CoV-2 replication which suggested the virus supportive role of eIF4E.Further, in a CHIP assay, we demonstrated that emetine inhibitsinteraction of the viral RNA with cap-binding protein eIF4E (Fig. 6b andFig. 8) . eIF4E activation is commonly mediated via RTK/ERK/MNK1/eIF4Esignalling axis (Kumar et al., 2018) .Blocking these upstream eIF4E kinases resulted in reduced SARS-CoV-2 1 8 replication which suggested that ERK/MNK1/eIF4E signalling is prerequisite for SARS-CoV-2 replication (Fig. 8) . Nevertheless previous studies suggest that coronaviruses induces eIF4E activation which plays a virus supportive role in coronavirus (including SARS-CoV-2)life cycle (Cencic et al., 2011; Gordon et al., 2020; Mizutani et al., 2004; Müller et al., 2018) .

Since virus replication requires the presence of the viral structural and nonstructuralproteins, their (viral polymerase), their restricted availability (in the presence of emetine) couldeventually block viral genome replicationwhich we have observed in our study in the form of reduced levels of viral RNA and mRNA. Alternatively, emetine may directly inhibit certain viral polymerases (Aguiar et al., 2007; Chaves Valadao et al., 2015) . However, in a cell free virion polymerase assay, we could not observe any direct effect of emetine on the function of viral polymerase (data not shown) which suggests that the major mechanism of action of emetine is mediated via targeting the cellular factor(s), one (eIF4E) which is described in this study. Like eIF4E, emetine may also target other host-dependency factors. Additional studies that involve transcriptome/proteome/phospho-proteome/lipidome analysis in emetine-treated anduntreated cells are required to precisely elucidate the molecular mechanism of the action of emetine.

The high selective index of emetine suggests its potential as anti-SARS-CoV-2 agent. However, since emetine also targets cellular factors, in vivo cytotoxicity may be a potential difficulty of its use. For the treatment of amebiasis, emetine is given at 1 mg/kg body weight daily for up to 10 days without any side effects (Mastrangelo et al., 1973) .

As an anti-cancer agent (clinical trials), emetine was shown to be well tolerated when delivered intravenously at 1.5 mg/kg doses twice a week (Panettiere and Coltman, 1971 ). In a mouse CMV (MCMV) model, emetine inhibited virus replication at an oral dose of 0.1 mg/kg body weight (Mukhopadhyay et al., 2016) which is somewhat in the similar range required to protect chicken embryos against virulent IBVchallenge in this study (considering an embryo weight of 20 gram and an EC 50 of 2.3 ng/egg). Although the route of administration and potential cumulative cytotoxicity needs to be determined in the case of COVID-19, the doses required for virus inhibition are substantially lower than the traditional emetine doses used to treat amebiasis and other ailments.

Considering these facts in view, it is apparent that emetine has the potential to treat COVID-19.

In vitro antiviral efficacy against SARS-CoV-2 and its ability to protect chicken embryos against virulent IBV suggests that emetine could be repurposed to treat COVID-19. In addition, we provide a novel mechanistic insight into the antiviral activity of emetine. Emetine targets SARS-CoV-2 protein synthesis which is mediated by inhibiting the interaction of SARS-CoV-2 mRNA with the cellular cap-binding protein eIF4E. 

replication.Vero cells, in triplicates, were infected with SARS-CoV-2 in the presence of the indicated inhibitors viz; ERK inhibitor (FR180204: 0.2 µg/ml), MNK1 inhibitor (CGP57380: 0.5 µg/ml), eIF4E inhibitor (0.5 µg/ml) and Apigenin (2.5 µg/ml).The virus yield in the infected cell culture supernatants at 24 hpi was quantified by plaque assay. 

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