1 Can Plitidepsin Be Used as an Antiviral Against RSV?

2

3 Charlotte Estampes¹* (charlotte.estampes@griffithuni.edu.au), Jenna

4 Fix¹*(jenna.fix@inrae.fr), Julien Sourimant¹ (julien.sourimant@inrae.fr), Priscila Sutto-Ortiz²

5 (priscila.SUTTO-ORTIZ@univ-amu.fr), Charles-Adrien Richard¹ (charles-

6 adrien.richard@inrae.fr), Etienne Decroly² (etienne.decroly@univ-amu.fr) Marie Galloux¹

7 (marie.galloux@inrae.fr), and Jean-François Eléouët¹# (jean-francois.eleouet@inrae.fr)

8

9 ¹Unité de Virologie et Immunologie Moléculaires (VIM), INRAE, Université Paris Saclay,

10 78350 Jouy en Josas, France.

11 ²Aix Marseille Université, CNRS, AFMB UMR 7257, Marseille, France.

12 #Corresponding author. Email: [jean-francois.eleouet@inrae.fr](mailto:jean-francois.eleouet@inrae.fr)

13 *Equally contributed to this work

14

15 Keywords: Respiratory Syncytial Virus Plitidepsin eEF1A Inhibition

16

17 The authors declare no conflict of interest

18

19

20 Abstract

21 Human respiratory syncytial virus (HRSV) is a main cause of acute lower respiratory tract

22 infections in infants, the elderly and the immunocompromised patients. Although vaccines

23 have recently been approved for the elderly and for pregnant women, there is no curative
treatment for HRSV. HRSV replicates in the cytoplasm of infected cells, and transcription and
replication of the viral genome depend on the viral RNA polymerase complex, which recruits
cellular factors for RNA synthesis. Among them, the eucaryotic translation elongation factor
1A (eEF1A) was previously shown to be critical for HRSV replication. eEF1A activity can be
inhibited by plitidepsin (Aplidin), a cyclopeptide extracted from ascidian Aplidium albicans,
which was shown highly potent against SARS-CoV-2, with a 50% inhibitory concentration
(IC90) of 0.70 to 1.62 nM depending on the cell line. Here, we investigated whether
plitidepsin could also inhibit HRSV replication. We found that plitidepsin inhibited HRSV
replication with an IC50 of ≈3 nM in cell cultures. However, further investigation revealed
that plitidepsin has pleiotropic effects, affecting the translation of both cellular and viral
proteins in a similar manner. Overall, our results show that plitidepsin blocks cellular
translation and indicate that plitidepsin induces a proteasome-mediated degradation of
eEF1A, also showing the dependence of HRSV replication to cellular factors such as eEF1A.
These results thus highlight an original mechanism of action of plitidepsin on eEF1A, which
render the use of this compound for antiviral therapy very risky.

39

## 40 Introduction

Human respiratory syncytial virus (HRSV) is the commonest cause of lower respiratory tract
infection in young children worldwide and the first cause of their hospitalization (1). HRSV is
estimated to infect about 33 million children leading to more than 3 million hospitalizations,
and 26,000–152,000 deaths in children under 5 years, each year (2). The global healthcare
costs of HRSV-associated infections in young children in 2017 were estimated to be US$5.45
billion (3). In a systemic multisite study, HRSV was shown to be the first etiological agent
47 responsible for severe pneumonia (more than 30%) in hospitalized children in Asia and Africa
48 (4). HRSV infections are also associated with significant morbidity and mortality in the elderly
49 and immunocompromised people (5),(6). The true burden of disease in adults is likely
50 significantly under-recognized, and recent studies indicate that the HRSV impact is similar to
51 that of seasonal influenza in adults older than 65 years (7),(8),(9),(10),(11),(12).

52 HRSV vaccine research has been ongoing for nearly 60 years without success, and
53 only in 2023, two vaccines based on stabilized HRSV prefusion F protein, Arexvy (GSK) and
54 Abrysvo (Pfizer), were approved for medical use for adults aged 60 or older both in the USA
55 and Europe. In September 2023, Abrysvo was also approved in the USA for pregnant women
56 at 32-36 weeks' gestation to prevent HRSV infection in newborn children. Finally, Moderna
57 received U.S. FDA and E.U. approval for RSV Vaccine mRESVIA (13). The only pharmaceutical
58 intervention since 1998 has been passive prophylaxis with Palivizumab, a monoclonal
59 antibody targeting the fusion protein thereby limiting the HRSV entry. However, its use was
60 limited to high-risk infants because of the elevated cost and moderate efficacy (14). But
61 recently, nirsevimab (Beyfortus®), a long-acting monoclonal antibody, was approved by
62 several regulatory agencies around the world for the prevention of HRSV infections in
63 newborns and infants (15). A single intramuscular injection of this antibody should protect
64 infants for an entire season compared with monthly doses, and reduced costs (vaccine-like
65 pricing expected) allowing for administration to all infants. However, there is still no specific
66 curative treatment against HRSV.

67 HRSV is a nonsegmented single-stranded negative-sense RNA virus of the
68 *Mononegavirales* order, *Pneumoviridae* family, and *Orthopneumovirus* genus (16). The HRSV
69 genome is approximatively 15.2 kb long, and contains 10 genes encoding 11 proteins (17).
70 Replication and transcription rely on four of these proteins: the nucleoprotein N involved in
71 genome and antigenome encapsidation, forming the ribonucleoprotein complex (NC), the
72 RNA-dependent RNA polymerase L which exhibits all the enzymatic activities required for
73 viral replication, transcription and RNA capping, its cofactor the phosphoprotein P
74 responsible for recruitment of L on the NC template, and the transcription factor M2-1 that
75 has been described as an "antiterminating" factor during transcription and interacts with P
76 and viral mRNAs (18). All viral RNA synthesis take place in viral factories also called
77 cytoplasmic inclusion bodies (IBs) (19), which concentrate the viral proteins L, N, P, and M2-
78 1, but also cellular proteins, such as the protein phosphatase 1 (PP1), HSP70 or the human
79 translation elongation factor eEF1A, all involved in HRVS replication (20),(21),(22).
80 Interestingly, it was found that many RNA viruses utilize eEF1A for replication, although the
81 mechanisms by which they do this differ (23). In a previous study in which eEF1A was
82 knocked down or inhibited with didemnin B, it was suggested that eEF1A plays a key role in
83 the regulation of F-actin stress fiber formation required for HRSV assembly and release, but
84 with no effect on HRSV genome replication (24). Furthermore, eEF1A was found associated
85 with the N and P proteins in infected cells by using a proximity ligation assay and co-
86 immunoprecipitation (22). Recently, plitidepsin (Aplidin), an analog of didemnin B and a
87 potent anti-cancer agent targeting eEF1A2 ($K_D=80\mu M$) (25), was shown highly potent
88 against SARS-Cov-2 by targeting eEF1A, with a 90% inhibitory concentration ($IC_{90}$) of 0.88
89 nM (26). As suggested by the authors, since HRSV also uses eEF1A for viral replication,
90 inhibition of eEF1A could be a new strategy to limit HRSV propagation/infection. In this
91 work, we thus investigated the effect of plitidepsin on HRSV replication. We show that
92 plitidepsin inhibits HRSV replication in infected cells as well as a minigenome reporter
93 system with an $IC_{50}$ of ≈3 nM in cultured cells. Further mechanistic investigation revealed
94 that plitidepsin induces the degradation of eEF1A, inhibiting the translation of both viral and
95 cellular proteins in a similar range of concentration.

96

97 **Methods**

98 **Cells**

99 BSRT7/5 cells (BHK-21 cells that constitutively express the T7 RNA polymerase) (27) and HEp-
100 2 cells (ATCC: CCL-23) were maintained respectively in DMEM and MEM supplemented with
101 10% heat-inactivated fetal calf serum (FCS), with 2 mM glutamine, 100 µg/ml penicillin and
102 100 U/ml streptomycin. Cells were grown in an incubator at 37 °C in 5% CO2. Transfection
103 were performed with 2.5µL of Lipofectamine 2000 (Thermofisher) per 1 µg of DNA according
104 to the manufacturer instructions.

105

106 **Viruses**

107 Recombinant human RSV rHRSV-mCherry corresponding to HRSV Long strain expressing the
108 mCherry protein was amplified on HEp-2 cells and titrated using a plaque assay procedure as
109 previously described (28),(29).

110

111 Plitidepsin antiviral activity

112 Plitidepsin (MedChemExpress) and Carfilzomib (Cell Signaling #15022) were solubilized in
113 DMSO at 1 mM and 5 mM as stock solutions, respectively. HEp-2 cells in 96-well plates were
114 infected for two hours with rHRSV-mCherry at an MOI of 0.2, in the absence of FCS. The
115 medium was then changed by the same medium with 2% FCS and containing serial dilution
116 of plitidepsin, with a 1% final concentration of DMSO in the culture medium. At 48 h
117 postinfection, the red fluorescence intensity of mCherry was quantified using a Tecan infinite
118 M200Pro spectrofluorometer with excitation and emission wavelengths of 580 nm and 620 nm, respectively. Values obtained for nontreated infected and noninfected cells were used for standardization and normalization. In parallel, the toxicity of the treatment was assessed on non-infected cells using the CellTiter-Glo Luminescent cell viability assay (Promega). The half-maximal inhibitory concentration (IC₅₀) and cytotoxic concentration (CC₅₀) were determined by fitting the data to the dose-response curve implemented in GraphPad version 8 software.

125

126 Fluorescence microscopy

127 HEp-2 cells infected with rHRSV-mCherry were fixed with PBS-paraformaldehyde 4% for 20 min at room temperature, rinsed with PBS, and permeabilized with PBS-BSA 1% - Triton X-100 0.1% for 10 min. Nuclei were stained with Hoechst 33342 (1 µg/ml) for 5 min, washed with PBS, examined under a Nikon TE200 microscope equipped with a CoolSNAP ES2 (Photometrics) camera, and images were processed with Meta-Vue software (Molecular Devices).

133

134 Minigenome assay

135 BSRT7/5 cells at 90% confluence in 96-well dishes were transfected with a plasmid mixture containing 125 ng of pM/Luc, 125 ng of pN, 125 ng of pP, 62.5 ng of pL, and 31 ng of pM2-1 as well as 31 ng of pRSV-β-Gal (Promega) to normalize transfection efficiencies as described previously (30),(31). After 6 h, the transfection mix was removed and serial dilutions of plitidepsin were added for 14 h. Transfections were done in triplicate, and each independent transfection was performed three times. Cells were harvested 24 h post-transfection, then lysed in luciferase lysis buffer (30 mM Tris pH 7.9, 10 mM MgCl₂, 1 mM DTT, 1% Triton X-100,
142 and 15% glycerol). The luciferase and β-galactosidase (β-Gal) activities were determined for
143 each cell lysate with an Infinite 200 Pro (Tecan, Männedorf, Switzerland) and normalized
144 based on the values obtained for cells treated with DMSO only.

145

146 Fluorescence-based nucleotide-incorporation assay

147 Each reaction contained 0.2 µM recombinant RSV L-P, 2 µM of oligonucleotide template and
148 2 µM of a 5'-6-FAM primer, mixed in a buffer containing 20 mM Tris-HCl pH 7.5, 10 mM KCl,
149 2 mM DTT, 0.01% Triton X-100, 5% DMSO, 0.2 U/mL RNasin (Ambion), and 6 mM MgCl₂.
150 Reactions were started by adding specific NTPs at 100 µM and incubated for 2h at 30°C.
151 Reactions were quenched by adding an equal volume of gel-loading buffer. Samples were
denatured at 70°C for 10 minutes and run on a 20% polyacrylamide urea sequencing gel for
152 2.5 hours at 45 W. The gel scanned on a Typhoon imager (GE Healthcare). (Unpublished
153 methodology Sutto et al. 2024)

155

156 Western blotting

157 Cells were lysed in 2X Laemmli buffer, run on a 12% polyacrylamide gel and transferred to a
158 nitrocellulose membrane using the Trans-Blot Turbo system (Bio-Rad). The blots were
159 blocked with 5% nonfat milk in PBS Tween20 0.2%, and probed with either a polyclonal
160 rabbit anti-N serum (32), a rabbit anti-GFP (Invitrogen, Whaltham, MA, USA), a mouse
161 monoclonal anti-EF1A antibody (Santa Cruz G-8, sc377439), a mouse monoclonal anti-
162 GAPDH antibody (Sigma-Aldrich MAB374) or a mouse anti-alpha-tubulin (DM1A, Sigma) and
163 further incubated with HRP-coupled anti-mouse or anti-rabbit antibodies (Life Science).
164 Membranes were revealed with the Clarity Western ECL kit (Bio-Rad) and analyzed with a
165 ChemiDoc Touch Imaging System and Image Lab software (Bio-Rad).
166

167 **Pulse labelling of newly synthesized proteins with L-azidohomoalanine (AHA)**

168 HEp-2 cells were infected with rHRSV-mCherry at MOI = 1 in 6-wells plates (35 mm in diameter) and incubated in MEM supplemented with 2% heat-inactivated FCS, 2 mM glutamine, 100 µg/ml penicillin and 100 U/ml streptomycin. 24 h later, serial dilutions of plitidepsin or DMSO were added to the cells for 2 h. Cells were washed twice with methionine-free (Met-) MEM (Gibco), then live-labeled (pulsed) at 37°C for 4 h with 50 µM Click-iT L-azidohomoalanine (AHA; Jena Bioscience) in Met-MEM supplemented with 5-10% dialyzed FBS (Gibco) and plitidepsin. Cells were lysed in 1 ml of in RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Triton X-100, 2 mM, DTT, 10 mM of freshly prepared iodoacetamide [Merck] and complete protease inhibitor cocktail [Roche]). After centrifugation for 10 min at 10,000 rpm at 4°C, the supernatants were mixed with 5 µM AFDye 488-DBCO (Click Chemistry Tools, Jena Bioscience) and incubated for 2 h at 21°C. Proteins were immunoprecipitated with specific antibodies coupled to protein A sepharose beads (GE Healthcare) and analyzed by SDS-PAGE, together with the supernatants. Fluorescence was analyzed with a Fuji FLA-3000 scanner and quantified using the AIDA or Fiji softwares.

183

184 **RESULTS**

185 **Antiviral potency of plitidepsin against HRSV**

186 By using an siRNA approach, it was demonstrated ten years ago that downregulation of eEF1A correlates with a reduction in the amount of infectious HRSV release, accompanied by a reduction in viral genome expression, but not mRNA transcription or protein expression
189 (22). We therefore tested whether plitidepsin, known to inhibit the eEF1A activity, displays
190 antiviral activity against HRSV. For this purpose, HEp-2 cells were infected with rHRSV-
191 mCherry at MOI = 0.2 in a culture medium containing increasing concentration of plitidepsin.
192 Viral replication was quantified by mCherry fluorescence measurement on live cells 48 h
193 post infection using the Tecan spectrofluorometer. Values were standardized and
194 normalized by those of obtained for nontreated infected and noninfected cells. The IC₅₀ was
195 determined by dose-response curve fitting. In parallel, the toxicity of plitidepsin was
196 evaluated on non-infected HEp-2 cells. As shown in Figure 1A, plitidepsin inhibited rHRSV-
197 mCherry replication in a dose-dependent manner, with an IC₅₀ of 3.5 nM. These data were
198 confirmed by direct observation of mCherry fluorescence and nuclei staining on fixed rHRSV-
199 mCherry-infected cells (Figure 1B). Noteworthy, a cytotoxic activity of plitidepsin was
200 observed, with a calculated half-maximal cytotoxic concentration (CC₅₀) close to 145 nM
201 (Figure 1A). With a calculated selective index >30, plitidepsin thus appeared to be a potent
202 inhibitor of HRSV replication in HEp-2 cells.

203

204 Effect of plitidepsin on a HRSV minigenome

205 To determine whether plitidepsin could affect the RNA polymerase replication/transcription
206 machinery of HRSV, we used a well-established HRSV-specific minigenome assay system
207 (30). The pM/Luc plasmid, which contains the authentic M/SH gene junction and the Luc
208 reporter gene downstream of the gene start sequence inserted in this gene junction, was co-
209 transfected in BSR-T7/5 cells together with p-β-gal, pL, pP, PN, and pM2-1. Luciferase activity
210 was determined and normalized based on the signal obtained for cells incubated in the
211 similar media with 1% of DMSO. In parallel, β-galactosidase (β-Gal) activity was determined
212 for each cell lysate in order to normalize transfection efficiency. Figure 2A shows that
213 plitidepsin inhibits the function of the HRSV polymerase complex in a dose-dependent
214 manner with an IC₅₀ of ~5 nM while showing no toxicity towards BSR-T7/5 cells. However,
215 plitidepsin also inhibited, to a lesser extent, the β-Gal activity, the effect being visible from
216 3.3 nM of plitidepsin.

217 Since plitidepsin targets the eEF1A cellular translation factor, the inhibition of HRSV
218 RNA polymerase activity could result from either the direct inhibition of polymerase activity
219 of the HRSV L protein or from the inhibition of translation of the viral mRNAs coding for the
220 viral proteins. To assess whether the plitidepsin treatment had a specific or global impact on
221 proteins expression, cells were co-transfected either with the minigenome system or with a
222 plasmid encoding EGFP, in the presence of increasing concentrations of plitidepsin. The
223 expression of proteins was analyzed by Western blot. As shown on Figure 2B, a similar
224 decrease in N and EGFP expression was observed in the presence of plitidepsin, indicating
225 that plitidepsin had a general inhibitory effect on viral or non-viral proteins expression. In
226 contrast, the signal corresponding to alpha-tubulin did not decrease significantly, which can
227 easily be explained by the half-life of this cellular protein (~8 days) (33).

228

229 **Effect of plitidepsin on *in vitro* HRSV RdRp activity**

230 To test whether plitidepsin could also affect directly the HRSV RNA polymerase activity, we
231 performed an *in vitro* RNA polymerase assay using a recombinant L-P complex (34),(35),(36)
232 in the presence of increasing plitidepsin concentrations. As shown on Figure 3, no inhibitory
233 effect of plitidepsin on the RNA synthesis was observed, even for a concentration of 100 µM
234 of plitidepsin. These results strongly suggest that the effect of plitidepsin on viral replication
235 was not due to a direct effect of the RdRp complex but mainly due to its impact on protein
236 expression.
237

238 **Effect of plitidepsin on cellular and viral proteins neosynthesis**

239 To further investigate the impact of plitidepsin on viral and cellular RNA translation, we used
240 a complementary approach, consisting in a pulse labelling of newly synthesized proteins with
241 the methionine analog L-azidohomoalanine (AHA) revealed by the fluorescent dye AFDye
242 488-DBCO based on Click chemistry (37). At 24 h postinfection, HEp-2 cells were incubated
243 with serial dilutions of plitidepsin and then labelled with AHA for 1 h. Then, cells were lysed
244 and the HRSV N protein or the cellular α-tubulin protein were immunoprecipitated.
245 Immunoprecipitated proteins and total cell lysates were analyzed by SDS-PAGE and the
246 fluorescence was quantified. As shown on Figure 4A and 4B, a similar drop in protein
247 synthesis was observed for N and α-tubulin, but also for the total proteins present in the cell
248 lysates for concentrations ≥ 15 nM.

249

250 Plitidepsin induces proteasome-mediated degradation of eEF1A

251 The mechanism by which plitidepsin inhibits the activity of the host factor eEF1A is still
debated. It was shown that overexpression of a negative dominant mutant an Ala399 → Val
252 (A399V) of eEF1A reduced sensitivity of cancer cells to didemnin B (38) as well as SARS-CoV-
253 2 to plitidepsin by a factor >10 (26). Interestingly, this A399V substitution also confers
254 resistance to the structurally unrelated ternatin-4 (38), suggesting that these molecules
255 interact with the same binding site on eEF1A (26). However, the mechanism of action of
256 ternatin-4 was recently studied in more details and revealed that it was mediated by
257 ubiquitination and proteasome degradation of eEF1A (39). We thus wondered whether
258 plitidepsin could also induce degradation of eEF1A by a similar mechanism. BSRT7 cells were
259 treated with serial dilutions of plitidepsin in the presence or absence of the proteasome
260
261 inhibitor carfilzomib and the expression of eEF1A or the control GAPDH were analyzed by
262 Western blot. As shown on Figure 5, at the RSV inhibitory concentration of plitidepsin,
263 minigenome activity or translation inhibition, eEF1A expression was impaired. However,
264 expression of eEF1A was restored upon treatment in the presence of the proteasome
265 inhibitor Carfilzomib. These results thus strongly suggest that plitidepsin induces the
266 degradation of eEF1A by ubiquitination and proteasome degradation in a similar way to
267 ternatin-4.

268

269 DISCUSSION

270 The mammalian translation elongation factor eEF1A is an essential GTPase and the second
271 most abundant intra-cellular protein after actin (3% of the total cellular protein). It is
272 localized extensively in the cytoplasm and nucleus (40),(41),(23). The canonical function of
273 eEF1A is to deliver amino acyl tRNAs to the ribosomal A site during the elongation stage of
274 protein synthesis. In addition to its canonical functions in transporting aa-tRNA to the
275 ribosome, eEF1A is found to be involved in cellular mechanisms such as regulation of
276 cytoskeleton organization by interacting with actin and tubulin, protein degradation
277 mediated by the proteasome, nuclear aa-tRNAs and protein export, signaling transduction
278 pathway concerning apoptosis and oncogenesis, or binding to viral RNA (42),(41),(43). There
279 are two variants of eEF1A, eEF1A1 and eEF1A2, that share 92% amino acid identity (44). In
280 contrast to the ubiquitous expression of eEF1A1 in many cell types, eEF1A2 expression is
281 limited to the terminally differentiated cells of the brain, heart, and skeletal muscle (45). The
282 two eEF1A variants have similar translation activity but may differ with respect to their
283 secondary, "moonlighting" functions. eEF1A1 also plays an important role in the process of
284 heat shock stress response. eEF1A2 activates Akt in a PI3K-dependent fashion, stimulating
285 cell migration, actin remodeling and invasion, and inhibiting apoptosis (23).

286 Like for many viruses (23),(41), eEF1A has been identified as an important cellular
287 factor for HRSV replication (22). As an abundant, multifunctional protein, it is not surprising
288 that many viruses have adapted to use eEF1A as a cofactor for viral transcription,
289 translation, assembly, and pathogenesis. So, eEF1A was shown to bind to some viral RNA
290 structures, some viral structural and nonstructural proteins, or to interact with viral
291 polymerase complexes, such as the one of Vesicular Stomatitis Virus (VSV) (46),(41). Like for
292 HRSV, VSV forms two different viral RNA polymerase complexes in infected cells: the
293 transcriptase and the replicase. The transcriptase complex synthesizes capped mRNAs,
294 whereas the replicase complex initiates genomic minus-strand RNA synthesis at the precise
295 3' ends of the plus-strand antigenomic and negative-strand genomic RNAs. The transcriptase
296 was described as a protein complex containing the L and P proteins, the cellular eEF1A and
297 heat shock protein 60, and a submolar amount of cellular mRNA cap guanylyltransferase
298 (47). The replicase complex was described as containing the viral proteins L, P, and
299 nucleocapsid (N) but not eEF1A, heat shock protein 60, or the guanylyltransferase. Hence, it
300 was proposed that eEF1A is important for transcription of viral mRNAs but not for genomic
301 RNA replication in VSV.

302 Disentangling the canonical and noncanonical roles of eEF1A and the extent to which
303 each contributes to viral function is essential if eEF1A is to be targeted therapeutically. It was
304 previously shown that didemnin B treatment, a drug targeting eEF1A, protected cells from
305 HRSV-induced cell death (22). Didemnin B did not significantly affect HRSV transcription and
306 replication, especially at 24 h post-infection, but significantly reduced infectious virus
307 production and release, possibly as a consequence of changes in actin stress fiber formation
308 (24). More recently, plitidepsin (Aplidin), an analog of didemnin B that was approved in 309 Australia for the treatment of myeloma (48), was shown to be very efficient against SARS- 310 CoV-2 in cultured cells as well as in a mice model (26). Since it has entered clinical trials as an 311 anti-SARS-CoV-2 drug and was shown as a favorable long-term safety profile in adult patients 312 hospitalized for COVID-19 (49),(50),(51),(52), we wondered whether this compound could 313 also be efficient against HRSV. Plitidepsin is a cyclic depsipeptide that was first isolated from 314 a Mediterranean marine tunicate (Aplidium albicans) and, at present, is manufactured by 315 total synthesis and commercialized by PharmaMar, S.A., as Aplidin®(53). Plitidepsin has 316 antitumoral, and immunosuppressive activities (54). Although not fully clarified, the 317 molecular mechanisms of action of plitidepsin against tumor cells and SARS-CoV-2 have been 318 investigated. Plitidepsin induces cell-cycle arrest and apoptosis (55). These effects rely on 319 the induction of early oxidative stress, the rapid activation of Rac1 GTPase, and activation of 320 c-Jun N-terminal kinase (JNK), ERK and p38 mitogen-activated protein kinases (p38/MAPK), 321 which finally result in caspase-dependent apoptosis (56),(57). It was determined that eEF1A 322 is the primary target of plitidepsin, which can bind to eEF1A at the interface between 323 domains 1 and 2 of this protein in the GTP conformation with a measured KD of 80 nM (25). 324 However, it was proposed that the antitumoral effect of plitidepsin is not due to translation 325 inhibition but inhibition of eEF1A binding to double-stranded RNA-dependent protein kinase 326 (PKR). In the presence of plitidepsin, PKR would disengage from eEF1A, thereby regaining its 327 kinase activity to initiate extrinsic apoptosis through activation of MAPK and NF-κB signaling 328 cascades (58). Plitidepsin was also reported as an ER stress inducer by activating the 329 unfolded protein response (59). In parallel, plitidepsin was also shown to induce the 330 phosphorylation of eIF2α, resulting in the arrest of protein synthesis at the initiation step 331 (59),(60). For SARS-CoV-2, it has been determined that the effect of plitidepsin is also
332 mediated by eEF1A by using a mutated (A399V) version of eEF1A1 in 293T cells (26). It is
333 likely that this effect is mediated by inhibition of translation of the viral proteins (26),
334 (61),(55). Very recently, the mechanism of SARS-CoV-2 inhibition was revisited (62). In this
335 paper, using Vero E6 cells, the authors found that plitidepsin at 50 nM reduced the
336 translation of RNAs, including cellular RNAs, but with a higher impact on viral mRNAs
337 translation and without affecting cellular viability. The molecular mechanisms involved cell's
338 proteostatic response to eEF1A blockade, namely a shift from cap or internal ribosome entry
339 sites (IRES)-mediated translation towards a N-6-methyladenosine (m6A)-dependent
340 translation, that could explain cell survival. They also tested plitidepsin against several other
341 viruses including HRSV, and found an IC50 of 27 nM in Vero E6 cells, about ten times more
342 than what we found using HEp-2 cells. For in vivo effects, it was also shown that treatment
343 of a monocyte-derived macrophage cell line by plitidepsin in the same range than ours (2.5-
344 10 nM) reduced the production of the proinflammatory cytokine IL-6 in the presence of
345 SARS-CoV-2 virions, and that was associated with a reduction of NF-κB p65 subunit
346 phosphorylation and of its transcription activity in the inflammatory cascade (63). This effect
347 was also observed in vivo in SARS-CoV-2 or Influenza virus infected mice.

348 In our experiments, we observed that plitidepsin had an inhibitory effect on viral
349 replication and minigenome activity with an IEC50 ≈ 3-5 nM, and a toxic effect on cells with
350 an IC50 ≈ 150 nM. However, further investigation revealed that this inhibitory effect was due
351 to a general inhibition of translation in the host cell, with no significant difference between
352 cellular and viral proteins expression. We then wondered whether eEF1A, which is essential
353 for cellular translation, could be actively degraded by plitidepsin treatment, as it was
354 observed recently with ternatin-4, another eEF1A-targeting drug (39). We found that eEF1A
355 was degraded after plitidepsin treatment for 18H at concentrations as low as 1 nM, which
356 was inhibited by proteasome inhibitor carfilzomib, indicating that a eEF1A ubiquitination
357 mechanism occurred, that remain to be determined. During this study it was published by
358 Molina et al. that cellular cap-dependent translation was inhibited by plitidepsin (see above).
359 However, using pulse-chase experiments, we found that cellular translation was mainly
360 affected (see Figure 4).

361 In conclusion, we show here for the first time that treatment of cells with plitidepsin
362 induces the degradation of eEF1A by the proteasome pathway, which is correlated with a
363 global extinction of translation. These experiments also show the dependance of HRSV
364 replication to the cellular factor eEF1A. Although effective concentrations of plitidepsin
365 against HRSV are in the same range that those found for SARS-CoV-2, the side effects of
366 plitidepsin on cells and its potential toxicity raises the question of its use in vivo for antiviral
367 assays against HRSV.

368

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627

628

629 **Figures legend**

630 **Figure 1.** Impact of plitidepsin treatment on HRSV replication on HEp-2 cells. (A) Cells were
631 infected for 2 h with rHRSV-mCherry at MOI 0.2 and the medium was then replaced to
632 incubate cells in the presence of serial dilutions of plitidepsin for 48 h (red curve). The viral
633 replication was quantified by measurement of the mCherry fluorescence. In parallel, cell
634 viability upon treatment with plitidepsin was quantified in non-infected HEp-2 cells (black
635 curve). Error bars are standard deviations from duplicates. Data are representative of three
636 experiments. The curves were fitted in Graph Pad 8 software using a four parameters logistic
637 (4PL) regression. Both IC50 and CC50 are indicated. (B) Representative images of HEp-2 cell
638 cultures infected with rHRSV-mCherry at MOI 0.2, 48 hours post-infection in the absence or
639 presence of plitidepsin at 12, 4, and 1.4 nM. Nuclei were colored with Hoechst 33432. Scale
640 bars, 250 µm.

641 **Figure 2.** Plitidepsin effect on a HRSV minigenome. (A) BSRT7/5 cells were transfected with
642 plasmids encoding the N, P, M2–1, and L proteins; the pMT/Luc minigenome, together with
643 pCMV-beta-gal for transfection standardization. Serial dilutions of plitidepsin were added 6 h
644 post-transfection after removing transfection reagents. Viral RNA synthesis was quantified
645 by measuring the luciferase activity after cell lysis 24 h post-transfection. Each luciferase
646 minigenome activity value was normalized based on DMSO treated cells and is the average
647 of three independent experiments performed in triplicate. Error bars represent standard
648 deviations calculated based on three independent experiments made in triplicate. (B) BSRT7
649 cells were transfected with the complete minigenome system (see above) or with the pEGFP
650 plasmid coding for EGFP. Six hours later, the transfection mix was removed and medium
651 containing plitidepsin at different concentrations was added. Then, 24h post-transfection,
652 cells were lysed and analyzed by Western blotting using anti-N, anti-GFP or anti-alpha
653 tubulin antibodies.

654 **Figure 3.** *In vitro* effect of plitidepsin on HRSV RNA polymerase (L-P complex) activity. Primer
655 extension polymerase assay was performed in the presence of increasing concentration of
656 plitidepsin. Briefly, 200 nM of RSV L/P was incubated with 2 µM of 11 nucleotide length RNA
657 template and 4 nucleotide primer in presence of increasing concentrations of plitidepsin.
658 The reactions were incubated for 30' and 120 min at 30°C and the reaction products were
659 separated on a 20 % UREA-PAGE gel before gel scanning on a Typhoon imager.

660 **Figure 4.** Compared effect of plitidepsin on viral or cellular proteins synthesis. HEp-2 cells in
661 6 well plates were infected with rHRSV-mCherry at MOI = 1. 24 h later, medium was changed
662 for protein labelling with AHA with serial dilutions of plitidepsin. After 4 h, cells were lysed
663 and AHA-labelled proteins were revealed with AFDye 488-DBCO, then immunoprecipitated
664 with anti-N or anti-alpha-tubulin antibodies. Immunoprecipitated proteins or supernatants
665 were resolved by SDS-PAGE (A) and fluorescence in the gels was measured using a Fuji FLA-
666 3000 scanner (B).

667 **Fig. 5.** Antiviral mechanism of action of plitidepsin is mediated through proteasome-mediated degradation of eEF1A. BSRT7/5 cells were treated with serial dilutions of
669 plitidepsin or DMSO for 18H in the presence or not of the proteasome inhibitor Carfilzomib

670 (500 nM) and the expression of eEF1A or GAPDH were analyzed by WB.

671
<figure><img src="image_1.png" /><figcaption>672 Figure 1</figcaption></figure>

<figure><img src="image_2.png" /></figure>

673

674

<figure><img src="image_3.png" /><figcaption>675 Figure 2</figcaption></figure>

676

677

678
679 Figure 3

680

<figure><img src="image_4.png" /></figure>

681

682

683

684

685 Figure 4

A

<figure><img src="image_5.png" /></figure>

686

687

B

<figure><img src="image_6.png" /></figure>
688 Figure 5

689

<figure><img src="image_7.png" /></figure>

690