# The SARS-CoV-2 envelope PDZ binding motif acts as a virulence factor disrupting host's epithelial cell-cell junctions [brief report]

Flavio Alvarez, Guilherme Dias de Melo, Florence Larrous, Lauriane Kergoat, Batiste Boëda, Vincent Michel, Danielle Seilhean, Magali Tichit, David Hing, David Hardy, et al.

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To cite this version:

Flavio Alvarez, Guilherme Dias de Melo, Florence Larrous, Lauriane Kergoat, Batiste Boëda, et al. The SARS-CoV-2 envelope PDZ binding motif acts as a virulence factor disrupting host's epithelial cell-cell junctions [brief report]. Cellular and Molecular Biology Letters, 2025, 30 (1), pp.80. 10.1186/s11658-025-00758-y. http://hal-05162178

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BRIEF REPORT

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# The SARS-CoV-2 envelope PDZ binding motif acts as a virulence factor disrupting host's epithelial cell-cell junctions

Flavio Alvarez<fnref n="1" />,<fnref n="2" />* , Guilherme Dias de Melo<fnref n="3" />†, Florence Larrous<fnref n="3" />†, Lauriane Kergoat<fnref n="3" />, Batiste Boëda<fnref n="4" />, Vincent Michel<fnref n="5" />, Danielle Seilhean<fnref n="6" />, Magali Tichit<fnref n="7" />, David Hing<fnref n="7" />, David Hardy<fnref n="7" />, Etienne Kornobis<fnref n="8" />, Hervé Bourhy<fnref n="3" />†, Nicolas Wolff<fnref n="1" />† and Célia Caillet-Saguy<fnref n="9" />†

## Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), disrupts the alveolar epithelial barrier and exacerbates airway inflammation, leading to acute respiratory failure. The Envelope (E) protein is key to virulence, notably through its PDZ-binding motif (PBM), which interacts with host PDZ proteins, affecting signaling pathways and pathogenicity. This study investigates the PBM's role in virulence by generating PBM-deficient mutant viruses and assessing their impact in vitro and in vivo. The mutants showed delayed replication and reduced cytopathic effects in vitro. In vivo, infected hamsters exhibited less weight loss, lower viral loads, and reduced inflammation, indicating attenuated pathogenicity. Histological analysis confirmed milder airway damage. Additionally, PBM-deficient viruses had impaired interactions with tight junction proteins like ZO-1, a PDZ-containing protein essential for epithelial integrity. Although the PBM played a key role in airway pathology, its impact on neuroinvasion was minimal during the acute phase of infection. Thus, the E protein PBM plays a critical role in SARS-CoV-2's fitness, virulence, and pathogenicity, through the disruption of cell junctions and inflammation, underscoring its potential as a therapeutic target.

**Keywords:** SARS-CoV-2, E protein, PDZ binding motif, Tight junctions, Virulence, Epithelial barrier, Inflammation, ZO-1, Viral pathogenesis

## Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, has posed a significant threat to global health. This pandemic has highlighted the critical need to understand the molecular mechanisms governing the virus's pathogenicity and virulence. Among its various pathogenic effects, SARS-CoV-2 has been implicated in disrupting the alveolar epithelial barrier [1, 2] and exacerbating airway inflammation [3–5], which can lead to acute respiratory failure. However, the intricate molecular mechanisms governing these effects

<footnote>†Guilherme Dias de Melo and Florence Larrous have contributed equally.</footnote>

<footnote>Hervé Bourhy, Nicolas Wolff and Célia Caillet-Saguy have contributed equally.</footnote>

<footnote>*Correspondence: flavio.alvarez@pasteur.fr</footnote>

<footnote n="1">¹ Institut Pasteur, Signaling and Receptors Dynamics Unit, Université Paris Cité, 75015 Paris, France</footnote>

<footnote n="2">² Collège Doctoral, Sorbonne Université, 75005 Paris, France</footnote>

<footnote n="3">³ Institut Pasteur, Lyssavirus Epidemiology and Neuropathology Unit, Université Paris Cité, 75015 Paris, France</footnote>

<footnote n="4">⁴ Institut Pasteur, Cell Polarity Migration and Cancer Unit, 75015 Paris, France</footnote>

<footnote n="5">⁵ Institut Pasteur, Pathogenesis of Vascular Infections Unit, Université Paris Cité, 75015 Paris, France</footnote>

<footnote n="6">⁶ AP-HP, Hôpital Pitié-Salpêtrière, Département de Neuropathologie, Sorbonne Université, Paris, France</footnote>

<footnote n="7">⁷ Institut Pasteur, Université Paris Cité, Histopathology Platform, 75015 Paris, France</footnote>

<footnote n="8">⁸ Institut Pasteur, Université Paris Cité, Bionics C2RT, 75015 Paris, France</footnote>

<footnote n="9">⁹ Institut Pasteur, Humoral Immunology Unit, Université Paris Cité, 75015 Paris, France</footnote>
remain incompletely understood while they are essential for identifying potential therapeutic targets.

SARS-CoV-2 is an enveloped virus with a positive strand RNA genome encoding non-structural and structural proteins essential for its replication and pathogenicity. Among the latter, the multifunctional Envelope (E) protein emerges as a critical virulence factor [6–8], orchestrating crucial aspects of the viral life cycle such as virus assembly, budding, and release. Studies on both SARS-CoV-1 and SARS-CoV-2 have demonstrated that viruses lacking the E protein exhibit significantly reduced titers, underscoring the protein’s necessity for virus particle formation [9–11]. While extensive research has been made in understanding the roles of other structural proteins such as the Spike, the Membrane, and the Nucleocapsid proteins, the specific mechanisms and effects of the SARS-CoV-2 E protein on virulence, epithelial junctions, and mucosal inflammation remain less well understood and warrant further investigation [12, 13]. The E protein of SARS-CoV-2 contains a type 2 PDZ-Binding Motif (PBM) at its C-terminus (DLLV<sub>COOH</sub>) with the consensus sequence X-φ-X-φ<sub>COOH</sub> (where X represents any amino acid and φ a hydrophobic residue), identical to that of SARS-CoV-1 [7]. PDZ domains are common protein-protein interaction motifs found in many host cell proteins, including those crucial for maintaining cellular junctions and polarity. The SARS-CoV-1 and –2 PBMs are known to interact with host PDZ domain-containing proteins, modulating host cell signaling pathways and contributing to the virus’ pathogenic profile. Indeed, previous studies have underscored the harmful impact of high viral loads on airway epithelial tight junctions, involving for instance interactions with Proteins Associated with Lin Seven 1 (PALS1) and Syntenin [7, 14, 15]. The E protein PBM is highly conserved across the SARS-CoV-2 variants [16, 17] and the related coronaviruses [7]. This conservation suggests an evolutionary advantage, highlighting the PBM’s essential role in viral adaptation and host interaction. The consistent presence of this motif across diverse coronavirus species underscores its critical function in viral survival and pathogenesis.

In addition to airway damage, SARS-CoV-2 infection is characterized by a wide array of acute and chronic neurological symptoms, including headaches, seizures, attention and sleep disorders, short-term memory loss, and neuropsychiatric symptoms such as anxiety and depression [18–20]. These neurological aspects of the infection are particularly intriguing as the mechanisms of neuroinvasion are not well understood and may involve the crossing of cerebral barriers, such as the blood–brain barrier (BBB) and the blood–cerebrospinal fluid barrier (BCSFB) [21, 22]. These structures, especially the choroid plexus, are rich in PDZ-containing proteins such as ZO-1, which are essential for cellular junctions [23, 24]. The E protein of SARS-CoV-2 has been reported to upregulate inflammation pathways in the brain, accompanied by depression-like behaviors and dysosmia when injected directly into the brain [25]. The disruption of cell junctions remains controversial in literature [26, 27], but the PBM of the SARS-CoV-2 E protein might play a pivotal role in this process and raises questions concerning the specificity of epithelial disruptions during infection.

In our previous high-throughput interactomic screening, we identified ten human PDZ-containing proteins that bind to the SARS-CoV-2 E protein PBM [28]. Many of these proteins, such as ZO-1 (also called TJP1), PARD3, MLLT4 (also called Afa-din), LNX2, and MPP5 (also called PALS1), play crucial roles in cellular junctions and
polarity. These PDZ-PBM interactions were characterized by structural and functional approaches. These PBM-dependent interactions trigger the colocalization and sequestration of PDZ domains with the full-length E protein in cells, specifically in the Golgi compartment [29].

In this study, we aimed to elucidate the specific role of the E protein PBM in SARS-CoV-2 virulence and pathogenicity. We generated and characterized two recombinant viruses lacking the PBM, either through deletion (E-ΔPBM) or mutation (E-MutPBM). Both in vitro and in vivo experiments demonstrated that the absence of the PBM significantly impaired viral fitness, resulting in delayed replication and reduced cytopathic effects. In golden hamsters, PBM-deficient viruses led to milder symptoms, less weight loss, and reduced airway inflammation and lesions, as confirmed by histological and transcriptomic analyses. Using human bronchial epithelial models, we found that the E protein's subcellular localization is independent of the PBM, but the PBM is essential for interacting with the tight junction proteins ZO-1, thereby affecting epithelial barrier integrity during infection. While further research is needed to understand the mechanisms of neuroinvasion and epithelial barrier crossing, our findings suggest that the E protein PBM does not significantly affect neuroinvasion processes. Together, these results emphasize the essential role of the E protein PBM in SARS-CoV-2 fitness, virulence, and pathogenicity, highlighting its potential as a target for therapeutic strategies.

# Materials and methods

## Generation of SARS-CoV-2 recombinant viruses

The recombinant viral genomes were constructed as previously described by de Melo et al., 2023. Eleven overlapping fragments around 3 kb each and two fragments encoding for yeast-specific selection genes (His3 and Leu2) were assembled in the yeast centromere plasmid pRS416 to generate the complete genome of the recombinant virus. The T7 promotor was placed just before the 5'UTR sequence and a unique restriction site EAG1 was placed just after the poly(A) tail. Viral fragments were obtained either by RT-PCR on RNA extracted from Vero-E6 cells infected by SARS-CoV-2 using the SuperScript IV VILO Master Mix (11756050, Thermo Fisher Scientific) according to the manufacturer's protocol, or using synthetic genes (GeneArt). All PCR amplifications were performed using Phusion™ High-Fidelity DNA Polymerase (F530, Thermofisher). To produce recombinant viruses deleted or mutated in the PDZ Binding Motif (PBM) of the Envelope protein (Fig. 1), the PCR product of the fragment 9 which includes E protein sequence was cloned in Topo-TA vector, using TOPO™ TA Cloning™ kit (K465001, Invitrogen) according to the manufacturer's protocol. Deletion of the 4 last carboxy-terminal amino acids in rSARS-CoV2-E-ΔPBM (LNSSRVP-) or mutation in rSARS-CoV2-E-MutPBM (LNSRVPGGG) (Fig. 1A) were introduced by Site-Directed Mutagenesis following Site-Directed Mutagenesis Phusion Kit manufacturer's protocol (F541, Thermo Fisher Scientific). TOPO-TA-F9 plasmid was used as a template and mutagenesis PCRs were performed with specific primers: E_ΔPBM_FOR 5'-TAAACGAACTAA ATATTATAGTTTTTCTGTTTGG-3' and E_ΔPBM_REV 5'-AGGAACTCT AGAAGAATTCAGA-3' primers pair was employed for PBM deletion and E_MutPBM_FOR 5'-TTCTAGAGTTCCTGGTGGTGGAGGTTAAACGAAC-3' and
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Production and characterization of recombinant SARS-CoV-2 viruses lacking E protein PBM. **A** Sequences of the Envelope C-terminus end of the recombinant viruses generated by reverse genetics. The box highlights the PBM. rSARS-CoV-2-E-WT, rSARS-CoV-2-E-ΔPBM, and rSARS-CoV-2-E-MutPBM correspond to the wild-type recombinant virus, and respectively to the viruses that lack the PBM by deletion of the four last residues or by mutations in a quadruplex of glycine. **B** Plaque morphology of the recombinant viruses. Plaques were developed in Vero-E6 cells on day 4 (left) or 6 (right) after infection. Images are representative of at least 3 independent experiments. Scale bar = 1 cm. **C** In vitro viral growth curves of the different recombinant SARS-CoV-2 viruses along with the original clinical isolate Wuhan SARS-CoV-2 virus (Wuhan CoV-2) used as reference. Subconfluent monolayers of Vero-E6 cells were infected at different MOIs, primarily 0.01 (left) and 0.001 (right), to assess the effect of initial viral load on replication kinetics. Culture supernatants were collected at several time points postinfection and titrated by TCID₅₀ assay. Dots correspond to means with standard deviations and dashed lines indicate the limit of detection (n = 3 independent replicates/time-point, nd: not detected). **D** Resistance dose response in Vero-E6 cells at 72 hpi. Impedance was measured every minute over the course of 96 h in wells that were mock infected or infected with recombinant SARS-CoV-2 viruses in tenfold dilutions ranging from MOIs of 1 to 0.0001. Sigmoid curves were generated using AxIS Z software. Dots indicate the median and vertical lines indicate the interquartile range (n = 3). **E** Median time-to-death calculations based on raw resistance data for each MOI. It was noted that the median time to cell death did not show significant variation across different MOIs, particularly between 0.01 and 0.1, suggesting a potential saturation effect at higher MOIs. Horizontal lines indicate median with the interquartile range (n = 3). Kruskal-Wallis test followed by the Dunn's multiple comparisons test (the adjusted p value is indicated when significant)</figcaption></figure>
E_MutPBM_REV 5'-GAATTCAGATTTTTAACACGAGAGTAAACG-3' primers pair for the PBM mutation. Recombination of 11 overlapping viral DNA fragments and 3 yeast specific fragments (PRS416, Leucine-2 and Histidine-3) was performed into *Saccharomyces cerevisiae* BY4740 strand cultured for three days at 30 °C. For each recombination, subcultured clones were screened by Multiplex PCR Kit to verify the presence of the different fragments (data not shown) (206143, *QIAGEN*) according to the manufacturer's protocol, using specific subsets of primer pairs as previously described [30]. To perform purification of a large amount of recombinant plasmid, the yeast colony of interest was cultured overnight in 200 mL of SD-His-Ura-Leu- medium at 30 °C under agitation (200 rpm). DNA extraction was processed using the NucleoBond Xtra Midi Plus kit (740422, *Macherey-Nagel*). Manufacturer's protocol was followed with a beforehand lysing step in which harvested cells were incubated in 16 mL RES-Buffer complemented with 1.6 mg Zymolyase® 100-T (120493-1, *AmsBio*) and 160 µl of β-mercapthoethanol, and incubated for 1 h at 37 °C before the addition of LYS-Buffer. The YAC plasmids containing viral cDNA of rSARS-CoV2-E-WT, rSARS-CoV2-E-ΔPBM or rSARS-CoV2-E-MutPBM constructs were digested at the unique restriction site located downstream of the 3' end poly(A) tail using EagI-HF® enzyme (R3505, *New England Biolabs*) following the manufacturer's protocol. The cDNA purified by a classical phenol-chloroform process was then transcribed in vitro using the T7 RiboMax™ Large Scale RNA Production System (P1300, *Promega*). The synthesized RNA was finally purified using a classical phenol-chloroform method, precipitated, and resuspended in Dnase/Rnase free water. Then, 12 µg of complete viral mRNA and 4 µg of pCMV plasmid encoding the viral nucleoprotein (N) gene were electroporated into 8.10⁶ Vero-E6 cells (ATCC #CRL-1586) resuspended in 0.8 mL of Electroporation Solution (MIR50114, *Mirus Bio™ Ingenio™*) using the Gene Pulser Xcell Electroporation System (1652660, *BioRad*) with a pulse of 270 V and 950 µF. Cells were then transferred to a T75 culture flask with 12 mL of DMEM supplemented with 2% FCS (v/v) and cultured at 37 °C, 5% CO₂ for several days until the cytopathic effect (CPE) was observed. The supernatant that corresponds to a P0 stock was harvested, aliquoted and frozen at -80 °C until titration. The viral sequence was controlled by NGS sequencing.

## Viral stock titration

The titer of recombinant viruses from the P0 stock was determined by a lysis plaque assay. 1.10⁶ Vero-E6 cells were seeded into each well of 6-well plates and cultured overnight at 37 °C, 5% CO₂. The Virus was serially diluted in DMEM without FCS and 400 µl diluted viruses were transferred onto the monolayers. The viruses were incubated with the cells at 37 °C with 5% CO₂ for 1 h. After the incubation, inoculum was removed, and an overlay medium was added to the infected cells per well. The overlay medium per well contained MEM 1X (prepared from MEM 10X (21430020, *Gibco™*), distilled water (15230162, *Gibco™*), L-glutamine (25030123, *Gibco™*) and gentamicin 10 mg/ml (15710064, *Gibco™*)), 0.25% sodium phosphate (25080094, *Gibco™*), and 1X AVICEL (RC-581, *Dupont*). After a 4 to 6 days of incubation, the plates were stained with crystal violet (V5265, *Merck*) and lyses plaques were counted to assess virus titer.
## Virus growth kinetics

Vero-E6 cells were used to compare the replication kinetics of SARS-CoV-2 reference strain and rSARS-CoV-2-E-WT viruses with rSARS-CoV2-E-ΔPBM and rSARS-CoV2-E-MutPBM mutant viruses. On the day before infection, 7.10⁶ Vero-E6 cells were seeded into a T75 flask. Cells were washed once with PBS and inoculated with the different viruses in 4 mL of their respective cell culture media for 1 h at MOI of 0.01, 0.001, or 0.01. Inoculums were then removed, and 12 mL of their respective cell culture media were added to each flask. All cells were maintained at 37 °C in a humidified atmosphere in the presence of 5% CO₂. Cell-culture supernatants were collected at the indicated time points after infection (8, 24, 32, 48, 56, 72, and 80 hpi) and frozen at -80 °C. Viral titers were obtained by classical TCID₅₀ method on Vero-E6 cells after 72 hpi [31]. Every experiment was performed in triplicate (n = 3).

## Maestro Z impedance experiments

Impedance measurements were conducted using CytoView-Z 96-well electrode plates (Axion BioSystems, Atlanta, GA, USA) on a Maestro Z device (Axion BioSystems, Atlanta, GA, USA). Prior to cell plating, each well was coated with a 2 mg/mL solution of human fibronectin (F1141, Merck) for 1 h at 37 °C. Then, 100 µL of DMEM containing 5% FCS was added to establish a resistance baseline. Vero-E6 cells, at a density of 50 000 cells per well were seeded onto the fibronectin-coated plates in medium supplemented with 5% FCS. The plate was then inserted into the Maestro Z device and resistance at 10 kHz, as a component of impedance, was monitored to observe cell attachment and monolayer stabilization. Infections with recombinant SARS-CoV-2 viruses were performed across a range of MOIs from 0.0001 to 1 by incubating cells 1 h with 50 µL of inoculum volume. Inoculum was removed and 100 µL of DMEM without FCS were added per well. Resistance kinetics were tracked for up to 96 hpi. Data acquisition and analysis were performed using the Axis Z software. Every experiment was performed in triplicate (n = 3).

## Golden Syrian hamster SARS-CoV-2 infection

Male Syrian hamsters (Mesocricetus auratus) of 5–6 weeks of age (average weight 60–80 g) were purchased from Janvier Laboratories and handled under specific pathogen-free conditions. Hamsters were housed by groups of four animals (n=4/group) in isolators in a biosafety level-3 facility, with ad libitum access to water and food. Following an acclimation period of one week, animal infections were performed as previously described [30]. Briefly, hamsters were anesthetized with an intraperitoneal injection of ketamine (200 mg/kg, Imalgène 1000, Merial) and xylazine (10 mg/kg, Rompun, Bayer). Infections were performed intranasally by administering 100 µL of physiological solution containing 6.10⁴ PFU of SARS-CoV-2 recombinant viruses. Body weight variation and clinical signs (clinical score) were monitored for four days. Clinical scoring was based on the evaluation of four parameters: ruffled fur, slow movements, apathy, and absence of exploration activity, each scored from 0 (normal) to 1 (severe), for a maximum cumulative score of 4. At day 3 post-infection (dpi), animals underwent a food finding test to assess olfaction as previously described [30]. At day 4 post-infection, animals were euthanized with an excess of anesthetics (ketamine and xylazine) and exsanguination [32], and samples of nasal turbinates, lungs, olfactory bulbs, cortexes, brain stems, and
cerebellums were collected and immediately frozen at -80 °C. Lungs, complete head and brain were also collected and fixed in 10% neutral-buffered formalin for histopathological studies.

## SARS-CoV-2 detection in golden hamsters' tissues

Frozen lungs, nasal turbinates and brain parts (olfactory bulbs, cortexes, brain stems and cerebellums) were weighed and homogenized with 1 mL of DMEM supplemented with 1% penicillin/streptomycin (15140148, *Thermo Fisher Scientific*) in Lysing Matrix M 2 mL tubes (116923050-CF, *MP Biomedicals*) using the FastPrep-24™ system (*MP Biomedicals*). Two cycles of homogenization at 4.0m/s for 20 s were performed with a resting time of 2 min in between. The tubes were centrifuged at 10,000 g for 2 min at 4 °C, and the supernatants collected. A classical TCID₅₀ method on Vero-E6 cells after 72 hpi [31] allowed assessment of viral titers (n=1/organ and n=4/group). To quantify genomic and sub-genomic Viral RNA loads, RNA was extracted from 125 µL of the supernatants homogenized with 375 µL of Trizol LS (10296028, *Invitrogen*) using the Direct-zol RNA MiniPrep Kit (R2052, *Zymo Research*). Taqman one-step qRT-PCR on the E gene (Invitrogen 1 1732-020) was conducted in a 12.5 µL reaction volume in 384-wells PCR plates using a QuantStudio 6 Flex thermocycler (*Applied Biosystems*). Briefly, 2.5 µL of RNA were added to 10 µL of a master mix containing 6.25 µL of 2X reaction mix, 0.2 µL of MgSO₄ (50 mM), 0.5 µL of Superscript III RT/Platinum Taq Mix (2 UI/µL) and 3.05 µL of nuclease-free water containing 400 nM of primers and 200 nM of probe. E_sarbeco primers and probe were used to detect genomic RNA (E_Sarbeco_F1 5'-ACAGGTACGTTAATAGTTAATAGCGT-3'; E_Sarbeco_R2 5'-ATATTGCAGCAG TACGCACACA-3'; E_Sarbeco_Probe FAM-5'-ACACTAGCCATCCTTACTGCG CTTCG-3'-TAMRA). For sub-genomic SARS-CoV-2 RNA, detection was achieved by replacing the E_Sarbeco_F1 primer by the CoV2sgLead primer (CoV2sgLead-Fw 5'-CGATCTCTTGTAGATCTGTTCTC-3'). A synthetic gene containing the PCR target sequences was purchased from *Thermo Fisher Scientific*. PCR amplification was performed using Phusion™ High-Fidelity DNA Polymerase (F530, *Thermo Fisher Scientific*) and subsequently transcribed in vitro using the T7 RiboMax™ Large Scale RNA Production System (P1300, *Promega*). The RNA was quantified using the Qubit RNA HS Assay kit (15958140, *Thermo Fisher Scientific*), normalized, and employed as a standard for determining the absolute copy number of RNA. The amplification process involved an initial incubation at 55 °C for 20 min, followed by an initial denaturation step at 95 °C for 3 min. This was followed by 50 cycles of denaturation at 95 °C for 15 s and annealing/extension at 58 °C for 30 s, with a final extension step at 40 °C for 30 s. Data were processed using the QuantStudio Design Analysis software.

## Gene expression RT-qPCR

RNA from organs was extracted in Trizol using Direct-zol RNA Miniprep kit following the manufacturer's protocol (R2050, *Zymo Research*). RNA was reverse transcribed into cDNA using SuperScript™ IV VILO™ Master Mix (11766050, *Invitrogen*). qPCR was performed according to the manufacturer's protocols for either the TaqMan Fast Advanced Master Mix (444457, *Applied Biosystems*) or SYBR™ Green Master Mix (4309155, *Applied Biosystems*), using 2.5 µL of cDNA (12.5 ng) and golden hamster primer pairs
for the genes of interest, as reported in the literature [30, 33]. The qPCR reactions in a final volume of 10 µL were performed in 384-well PCR plates using a QuantStudio 6 Flex thermocycler (Applied Biosystems). The amplification conditions were as follows: 95 °C for 20 s followed by 45 cycles of 95 °C for 1 s and 60 °C for 20 s. Data were treated using the Design and Analysis v.2.8.0 software (Applied Biosystems) using the γ-actin gene as reference and comparing the fold change in expression in the tissues from the infected hamsters with the tissues of the mock-infected group using the 2-ΔΔCt method (n=1/organ and n=4/group).

## Histopathology and immunohistochemistry

Lung fragments, brains and heads fixed 7 days in 10% neutral-buffered formalin were embedded in paraffin. Decalcification treatment was performed on heads consisting of two baths of 4 days within an EDTA based OSTEOMOLL® solution (101,736, Merck). Four µm thick organ sections were cut and stained with hematoxylin and eosin staining. Lung sections immunohistochemistry was performed on a Bond RX (Leica™) using a rabbit polyclonal antibody against SARS Nucleocapsid Protein antibody (1:500, NB100-56576, Novus Biologicals) and biotinylated goat anti-rabbit Ig secondary antibody (1:600, E0432, Dako®, Agilent). Brain sections immunohistochemistry was performed on a Ventana BenchMark stainer (Roche™) using a mouse monoclonal antibody against GFAP (1:500, 6F2, Dako®, Agilent), a rabbit polyclonal antibody against fibrinogen (1:500, Dako®, Agilent) and a biotinylated secondary antibody included in the detection kit (Ventana DAB Detection Kit 250-001, Roche™). Diaminobenzidine (DAB), occasionally combined with alkaline phosphatase (ALP) for double labelling, was used as a chromogen. Slides were then scanned using Axioscan Z1 slide scanner (Zeiss), and images were analyzed with the Zen 2.6 software (Zeiss). Images correspond to multiple representative fields from 4 animals per group. Lung tissue sections were analyzed using images acquired in a double-blinded manner and lung injury was evaluated based on the severity of inflammation, presence of interstitial and alveolar syndromes, and alterations of the bronchiolar epithelium. Each parameter was scored semi-quantitatively on a 5-point scale: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe (see Table S2).

## MucilAir™ infection

The MucilAir™ model, a pseudostratified bronchiolar epithelium, was purchased from Epithelix (Saint-Julien-en-Genevois, France). The model was cultured at 37 °C under a 5% CO₂ atmosphere with 700 µl of medium in the basal compartment, maintaining the apical side exposed to air to mimic the air-liquid interface. Infections were performed in the apical compartment by incubating the epithelium with 250,000 plaque-forming units (~ MOI of 0.5) of recombinant viruses diluted in medium (EP04MM, Epithelix), for 4 h at 37 °C. After incubation, the viral inoculum was removed, and the epithelium was washed twice with 200 µL of PBS for 5 min at 37 °C and once with 200 µL of medium. The apical side was washed every 2–3 days by incubating it with 200 µL of medium for 20 min at 37 °C, and the basal medium was replaced every 2–3 days. All supernatants were collected for further analysis (n = 3).
## Immunofluorescence experiments

MucilAir™ cultures at 4 dpi and A549 ACE2 TMPRSS infected cells were washed twice with PBS and fixed in 4% PFA for 30 min. The fixed samples were washed in PBS for 5 min three times. MucilAir™ and A549 ACE2 TMPRSS cells were respectively permeabilized during 20 min with PBS-TritonX-100 0.5% and PBS-TritonX-100 0.1%. After 3 washes of 5 min with PBS-Tween 0.05%, MucilAir™ membranes were incubated in a blocking solution composed by PBS-Tween 0.05%, 1% BSA, 10% normal goat serum and A549 ACE2 TMPRSS cells were blocked with a solution of PBS-Tween 0.05% with 5% normal goat serum. The permeabilized samples were incubated overnight at 4 °C with primary antibodies diluted in blocking solution: rabbit anti-E 1:250 to 1:500 (HL1443, *Genetex*), mouse anti-GM130 1:250 to 1:500 (610,823, *BD Biosciences*), mouse anti-ZO-1 1:50 to 1:100 (610,966, *BD Biosciences*), mouse anti-hAfadin 1:100, (MAB78291, *Novus Biologicals*). The next day, after 3 washes of 5 min in PBST, samples were incubated 1 h at 4 °C with conjugated secondary antibody diluted in blocking solution: anti-rabbit antibody conjugated to AF488 1:500 to 1:1000 (A11035, *Invitrogen*) and anti-mouse antibody conjugated to AF594 1:500 to 1:1000 (A21125, *Invitrogen*). After 3 washes of 5 min, samples were mounted in Fluoroshield™ with DAPI (F6057, *Merck*). Stainings were observed with a LSM700 confocal microscope (*Zeiss*). Acquired images were processed using ZEN lite software (*Zeiss*). Images correspond to multiple representative fields from 3–5 experiments.

## Pull down assays

Subconfluent cultures of HEK 293 cells were transiently transfected with a plasmid encoding GFP or GFP-tagged ZO-1 full-length protein using the calcium phosphate method. In parallel, subconfluent monolayer cultures of Vero-E6 cells were infected with rSARS-CoV2-E-WT or rSARS-CoV2-E-MutPBM viruses for 48 h at a MOI of 0.01. Both cell cultures were then scraped, lysed using RIPA Lysis Buffer (sc-24948, *Santa Cruz*), and centrifuged at 13,000 rpm for 10 min at 4 °C to pellet cell debris. Soluble detergent extracts from the GFP constructs expressing cells were incubated with GFP resins for 2 h at 4 °C with gentle shaking, and then washed 3 times with a washing buffer containing PBS supplemented with 200 mM NaCl and 0.1% Triton X-100. The GFP construct-coated beads were then incubated with mock or infected cell lysates for 2 h at 4 °C, followed by three washes. The eluted proteins were processed for Western blot analysis using GFP antibody (NB600-313, *Novus Biologicals*) and envelope protein antibody (HL1443, *Genetex*).

## RNA libraries and sequencing

Libraries were built using a Illumina Stranded mRNA library Preparation Kit (Illumina, USA) following the manufacturer's protocol from the RNA extractions of brain-stem and lung samples from non-infected hamsters or infected with the recombinant viruses (E-WT, E-ΔPBM or E-MutPBM), producing four replicates per group. Quality control was performed on an BioAnalyzer (Agilent). Sequencing was performed on one NovaSeq X lane 10B300 (Illumina) to obtain 150 base paired end reads. Unfortunately,
the libraries for the four replicates of the E-MutPBM infected lung failed and had to be
performed a second time and sequenced in another NextSeq 2000 sequencing run to
obtain 100 base single-end reads.

RNA-seq analysis

The RNA-seq analysis was performed with Sequana 0.16.11 [34]. We used the RNA-seq
pipeline 0.19.2 (https://github.com/sequana/sequana_rnaseq) built on top of Snake-
make 7.32.4 [35]. Briefly, reads were trimmed from adapters using Fastp 0.22.0 [36] then
mapped to the golden hamster MesAur1.0.100 genome assembly from Ensembl using
STAR 2.7.10a [37]. FeatureCounts 2.0.1 [38] was used to produce the count matrix,
assigning reads to features using corresponding annotation from Ensembl with strand-
specificity information. Quality control statistics were summarized using MultiQC 1.17
[39]. Statistical analysis on the count matrix was performed to identify differentially
regulated genes comparing each recombinant viruses infected tissues versus the mock
group and including a batch effect to the model corresponding to the replicates. Clus-
tering of transcriptomic profiles were assessed using a Principal Component Analysis
(PCA). Differential expression testing was conducted using DESeq2 library 1.34.0 [40]
scripts indicating the significance (Benjamini-Hochberg adjusted *p*-values, false dis-
covery rate FDR<0.05) and the effect size (fold-change) for each comparison. Finally,
enrichment analysis from the differentially regulated gene lists was performed using
modules from Sequana. KEGG pathways enrichment uses GSEApy 1.1.1 [41], EnrichR
[42] and the KEGG database [43]. Programmatic accesses to online web services were
performed via BioServices 1.11.2 [44].

Statistics and reproducibility

Statistical analysis was performed using Prism 10 (GraphPad, version 10.1.1, San Diego, USA). Quantitative data were compared across groups using Kruskal-Wallis' test followed by Dunn's multiple comparisons test (horizontal lines indicate median with the interquartile range). Statistical significance was assigned when *p* values were < 0.05 and is indicated in the figures. Randomization and blinding were not possible for in vivo experiments due to pre-defined housing conditions (separated isolators between animals infected by each virus strains). Ex vivo and in vitro analyses were blinded (coded samples). The nature of statistical tests and the number of experiments or animals (*n*) are reported in the figure legends and Materials and Methods section.

Results

The alteration of the E protein PBM significantly affects the SARS-CoV-2 viral fitness

To characterize the role of the E protein PBM in the virulence and pathogenicity of SARS-CoV-2, we produced recombinant viruses lacking the E protein PBM using a reverse genetic method based on yeast recombination as previously described [30]. We generated a recombinant Wild-Type virus from the original clinical isolate (rSARS-CoV2-E-WT), a deleted construct missing the four last carboxy-terminal amino acids (rSARS-CoV2-E-ΔPBM), and a mutated construct with a quadruplex of glycine instead of the PBM (rSARS-CoV2-E-MutPBM) (Fig. 1A). The corresponding infectious RNAs of the full-length virus genomes were electroporated into Vero-E6 cells and rescue viruses
from the culture medium were collected and titrated (Fig. 1B). The viral sequences were controlled by Next Generation Sequencing (NGS) (table S1). All the mutations close to the PBM and elsewhere in the genome above a frequency of 10% in the viral population were analyzed (Table S1). Twelve percent of the viral population exhibit a T2087A amino acid substitution in the G2M domain of NSP3 coding sequence, a position in a domain that is weakly characterized in the literature, nor identified in SARS-CoV-2 variants. However, three percent of the viral population of the rSARS-CoV2-E-ΔPBM carried the P71L substitution, a mutation characteristic of the Beta variant B.1.351 [45]. This early appearance of the mutation suggests significant mutational pressure on this residue, located just one position upstream of the PBM (Fig. 1A). All the following experiments were conducted with the initial viral stock (P0 rescue) in which the genomic sequences were consistent.

Interestingly, a marked differential in lysis plaque size was observed between the mutants and the WT viruses in the lysis plaque titration assay (Fig. 1B). Indeed, PBM-deleted viruses induced the formation of smaller lysis plaques than those induced by the WT virus. This effect was even more drastic in the presence of the PBM-mutated viruses. To assess the involvement of the PBM in virus fitness in *cellulo*, viral growth kinetics of recombinant viruses lacking the PBM were compared with the ones of the WT viruses in Vero-E6 cells (Fig. 1C). We verified beforehand that viral growth curves of the original Wuhan SARS-CoV-2 clinical isolate (Fig. 1C, Wuhan CoV-2 curve) and the recombinant rSARS-CoV-2-E-WT, also generated on the Wuhan reference strain backbone (Fig. 1C, E-WT curve) were comparable. We conducted these experiments using a range of low MOIs (0.001 and 0.01) to account for different initial viral loads and their impact on viral kinetics. We observed replication defects for the E-ΔPBM and E-MutPBM viruses at a MOI of 0.01 during the early stages of infection (prior to 48 hours post-infection (hpi)) with a viral production around 2 logs lower than WT viruses. After 48 hpi, all the viruses reached similar titers. This replication delay was even more drastic for an infection at a MOI of 0.001, where, akin to the lysis plaque assay, the E-ΔPBM mutant appeared to exhibit an intermediate phenotype with a viral growth curve between those of the E-WT and the E-MutPBM viruses. While lower MOIs help delineate viral growth kinetics, it's important to note that at higher MOIs, such as 0.1, these differences may become less discernible due to more pronounced cytopathic effects. As SARS-CoV-2-induced cytopathic effects damage the cell monolayer, we performed impedance measurements in infected Vero-E6 monolayers using the Maestro Z platform (Axion BioSystems) (Fig. 1D). To differentiate levels of virus replication and cytolysis effects, confluent Vero-E6 monolayers in CytoView-Z plates were infected with recombinant viruses at multiple MOIs (0.0001 to 1), and resistance measurements were acquired over 96 hpi. The observed shift in sigmoid infectious dose-response curves for PBM-lacking mutants at 72 hpi (Fig. 1D) and the differential median time to cell death (Fig. 1E) agreed with a replication delay and attenuated cytopathic effects observed for the mutants. The E-ΔPBM mutant exhibited again an intermediate phenotype with a resistance dose response located between those of the E-WT and the E-MutPBM viruses. We also note that the median time to cell death across different MOIs, particularly between 0.01 and 0.1, did not show significant variation for the E-WT, which may indicate a saturation effect at higher MOIs. Together, these findings suggest that while the E PBM may not play an
essential role in viral replication, its absence leads to a delay in viral growth, consistent
with the known functions of the E protein in viral assembly, budding, and release.

The PBM does not impact E protein localization in cells and air-liquid reconstructed human bronchial epithelium

To investigate the localization of the E protein and the impact of the PBM, we infected A549 ACE2 TMPRSS2 cells and MucilAir™ model, an in vitro reconstructed human pseudostratified bronchiolar epithelium mimicking the Air–Liquid Interface (ALI) in the lungs (Fig. 2A), with E-WT, E-ΔPBM, and E-MutPBM viruses. The kinetics of viral replication in the apical compartment of the MucilAir™ model showed a replication defect for the E-ΔPBM and E-MutPBM viruses during the early stage of infection, with a viral production 3–4 logs lower than the one of E-WT virus (Fig. 2B). From day 4 to day 7 post-infection, the viral loads of the mutant viruses remain stable and never reach the levels observed with the E-WT virus. The E-ΔPBM mutant exhibits an intermediate viral replication kinetic, in line with those observed in Vero-E6 cells. Additionally, a lower viral load was detected for E-WT virus in the basolateral compartment (Fig. 2C), indicating that SARS-CoV-2 particles are predominantly released from the apical side of the epithelium. Interestingly, the E-MutPBM virus was not detected in the basolateral compartment, and the E-ΔPBM virus again exhibits an intermediate phenotype with 1 to 2 logs lower than the E-WT virus at 4 dpi. Immunofluorescence analysis was performed on A549 ACE2 TMPRSS2 cells and on the MucilAir™ model at 2 and 4 dpi respectively, after infection with E-WT, E-ΔPBM, and E-MutPBM viruses (Fig. 2D and E; Fig. S1). E

(See figure on next page.)

**Fig. 2** Subcellular localization of the Envelope protein and subversion of ZO-1 protein during SARS-CoV-2 infection in an ALI Transwell model. **A** Schematic view of the MucilAir reconstructed human bronchial epithelium. The model exhibits an air–liquid interface with different cell types (ciliated, goblet, and basal cells). **B, C** Kinetics of viral replication of the recombinant viruses in apical **B** and basal **C** compartment of the ALI Transwell model. Cells were infected apically with recombinant SARS-CoV-2 viruses with 250,000 pfu (~MOI of 0.5), and viral release was monitored over time. Apical washes were collected at 2, 4, and 7 dpi and basal medium was simultaneously harvested from the lower chamber to evaluate trans-epithelial viral passage. Viral titers in both apical and basal samples were determined TCID₅₀ assay on Vero-E6 cells. The “Day 0” time point corresponds to viral quantification from the third apical wash performed after the inoculum removal to assess residual input virus. Dots correspond to medians with interquartile range and dashed lines indicate the limit of detection (n = 3 independent replicates/time-point, nd: not detected). **D, E** Immunofluorescence assay showing E protein localization within apical ciliated cells **D** and basal cells **E** in the MucilAir model. Staining was performed at 4 dpi on a 250,000 pfu infected epithelium (~MOI of 0.5) with recombinant E-WT (top panel), E-ΔPBM (middle panel), or E-MutPBM (bottom panel) (scale bar = 10 µm). Images are reconstituted in 3D from Z stack acquisitions. Hoechst: nuclei (blue); GM130: Golgi apparatus (red); Envelope: recombinant SARS-CoV-2 envelope protein (green); Phalloidin: actin (cyan). Arrowheads indicate E-Golgi colocalizations. Images correspond to multiple representative fields from three independent experiments. **F** Visualization of ZO-1 protein network correlated with E protein localization in the MucilAir model using immunofluorescence staining. Staining was performed on either non-infected epithelium or on epithelium fixed at 4 days post-infection (dpi) with 250,000 pfu (~MOI of 0.5) of E-WT, E-ΔPBM or E-MutPBM recombinant viruses. Arrowheads indicate areas of E-ZO-1 colocalization. Images are reconstituted in 3D from Z stack acquisitions (scale bar = 20 µm). Hoechst: nuclei (blue); ZO-1: zonula occludens 1 (red); Envelope: recombinant SARS-CoV-2 envelope protein (green). Images correspond to multiple representative fields from 3 independent experiments. **G** Western blot of GFP pull-down. GFP or GFP-ZO-1 from transfected HEK293 cell lysates were immobilised on GFPtrap resin and incubated with Vero-E6 cell lysates non infected or infected 48 h at a 0.01 MOI with recombinant E-WT (top panel) or E-MutPBM viruses (bottom panel). The bound fractions were analysed by immunoblotting using anti-GFP (top panel) and anti-E (bottom panel) antibodies
<figure><img src="image_5.png" /><figcaption>**Fig. 2** (See legend on previous page.)</figcaption></figure>

protein co-stained with GM130, a marker for the Golgi apparatus. A cytoplasmic vesicular distribution of the E protein is also observed (Fig. 2D and E; Fig. S1), consistent with the secretory pathway of SARS-CoV-2 viral particles, mainly described in the literature
through peroxisomes and lysosomes [46–48]. Regardless of the virus, E proteins are detected on the apical surface of ciliated cells (Fig. 2D), distinctly identified from basal cells via actin staining (phalloidin) (Fig. 2E). Within the MucilAir™ model, E protein also exhibits intracellular localization, displaying a Golgi-like and vesicular distribution (Fig. 2D and E). Thus, the different subcellular localization observed with both E-WT and E mutant viruses indicates that E localization is independent of the PBM in infected cells or in the MucilAir™ model.

## The SARS-CoV-2 E protein interacts with ZO-1 in a PBM-dependent manner, leading to cell–cell junction alterations during infection

We then investigated the localization of ZO-1 and its interaction with the E protein upon infection with E-WT virus in A549 ACE2 TMPRSS cells (Fig. S2, A to E) and the MucilAir™ model (Fig. 2F; Fig. S2F and G). Immunofluorescence assays of mock-infected A549 ACE2 TMPRSS cells and MucilAir™ model showed the characteristic intact ZO-1 staining pattern at cell junctions (Fig. 2F; Fig. S2A). In A549 ACE2 TMPRSS cells, when the E-WT protein distribution was observed at the Golgi apparatus, the ZO-1 pattern remained conserved, exhibiting its expected localization. However, in regions where E protein presents a sporadic cytoplasmic distribution, meaning an advanced step in the viral cycle within the cell, ZO-1 staining is disrupted supporting epithelial reorganization (Fig. S2B). A similar behavior was observed in the MucilAir™ model (Fig. 2F; Fig. S2F). Colocalizations between E and ZO-1 proteins were observed in various subcellular locations, related to Golgi organelle, cytoplasmic vesicles, and cell surface (Fig. 2G; Fig. S2, C and F). Interestingly, cells infected with viruses lacking PBM exhibited significantly reduced disruption of ZO-1 (Fig. 2F; Fig. S2, D, E and G). To confirm the interaction between E and ZO-1 proteins in a PBM-dependent manner, *pull down* assays were performed. GFP-ZO-1, or GFP alone as control cell lysate, were incubated with lysates from cells infected with E-WT or E-MutPBM viruses. The E-WT protein is strongly precipitated with GFP-ZO-1 (Fig. 2G) while the E protein with a mutated PBM is not retained by GFP-ZO-1. These results indicate that E protein produced by viruses in cells interacts with full-length ZO-1 in a PBM-dependent manner. Together, these data support the role of the E protein/ZO-1 interaction in the tight junction disturbance and the impairment of epithelial barrier integrity during SARS-CoV-2 infection.

## PBM-defective viruses infection mitigates the severity of clinical disease associated with SARS-CoV-2 infection in hamsters

After validating the role of the E protein in SARS-CoV-2 pathogenicity in vitro, we further investigated the involvement of the E PBM in SARS-CoV-2 pathogenicity in vivo, by intranasally inoculating male golden hamsters with 6.0.10⁴ PFU of the three recombinant viruses (E-WT, E-ΔPBM and E-MutPBM) (Fig. 3A). The exclusive use of male hamsters was motivated by the aim to minimize biological variability and to maintain consistency with previous RNA-seq datasets, which identified peak infection and transcriptomic responses at day 4 post-infection specifically in males [49]. Moreover, prior studies have demonstrated that male hamsters exhibit more severe disease and higher levels of viral replication compared to females [50], thereby facilitating a more robust detection of pathological and molecular phenotypes. Daily monitoring of body weights
(Fig. 3B; Fig. S3A) and clinical signs (Fig. 3C; Fig. S3B) allowed for the assessment of disease progression. Clinical scoring was based on the evaluation of four parameters: ruffled fur, slow movements, apathy, and absence of exploration activity, each scored from 0 (normal) to 1 (severe), for a maximum cumulative score of 4. While E-WT infected hamsters presented a progressive weight loss and the onset of clinical symptoms, the animals infected with the PBM-lacking viruses displayed no or minor weight loss and clinical signs, comparable to non-infected hamsters. Lung-to-body weight ratio indicates a significant attenuation of lung inflammation in PBM-deficient infected animals, closely matching the profile observed in non-infected hamsters (Fig. 3D; Fig. S3C). In all these parameters, the E-ΔPBM mutant again displays an intermediate phenotype. As anosmia constitutes one of the clinical signs of COVID-19, olfactory performance was assessed at 3 dpi. Interestingly, half of the animals infected with the E-WT viruses exhibited anosmia (2/4, *p* value = 0.0007 from Chi square statistical test), whereas those infected with the two mutant viruses fully conserved their olfactory capacity (Fig. 3E; Fig. S3D). Altogether, these results demonstrate that removal of the E protein PBM drastically decreases pathogenicity in vivo highlighting that a functional PBM in E protein is essential for SARS-CoV-2 virulence.

To assess the effect of the E protein PBM on virus growth in vivo, viral loads were quantified by titration and RT-qPCR viral RNA quantification at 1, 2, and 4 dpi in the upper and lower airways (nasal turbinates and lung) (Fig. 3F to I; Fig. S3, E to H). While substantial viral charges were found in both airways' organs for E PBM-deficient viruses (10⁵–10⁷ TCID₅₀/100 mg of tissue), the E-WT virus replicated at a higher level (10⁷⁻⁸ TCID₅₀/100 mg of tissue). Lower quantities of viruses were recorded for E-ΔPBM and E-MutPBM viruses at the early stages post-infection mainly at 1 and 2 dpi. This tendency was more pronounced in the lung than in nasal turbinates, consistent with the direction of the viral spread into the respiratory tract. An intermediate profile was again observed for the E-ΔPBM virus located between those of the E-WT and E-MutPBM viruses (Fig. S3E to H). Genomic viral RNA in lung and nasal turbinates of all animals were detected

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**Fig. 3** Clinical profile and viral metrics of hamsters infected with recombinant wild-type SARS-CoV-2 (E-WT) and PBM lacking viruses (E-ΔPBM and E-MutPBM). **A** Schematic representation of the in vivo experimental design. Male golden hamsters were intranasally infected with 60,000 PFU per animal of each recombinant virus (n = 4/group). Animals were monitored and euthanized at defined time points post-infection for sample collection and analysis. Mock group received a control inoculation with vehicle solution. Tissues and organs were harvested for downstream virological, histopathological, and transcriptomic assessments. **B** Body weight at four days post-infection (4 dpi). **C** Clinical score at 4 dpi. The clinical score is based on a cumulative 0–4 scale: ruffled fur; slow movements; apathy; and absence of exploration activity. **D** Lung-to-body weight ratio measured at 4 dpi. Horizontal lines indicate median and the interquartile range (n = 8/group). Kruskal–Wallis test followed by the Dunn's multiple comparisons test (the adjusted *p* value is indicated when significant). **E** Olfactory performance loss measured at 3 days post-infection (dpi). The olfaction test is based on the hidden (buried) food finding test. Bars represent the percentage of anosmic animals (n = 4/group). Chi-square test for trend. **F, G** Infectious viral titers in nasal turbinates **F** and lung **G** at 4 days post-infection (dpi) expressed as TCID₅₀ per 100 mg of tissue. Horizontal lines indicate median and the interquartile range (n = 4/group). Kruskal–Wallis test followed by the Dunn's multiple comparisons test (the adjusted *p* value is indicated when significant). **H, I** Viral genomic and subgenomic RNA load detected in nasal turbinates **H** and lung **I** at 4 dpi. Horizontal lines indicate median and the interquartile range. Lines connect symbols from the same animals (n = 4/group). Kruskal–Wallis test followed by the Dunn's multiple comparisons test (the adjusted *p* value is indicated when significant). (n = 4/group)
<figure><img src="image_6.png" /><figcaption>**Fig. 3** (See legend on previous page.)</figcaption></figure>

with loads at 10⁸⁻⁹ copy number/µg of RNA associated with subgenomic RNA loads at 10⁵⁻⁹ copy number/µg of RNA (Fig. 3H and I; Fig. S3G and H), in agreement with the replicative capacity of all viruses in vivo.

To further explore the role of the E protein PBM beyond the respiratory tract, we assessed its involvement in viral infection within the brain, a site known to be affected by SARS-CoV-2, comparing its effects with those observed in the airways. Within the brain, titration in the different brain regions at 4 dpi (olfactory bulb, cortex, brainstem, and cerebellum) highlighted the detection of infectious viral particles only in the olfactory bulb of animals infected with E-WT virus (Fig. S4A). In contrast to the titration results, no differences were observed between the viruses when detecting genomic RNA in the olfactory bulb and cortex of the animals with high genomic RNA loads at 10⁷ and 10⁸ copy number/µg of RNA
respectively. More variability between replicates was observed in the brainstem and the cerebellum, where several RNA loads could not be quantified. Except for one positive hamster in the brainstem for the E-WT animal infected group, no subgenomic RNA was detected in the different parts of the brain (Fig. S4B). Thus, the absence of a functional PBM delayed the viral growth in the airway tract, while its implication in the neuroinvasion seems minor with progressive infection through the olfactory bulb, cortex, brainstem, and cerebellum.

## Damages to upper and lower airways epithelia are significantly reduced with PBM-lacking viruses compared with E-WT virus

Histological analysis was performed on nasal cavity and lungs sections from infected hamsters with E-WT, E-ΔPBM, and E-MutPBM viruses at 4 dpi (Fig. 4; Fig. S5; table S2). Examination of E-WT infected nasal epithelia sections reveals disorganization with important inflammation and desquamation. The bronchial epithelium shows substantial damage characterized by subsidence and desquamation (Fig. S5). Across all levels of airway epithelia, E-ΔPBM-infected samples exhibit moderate pathogenic signs, whereas E-MutPBM samples show only minor signs, comparable to those observed in non-infected animals (Fig. S5). These histological findings suggest that the observed attenuation in viruses lacking the E PBM is correlated with a decrease in airway pathology. Examination of lung sections from hamsters infected with E-WT exhibits significant edema, congestion, thickening of alveolar walls, and severe perivascular and peribronchiolar mononuclear cells infiltrates (Fig. 4A; Fig. S5). Additionally, viral antigen detection by immunostaining with SARS-CoV-2 nucleocapsid antibody reveals widespread distribution of the virus within the organ (Fig. 4B). In contrast, hamsters infected with viruses lacking E PBM show intermediate phenotypes between the mock-infected hamsters and those infected with E-WT, with significantly less damage and lung edema. The E-ΔPBM mutant displays an intermediate phenotype between those of E-WT and E-MutPBM (Fig. 4A; Fig. S5; table S2). SARS-CoV-2 nucleocapsid staining demonstrates limited viral propagation in the organ for E-ΔPBM and E-MutPBM compared with E-WT (Fig. 4B).

To focus on epithelial structures potentially disrupted by SARS-CoV-2, Hematoxylin-Eosin staining was performed on whole brain sections of hamsters at 4 dpi. We focused our investigation on brainstem and choroid plexus structures. No significant morphological changes were observed during infection, and the choroid plexus appeared similar across all groups (Fig. S4C). Immunohistochemical (IHC) staining for the glial fibrillary acidic protein (GFAP), an astrocyte marker, indicated an absence of inflammation when comparing mock-infected and E-WT-infected animals (Fig. S4C). To assess potential blood-brain barrier leakage, fibrinogen IHC staining was performed, revealing no differences at this time point of the infection (4 dpi) (Fig. S4C). Altogether, these findings suggest that SARS-CoV-2 neuroinvasion is highly restricted, with no evidence of blood-brain barrier leakage, microscopic brain damage, or astrocyte activation.

## The PBM drives a distinct transcriptomic signature, primarily modulating inflammatory signaling proteins and affecting adherens junction components

To investigate the impact of the PBM on SARS-CoV-2 virulence, we conducted a comparative RNA-seq transcriptomic analysis during the peak of infection, at 4 dpi, in the lungs and brainstem of hamsters infected with the E recombinant viruses (Fig. 5;
<figure><img src="image_7.png" /><figcaption>**Fig. 4** Histopathology and immunohistochemical study of lungs from hamsters infected with WT SARS-CoV-2 recombinant virus (E-WT) and PBM lacking viruses (E-ΔPBM and E-MutPBM) at 4 dpi.

**A** Representative images of Hematoxylin and Eosin (H&E) stained-whole lung sections (upper panels), alveoli (middle panels) and bronchiolar epithelium (bottom panels). Arrows indicate inflammation and arrowhead corresponds to epithelial damages. **B** Representative images of whole lung sections (upper panels), alveoli (middle panels) and bronchiolar epithelium (bottom panels) immuno-stained with SARS-CoV-2 Nucleocapsid antibody. Scale bars correspond to 2 mm for the whole-organ sections and 50 µm for the close-up views (n = 4/group)</figcaption></figure>

Fig. S6). Principal Component Analysis (PCA) and Venn diagrams (Fig. 5A and B) revealed that the transcriptomic responses (with an absolute fold change > 2) were significantly more pronounced in the lungs compared with the brainstem, consistent with the SARS-CoV-2's respiratory tropism. In the lungs, over 1,000 genes were differentially expressed compared with non-infected hamsters, while only a few dozen genes were impacted in the brainstem (Fig. 5B).
Notably, the expression of 530 genes in the lung was consistently altered across all three recombinant viruses compared with non-infected animals, reflecting a core SARS-CoV-2 infection signature that is independent of PBM alterations (Fig. 5B). This indicates that some pathways are commonly activated during infection, regardless of PBM presence. The different profiles observed in various infection parameters upon infection with the three recombinant viruses were reflected in the transcriptomic data. Indeed, only 32 genes differed between E-WT and E-ΔPBM, while 387 genes were distinct between E-WT and E-MutPBM (Fig. 5B). According to the KEGG analysis, several key inflammatory and immune pathways were consistently disrupted across all the three viruses, including “cytokine-cytokine receptor interaction,” as well as the “TNF,” “TLR,” “NF-κB,” and “chemokine” signaling pathways (Fig. 5C and D). In addition, pathways related to cell death mechanisms, including “apoptosis,” “necrosis,” “proteasome,” and “phagosome formation” were also dysregulated. In PBM-deficient viruses, although the same inflammatory pathways were enriched, they were less intensely modulated compared with the E-WT virus in terms of gene number and fold change. This matches the reduction in tissue inflammation observed through histopathological analysis (Fig. 4A; Fig. S5). To validate these findings, we conducted RT-qPCR at 1, 2, and 4 dpi in the upper and lower airways on common pro-inflammatory cytokines (MX2, CXCL10, IL-6, IFN-B and TNF-α), known to be upregulated during SARS-CoV-2 infection (Fig. S6A). By day 4, RT-qPCR showed that cytokine expression levels in mutant-infected animals were comparable to those of E-WT-infected animals. However, at day 1, viruses lacking the E protein PBM showed significantly lower expression (up to 4 Log₂ FC) compared with E-WT-infected animals. This reduction in cytokine expression was most pronounced in the lungs compared with nasal turbinates, notably for MX2 and CXCL10, which showed the highest over-expression at day 1. This decrease in inflammatory cytokine levels reflects delayed immune responses in agreement with the reduced viral fitness and pathogenicity of PBM-deficient viruses in vitro and in vivo (Fig. S6A).

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**Fig. 5** Transcriptomic analysis of lung and brainstem responses to PBM-lacking viruses' infection in hamsters. A Principal component analysis (PCA) of the general transcriptome characteristics. Transcriptomic analysis was conducted on lung and brainstem tissues from hamsters that were either non-infected (mock) or infected with E-WT, E-ΔPBM, and E-MutPBM viruses at 4 days post-infection (dpi). The first principal component (PC1) explained 95.34% of the total variance in the dataset, while the second principal component (PC2) accounted for an additional 3.23%. B Venn diagram showing the number of differentially expressed genes. The Venn diagram displays the number of differentially expressed genes, with numbers indicating both unique and shared DEGs when comparing the various recombinant-infected lung (left) and brainstem (right) tissues to non-infected controls (with an absolute fold change > 2). C KEGG enrichment analysis in lung tissue. The histogram shows the relevant KEGG pathways that are significantly up- or down-regulated in recombinant virus-infected lung tissue compared with non-infected controls, ranked by gene ratio. The gene ratio (x-axis) represents the proportion of significant genes relative to the total number of genes within each pathway. D Heatmaps of differentially expressed genes in key KEGG Pathways in lung tissue. Heatmaps display the expression levels of all differentially expressed genes in relevant KEGG pathways in the lungs of hamsters infected with E-WT, E-ΔPBM, and E-MutPBM, compared with mock-infected controls at 4 days post-infection (dpi) with an absolute fold change > 2. Asterisks (*) indicate genes with a Benjamini-Hochberg-adjusted *p*-value < 0.05 in comparisons between each recombinant virus group and the mock-infected group. The color gradient represents the log₂ fold change (Log₂ FC) in gene expression between infected and control animals. (n = 4/group)
<figure><img src="image_8.png" /><figcaption>**Fig. 5** (See legend on previous page.)</figcaption></figure>

Moreover, several crucial signaling pathways, such as “RAS,” “MAPK,” “WNT,” and “PI3K-AKT,” were downregulated in E-WT infected animals but unchanged in those infected with PBM-deficient E mutants (Fig. 5C). Pathways specifically associated with PBM effects, such as “lysosome function,” “neutrophil extracellular trap formation,” “antigen processing and presentation,” and “apoptosis,” were also identified. In the lungs of E-WT-infected animals, adherens junction proteins, notably those with PDZ
domains, such as ZO-1 (TJP1) and ERBB2, were downregulated compared to mock-infected controls.

As initial differences in viral load were observed in the brainstem, a key regulator of vital functions, we centered our transcriptomic analysis on this region. The results revealed no major pathway dysregulation, with only a few genes showing differential expression upon the infection (Fig. 5B). This finding aligns with the relatively limited impact of SARS-CoV-2, and specifically the PBM in the brainstem, indicating a finely tuned regulatory response in this region. Nonetheless, an inflammatory response was observed across all three groups, characterized by upregulation of key inflammatory genes, including *ISG15*, *IRF7*, *MX1* and *MX2* (Fig. S6, B and C). Differences in inflammatory response between E-WT and PBM-deficient viruses in the brainstem were highlighted as well as several genes such as *FOS*, *KCNV1*, *PLPPR4*, and *EGR3* (Fig. S6B and C). Notably, several PDZ-related proteins were identified, including *CNKSR2*, *SGK1* and *ERBIN*.

Overall, our findings demonstrate a marked difference in the transcriptional and inflammatory response between the lungs and the brainstem. The lungs showed extensive gene expression changes, while the brainstem's response is more limited, indicating a finely tuned immune regulation in this region.

## Discussion

In this study, we provided insights into the critical role played by the envelope protein's PBM in the virulence and pathogenicity of SARS-CoV-2, spanning from molecular characterization to in vivo infection in the hamster model. Using reverse genetics, we produced recombinant viruses expressing the E protein with either a deleted E-ΔPBM or a mutated E-MutPBM.

Viral titration and growth kinetics revealed a replication delay in viruses lacking a functional PBM, underscoring the importance of this motif in viral fitness. To assess the impact of PBM deletion more accurately, we conducted infections at low multiplicities of infection (MOIs of 0.001 and 0.01), where growth delays were more pronounced. In contrast, higher MOIs (0.1 and 1) might obscure subtle differences due to rapid cytopathic effects. The similarity in median time to cell death at MOIs 0.01 and 0.1 suggests a threshold effect that may limit the detection of fine-scale kinetic differences. This limitation should be considered in future studies exploring PBM's role in viral behavior. Nonetheless, the replication delay observed at lower MOIs aligns with previous reports attributing a critical role to this protein-protein interaction motif [51]. Indeed, the PBM of the SARS-CoV-2 E protein plays a role in viral replication and virulence by interacting with host PDZ-containing proteins, inducing an excessive proinflammatory response, and contributing to alveolar edema and acute respiratory distress syndrome.

The pathogenicity is marked by a disruption at the epithelial level of the respiratory tract [1, 52–54]. Airway barriers are characterized by a complex interplay of tight junctions, mucociliary clearance mechanisms, and immune surveillance. Their disturbance highlights a critical aspect of viral pathogenesis: viruses often target epithelium and cellular junctions and polarity components to hijack cellular machinery [55–57]. Respiratory virus infections are known to dysregulate tight junction proteins, compromising barrier function, and facilitating pathogen invasion into the subepithelial space.
PDZ domain-containing scaffold proteins are key components of paracellular barriers and innate immunity by linking transmembrane proteins of epithelial junctions to the cell cytoskeleton [55, 58, 59]. The E protein PBM of SARS-CoV-1 has been identified as targeting the PDZ domain of PALS-1, leading to disruption of cellular structures [14]. We previously identified PDZ-containing proteins, partners of the PBM involved in cell junction and polarity and characterized these interactions at structural and functional levels [29]. ZO-1, a protein observed to be mislocalized and disrupted during SARS-CoV-2 infection [1, 26, 60–62], emerged as a key partner for the E PBM.

Before investigating the disturbance of PDZ-containing proteins, we focused our study on the cellular localization of the E protein. Immunostaining in infected A549 ACE2 TMPRSS2 lung epithelial cells and infected ALI Transwell model (MucilAir) revealed that E protein localization depends on an upstream Golgi targeting sequence in the C-terminal tail, unaffected by the PBM [63, 64]. As reported in the literature for SARS-CoV-1 and 2, regardless of whether the viral construct includes the PBM or not, the E protein accumulates in the Golgi apparatus but also exhibits a vesicular cytoplasmic distribution. This generic term might encompass endosomes, lysosomes, peroxisomes, and other vesicular organelles involved in the exocytosis pathway and viral egress [11, 46–48, 65]. Moreover, studies have reported Golgi enlargement in response to the infection, indicating structural changes that may facilitate viral assembly and release [48, 66]. While our imaging approach provided valuable insights into E protein localization independently of its PBM in relevant lung epithelial models, additional staining strategies targeting specific cellular organelles and epithelial cell subtypes would be beneficial to further enhance the resolution of E protein subcellular localization. Such approaches would enable a more precise characterization of the intracellular compartments involved and clarify the differential impact of infection across distinct epithelial lineages.

Focusing on the cell junction disturbance, we previously demonstrated that the E protein PBM interacts with the PDZ2 domain of ZO-1 [28] and that transfected E protein, when expressed at the Golgi apparatus, can recruit PDZ2 and full-length ZO-1 to this organelle [29, 53]. During infection, ZO-1 staining revealed mislocalization and degradation in A549 cells and the MucilAir model. This phenomenon is directly associated with the PBM, as reported by immunofluorescence colocalization and co-immunoprecipitation experiments. Interestingly, this perturbation depends on the localization of the E protein. No disturbance is observed when the E protein is localized at the Golgi apparatus, suggesting that viral entry is not a critical step for epithelial disruption. However, when the E protein is engaged into the exocytosis pathway and exhibits vesicular localization associated with endosomal and lysosomal organelles, ZO-1 appears disrupted. These findings indicate that the disruption of cellular structures by the E protein is not solely dependent on the PBM but also on the protein's subcellular localization. It also supports the conclusions drawn from studies on PALS-1 during SARS-CoV-1 infection, where the E protein might impact the trafficking of cell junction components to the membrane, leading to leakage between adjacent epithelial cells, disruption of the epithelial barrier, and ultimately promoting viral spread [14]. ZO-1, as a key tight junction scaffolding protein, interacts not only with occludin [67–69] but also with a broader network of junctional proteins, including claudins, connexins, and other ZO family members [70–74]. Investigating these interconnected components may uncover a cascade of
molecular interactions within the cell junction complexes that modulate or amplify barrier dysfunction. While the present study emphasizes the role of the PBM in this process using cellular models, additional in vivo analyses, such as co-immunoprecipitation and co-immunofluorescence, are essential to confirm the E-ZO-1 interaction in infected animal tissues and to further substantiate its relevance to SARS-CoV-2 pathogenesis.

While quantifying the ZO-1 disruption could further support the PBM-dependent interaction between the E protein and ZO-1, the combination of qualitative immunofluorescence observations and biochemical evidence from pull-down assays provides compelling evidence for this conclusion. Indeed, our results consistently show a reduced disruption of ZO-1 in cells infected with PBM-deficient viruses, aligning with the known role of the PBM in epithelial barrier impairment and is further confirmed by the pull-down assays, in which the mutated PBM virus fails to interact with ZO-1. However, its preservation is not complete, as the perturbation of epithelial integrity results from multiple mechanisms involving several viral proteins. While the E PBM is widely recognized as a factor of pathogenicity, the Orf3a and Nucleocapsid proteins also possess a PBM, interacting with specific PDZ domain-containing proteins, with a limited literature [28, 75-77]. Interestingly, our previous in vitro high-throughput screening of the Orf3a PBM against the full human PDZome showed that ZO-1 is also a potential interactant [28]. Deletion of the SARS-CoV-1 Orf3a PBM has minimal impact on replication in infected cells and only a modest effect on weight loss or mortality in mice [76]. While its role in viral pathogenesis remains unclear, the Orf3a PBM may partially compensate for a PBM-deficient E protein.

A striking attenuation of pathogenicity was observed in vivo when hamsters were infected with viruses lacking the E PBM, as previously reported for SARS-CoV-1 and 2 in mice [7, 8, 51]. In our study, the animals infected with PBM-deficient viruses exhibited no weight loss and no clinical signs of the disease. Quantification of viral infectious particles revealed substantial viral loads in the airways for PBM-deficient viruses, though at lower levels compared with E-WT infection at the early time points of the infection. This phenomenon was more pronounced in the lower airways, consistent with the virus's pathway in the respiratory tract and propagation defects observed for the PBM-deficient viruses. Interestingly, the viruses expressing deleted or mutated PBM exhibited different behaviors following various infection parameters. Indeed, the PBM-mutated virus showed a stronger attenuation of phenotypes compared with the PBM-deleted virus. These results suggest a differential host response to the distinct envelope C-terminus sequences resulting from these modifications. Several hypotheses can be formulated concerning this effect. The deletion induces a change in E protein size that might impact various mechanisms in which E is involved. From this perspective, literature highlights several crucial motifs within the cytoplasmic domain of the E protein involved in protein and lipid interactions, folding, and post-translational modifications [78-81]. Moreover, this deletion could create a potential alternative internal PBM. Indeed, the deletion provides the sequence -NLNSSRVP<sub>COOH</sub> with a potential internal type I PBM (-X-S/T-X-φ) instead of the type II PBM (-X-φ-X-φ<sub>COOH</sub>) found in the E-WT (-NLNSSRVPD<sub>LLV</sub><sub>COOH</sub>). This new PBM might interact with a subset of PDZ-containing proteins targeted by E-WT, as it has been demonstrated that different PBM types in coronaviruses can have overlapping targets [28]. It is also possible that the new
PBM recruits other PDZ-containing proteins, partially compensating for the loss of the original interactions. Another hypothesis might be that the mutation and/or deletion of the PBM in viruses differentially alter the RNA structure in a different way in a region that plays a role in viral translation and/or replication. Further investigation is required to fully understand these changes and their implications.

In addition, our study focused on examining the role of the PBM in anosmia and neuroinvasion. The brain barriers, such as the choroid plexus, are reported to be highly enriched in PDZ-containing proteins [23, 24], which tightly regulate the central nervous system (CNS) homeostasis as well as protect the CNS from toxins, inflammation, injury, and pathogens [82]. This also prompts a broader consideration of epithelial barriers, raising the question of whether the effect of the PBM is specifically linked to the disruption of certain tissues. Unlike the animals infected with the E-WT virus, the hamsters infected with viruses lacking the PBM maintained their olfactory performance. Moreover, the genomic viral RNA quantification revealed similar viral loads regardless of the virus. These data support the notion that anosmia and neuroinvasion are independent phenomena during infection [30]. Indeed, anosmia is predominantly associated with the effects of the infection on the olfactory epithelium, where disorganization, inflammation, apoptosis, and damages to the ciliary layer occur [83, 84].

In this perspective, we focused our analysis on the impact of the PBM on airways lesions. Notable histological phenotypes associated with the infection and respiratory failure were observed. The hamster lungs infected with E-MutPBM present a dramatic reduction in edema and inflammation compared with animals infected with the E-WT, and E-ΔPBM virus again displays an intermediate phenotype exhibiting moderate inflammation. Altogether these data support literature on the involvement of the PBM and more largely the E protein in the inflammatory process characterizing the COVID-19 [85–88]. Furthermore, the conserved sequence of the PBM across all SARS-CoV-2 variants strongly supports its pivotal role in pathogenesis, reinforcing the relevance of our findings on epithelial barrier disruption and systemic inflammation, which may be applicable to emerging variants. Histopathological imaging of the upper airways, showing a drastic reduction in lesions, is consistent with the preservation of olfactory capabilities in animals infected with the PBM-deficient viruses. Although the olfactory bulb is a common structure in the SARS-CoV-2 cerebral infectious process, literature also reports infection through the blood brain barriers, including the choroid plexus [22, 89–92]. At 4 dpi, no morphological changes at the macroscopic scale were recorded in the histopathological analysis, nor was there any evidence of astrocyte activation or barrier leakage.

While COVID-19 infection is known to induce a severe inflammatory response, particularly in the early stages, which can lead to respiratory failure and death [4, 93, 94], our transcriptomic analysis further supports that the E PBM plays a critical role in amplifying this response. Indeed, in PBM-deficient cases, inflammatory pathways were enriched in our RNA-seq but with notably less intensity in terms of gene number and fold change. A significant decrease in the expression of genes involved in inflammatory pathways was observed, supporting the role of the PBM in driving the unrestrained immune response typically triggered during SARS-CoV-2 infection [5, 95, 96]. These data demonstrate that the exacerbated host innate immune response was impaired and
delayed in the absence of PBM, aligning with the viral replication defect observed in air-way organs. These findings may explain the attenuated pathogenicity observed for these viruses. The downregulation of key signaling pathways, including “WNT,” “MAPK,” and “PI3K-AKT,” observed exclusively in E-WT-infected lungs suggests a PBM-specific role in modulating these critical cellular processes. This finding warrants further investigation to elucidate potential mechanistic connections, as PBMs in other viruses are known to influence similar pathways [97–100]. Additionally, these findings correlate with the observed downregulation of adhesion junction proteins, including PDZ-containing proteins such as ZO-1, suggesting that the PBM may impact epithelial integrity not only at the proteomic level but also through gene expression regulation. However, transcriptionic changes in the brainstem, were minimal between PBM-deficient and E-WT virus infections, with the identification of three PDZ-related proteins: *CNKSR2*, which features both a PBM and a PDZ domain [101], as well as *SGK1*, recognized for its interaction with the PDZ-containing NHERF family [102] previously reported as a partner of the E PBM [28], and ERBIN, a basolateral epithelial protein [103] related to Ras-Raf and NF-kB signaling pathways [104–107]. Although the presence of PDZ-related proteins was notable, further investigations are needed to fully understand the molecular mechanisms through which the E protein PBM modulates brain function and the pathological consequences.

## Conclusions

Thus, the E protein serves as a virulence factor that influences viral fitness, spread, and pathogenicity in vivo. Altogether, these observations underscore the critical role of the SARS-CoV-2 E protein in disrupting epithelial barrier function, highlighting the significant and innovative therapeutic potential of targeting the E protein's PBM and its interactions with PDZ proteins.

## Abbreviations

<table><tr><td>A549</td><td>Human alveolar basal epithelial cell line</td></tr><tr><td>ACE2</td><td>Angiotensin-converting enzyme 2</td></tr><tr><td>AF-6</td><td>Afadin</td></tr><tr><td>AJs</td><td>Adherens junctions</td></tr><tr><td>ARDS</td><td>Acute respiratory distress syndrome</td></tr><tr><td>BBB</td><td>Blood-brain barrier</td></tr><tr><td>BCSFB</td><td>Blood-cerebrospinal fluid barrier</td></tr><tr><td>CNKSR2</td><td>Casein kinase 2 interacting protein 1</td></tr><tr><td>CoV-2</td><td>Coronavirus 2</td></tr><tr><td>COVID-19</td><td>Coronavirus disease 2019</td></tr><tr><td>E</td><td>Envelope protein</td></tr><tr><td>E-ΔPBM</td><td>E protein with deleted PBM</td></tr><tr><td>E-MutPBM</td><td>E protein with mutated PBM</td></tr><tr><td>E-WT</td><td>Wild-type E protein</td></tr><tr><td>ERBIN</td><td>Erb-B2 receptor tyrosine kinase 3 interacting protein</td></tr><tr><td>FCS</td><td>Fetal calf serum</td></tr><tr><td>Golgi</td><td>Golgi apparatus</td></tr><tr><td>IFN</td><td>Interferon</td></tr><tr><td>IL-6</td><td>Interleukin 6</td></tr><tr><td>MAPK</td><td>Mitogen-activated protein kinase</td></tr><tr><td>MLLT4</td><td>Myeloid/lymphoid or mixed-lineage leukemia translocated to 4 (Afadin)</td></tr><tr><td>MucilAir</td><td>Air-liquid interface (ALI) transwell model</td></tr><tr><td>NF-kB</td><td>Nuclear factor kappa-light-chain-enhancer of activated B cells</td></tr><tr><td>Occludin</td><td>Tight junction protein</td></tr><tr><td>PALS1</td><td>Protein associated with lin seven 1</td></tr><tr><td>PARD3</td><td>Partitioning defective 3</td></tr><tr><td>PBM</td><td>PDZ-binding motif</td></tr></table>
<table><tr><td>PDZ</td><td>PSD-95/Dlg/ZO-1</td></tr><tr><td>RNA</td><td>Ribonucleic acid</td></tr><tr><td>RNA-seq</td><td>RNA sequencing</td></tr><tr><td>SARS</td><td>Severe acute respiratory syndrome</td></tr><tr><td>SARS-CoV-1</td><td>Severe acute respiratory syndrome coronavirus 1</td></tr><tr><td>SARS-CoV-2</td><td>Severe acute respiratory syndrome coronavirus 2</td></tr><tr><td>SGK1</td><td>Serum- and glucocorticoid-inducible kinase 1</td></tr><tr><td>TJP1</td><td>Tight junction protein 1 (ZO-1)</td></tr><tr><td>TJs</td><td>Tight junctions</td></tr><tr><td>TNF-α</td><td>Tumor necrosis factor alpha</td></tr><tr><td>ZO-1</td><td>Zonula occludens-1</td></tr></table>

## Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s11658-025-00758-y.

Supplementary Material 1. The SARS-CoV-2 envelope PDZ Binding Motif acts as a virulence factor disrupting host's epithelial cell-cell junctions.

## Acknowledgements

We would like to acknowledge Caroline Demeret (Interactomics, RNA and immunity Laboratory, Institut Pasteur) for the fruitful discussions about SARS-CoV-2 protein interactome. We thank Remy Robinot and Lisa Chakrabarti (Virus and Immunity Unit, Institut Pasteur) for their advice on the ALI Transwell model. We would like to thank Axion Biosystem for giving us the opportunity to experiment with the Maestro machine, and especially Emma David and Nicolas Roy for their support and assistance. Part of this work was performed at the Histological core facility, UtechS Photonic Biomaging (PBI) and Biomics platforms of the Institut Pasteur. We acknowledge the work performed in transcriptomic experiments by Elodie Turc and Iakov Vintrenko, members of the Biomics Platform directed by Marc Monot (C2RT, Institut Pasteur).

## Author contributions

Conceptualization: FA, GDM, FL, NW, CCS. Methodology: FA, LK, GDM, FL, DH, DS. Investigation: FA, GDM, FL, LK, BB, VM, DS, MT, DH, EK. Funding Acquisition: FA, GDM, FL, HB, NW, CCS. Supervision: NW, CCS. Writing—original draft: FA, CCS. Writing—review and editing: all authors.

## Funding

Institut Pasteur's TASK FORCE COVID-19. Institut Pasteur's Programme Fédérateur de Recherche 5 (PFRS-Functional Genomics of the Viral Cycle funded by Fondation d'entreprise Michelin donation). Institut Pasteur's Programme Fédérateur de Recherche 1 (PFR-1-Reverse Genetics). Institut Pasteur's Emerging Infection Desease (EID) Junior Call. France Génomique (ANR-10-INBS-09) and IBISA.

## Availability of data and materials

All data are available in the main text or the supplementary materials.

## Declarations

### Ethics approval and consent to participate

All animal experiments were performed according to the French legislation and in compliance with the European Communities Council Directives (2010/63/UE, French Law 2013–118, February 6, 2013) and according to the regulations of Institut Pasteur Animal Care Committees. The Animal Experimentation Ethics Committee (CETEA 89) of the Institut Pasteur approved this study (200023; APAFIS#25326-2020050617114340 v2) before experiments were initiated. Hamsters were housed by groups of 4 animals in isolators and manipulated in class III safety cabinets in the Pasteur Institute animal facilities accredited by the French Ministry of Agriculture for performing experiments on live rodents. All animals were handled in strict accordance with good animal practice.

### Consent for publication

Not applicable.

### Competing interests

All authors declare they have no competing interests. Received: 19 March 2025 Accepted: 23 June 2025

Published online: 11 July 2025

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