Biology subjects

Legros, V.

Publications and source records attributed to Legros, V..

18 recordsLinked to original sources

bioRxiv2026

Cyclin B phosphorylation by PKA safeguards the G2-arrest in vertebrate oocytes

Entry into meiotic M-phase in vertebrate oocytes requires activation of Cdk1-Cyclin B, which is restrained by the cAMP-PKA signalling during prophase arrest. Although Cdc25C and ARPP19 are established PKA substrates in this context, they do not fully explain how PKA represses Cdk1 activation. Here we used Turbo-ID approach using catalytic PKA (PKAc) as the bait to identify substrates in Xenopus laevis oocytes. Unexpectedly, this screen identified Cyclin B2, the regulatory subunit of Cdk1, as a PKA-proximal protein. Although Cyclin B2 does not stably associate with PKAc, it harbors a conserved PKA motif around Serine 271 within Cyclin Box 2. We show that PKAc phosphorylates Cyclin B2 at S271 both in vivo and in vitro. Functionally, a phosphomimic Cyclin B2 mutant at this site fails to induce meiotic maturation and Cdk1 activation. S271 phosphorylation neither alters Cyclin B2 stability nor its binding to Cdk1 in vivo. S271 phosphorylation does not impair Cdk1 activity toward a single-site substrate, such as PP1, but slows Cyclin-B-dependent multisite phosphorylation of Cdc25C, a key regulator of the Cdk1 activation network. These findings identify Cyclin B as a direct and conserved PKA target, revealing the mechanistic link between PKA activity and Cdk1-Cyclin B repression that maintains oocyte prophase arrest.

cell biology↗

bioRxiv2026

Low-input proteomics enables proteome and phosphoproteome-scale molecular phenotyping of separase-deficient oocytes

We performed a comprehensive quantitative proteomic analysis of mouse oocytes using as few as 40 oocytes per condition, comparing wild-type and separase knockout oocytes at metaphase I and metaphase II. To this end, we generated a deep proteomic library spanning oocyte cell cycle stages, enabling the identification of numerous phosphosites without phosphopeptide enrichment. We further combined data-dependent (DDA) and data-independent (DIA) acquisition strategies, analyzed through multiple software pipelines in both library-based and library-free modes. Our results reveal extensive proteome remodeling during the metaphase I to metaphase II transition in wild-type oocytes, consistent with dynamic regulation of meiotic processes. As a proof of concept for our workflow, we asked whether separase knockout oocytes--unable to separate chromosomes in meiosis I--progress into meiosis II. Direct comparison of wild-type and separase knockout oocytes at the metaphase II stage revealed minimal global differences, supporting the idea that both conditions converge toward a comparable metaphase II-like cellular state despite distinct chromosomal configurations. However, at a finer scale, specific alterations were detected among chromosome-associated proteins. Notably, Meikin was enriched in separase-deficient metaphase II oocytes, consistent with defective separase-dependent cleavage and subsequent turnover. More broadly, several proteins involved in chromosome organization displayed behavior similar to Meikin, suggesting that separase activity regulates multiple substrates to orchestrate chromosome segregation during female meiosis.

Cell Biology↗

bioRxiv2026

An RNA-centric interactomics screen identifies novel proviral and antiviral genome binding interactors for tick-borne encephalitis virus

The tick-borne encephalitis virus (TBEV), which belongs to the Orthoflavivirus genus, is a medically significant arbovirus in Europe and Asia. Despite distinct pathobiological outcomes, arboviruses have evolved specific strategies to co-opt cellular factors for replication and immune evasion, that involve both viral proteins and viral RNA. The RNA genome of TBEV, as both the substrate for genome replication and translation and a designated target for innate antiviral immunity, is a nexus for interactions with the proteome of host cells. Nonetheless, the molecular bases of these interactions and their implications for the replicative cycle of TBEV remain poorly understood. To create an inventory of such interactions and gain understanding of their functional import, we have resolved the set of cellular proteins bound to TBEV RNA in infected human cells using an agnostic RNA-centric approach. Functional annotation of the resulting core interactome of 215 human host factors showed that viral RNA is deeply embedded in multiple cellular pathways, including those related to RNA and protein metabolism, cytoskeletal scaffolding, vesicle trafficking and innate antiviral immunity. For selected human interactors, we addressed their impact on TBEV infection in gene knockdown experiments, thereby identifying multiple restriction and dependency factors. These included sensors and effectors of innate immune pathways, as well as epitranscriptomic modifiers. Among the former, the dynamin-like GTPase MX2 protein, an interferon-stimulated gene with antiviral activity against multiple viruses including mosquito-borne orthoflaviviruses, displayed unexpected proviral activity against TBEV and a second tick-borne orthoflavivirus. Among the latter, WDR4, the non-catalytic component of the METTL1-WDR4 methyltransferase complex, emerged as a restriction factor with broad-spectrum activity against arboviruses belonging to multiple families of positive-strand RNA viruses. In conclusion, this first description of the RNA interactome of a tick-borne orthoflavivirus illuminates the molecular interactions that underpin TBEV infection of human cells, which taken together reflect both the common ancestry of tick- and mosquito-borne orthoflaviviruses and their considerable evolutionary divergence.

molecular biology↗

bioRxiv2026

Molecular dialogue between Orthonairovirus and tick: RNA-protein interactome of Hazara virus, a BSL2 model of Crimean-Congo Hemorrhagic Fever virus, in Hyalomma cells

Climate change and ecosystem collapse promote geographic expansion of vector-borne diseases, as witnessed by the recent incursions into Spain of the virus responsible for Crimean-Congo hemorrhagic fever (CCHFV). CCHFV is maintained in a tick-vertebrate cycle, principally involving ticks of the genus Hyalomma. Faced with the spread of Hyalomma ticks, and therefore the threat of a natural introduction of CCHFV into Western Europe, appropriate surveillance tools and control measures need to be implemented. It is both within and by the tick that CCHFV is maintained and spread in the environment. Despite prolonged portage of the virus, the tick is not overtly affected by CHFV infection. One of the prerequisites in conceiving control strategies is to understand the molecular mechanisms that intimately link the virus to its arthropod host. Despite the central role of the tick in the biology of CCHFV, these mechanisms are ill-defined, owing in part to the constraints associated with handling CCHFV-infected ticks in biosafety level 4 containment. In this study, we established the network of interactions between the S segment of the RNA genome Hazara virus (HAZV), a BSL-2 model of CCHFV, and Hyalomma proteins using ChIRP-MS technique. We identified 166 tick proteins, 21 of which have been described as RNA-binding proteins. Gene ontology and pathway enrichment analyses revealed that the S segment RNA interacts predominantly with mitochondrial proteins that belong to various mitochondrial metabolic pathways.

microbiology↗

bioRxiv2025

EMILIN1 emerges as a TGFbeta/SETDB1-regulated secreted biomarker in Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is an incurable muscle-wasting disorder characterized by chronic membrane damage, inflammation, and progressive fibrosis. Fibrosis in DMD is driven by sustained TGF{beta} signaling, which promotes extracellular matrix (ECM) accumulation. We previously showed that SETDB1 sustains the TGF{beta}-induced fibrotic response in DMD myotubes. Here, we further show that SETDB1 modulates the TGF{beta}-induced secretome, particularly by regulating ECM-related proteins. Comparison of the basal secretome from DMD patient-derived myotubes and healthy controls revealed a distinct disease-specific profile. Integrating both secretome analyses, we identified EMILIN1, an ECM glycoprotein not previously studied in skeletal muscle, as a robust shared candidate; EMILIN1 is enriched in the DMD secretome, further upregulated by TGF{beta}, and downregulated upon SETDB1 depletion. We confirmed EMILIN1 overexpression in DMD patient muscle biopsies, validating its pathological relevance. Functionally, EMILIN1 depletion modulated myogenic differentiation and reduced expression of the fibrotic marker SERPINE1. These findings establish EMILIN1 as a novel secreted regulator of myogenesis and fibrosis, and implicate SETDB1 in shaping the TGF{beta}-dependent secretome in DMD. Our integrative proteomic approach provides new insights into the molecular drivers of impaired regeneration in DMD and highlights potential therapeutic targets.

genetics↗

bioRxiv2025

Greatwall depletion from Xenopus oocytes reveals a key role of the cyclin B/CDK1-PP2A-B55 balance in the coordination of meiotic events

Meiotic progression relies on maintaining a precise balance between cyclin B/CDK1 activity and the phosphatase PP2A-B55. The latter is negatively regulated by the Greatwall kinase (Gwl). In Xenopus oocytes, we show that the loss of Gwl and the subsequent hyperactivation of PP2A-B55 severely disrupt the transition from meiosis I to meiosis II. This disruption prevents phosphorylation of both Wee1/Myt1 and the APC/C complex, thereby blocking APC/C activation. As a consequence, APC/C remains inactive during the MI-MII transition, which impairs cyclin degradation and the partial CDK1 inactivation that is normally required at this stage. Additionally, the mos-MAPK-Rsk1/2 pathway fails to activate due to insufficient Mos accumulation, thereby preventing metaphase II arrest. Finally, the lack of APC/C activation during meiosis I inhibits the degradation of its inhibitor Erp1, revealing a critical feedback loop between APC/C and Erp1. Overall, our findings reveal that Gwl is a key coordinator of both meiotic divisions, acting through dual regulation of APC/C and the Mos/MAPK/Rsk1/2 pathways by modulating PP2A-B55 activity.

biochemistry↗

bioRxiv2025

A genetic screen reveals a key role for Reg1 in 2-deoxyglucose sensing and yeast AMPK inhibition

The yeast Saccharomyces cerevisiae thrives in sugar-rich environments by rapidly consuming glucose and favoring alcoholic fermentation. This metabolic strategy is tightly regulated by glucose signaling pathways, particularly the glucose repression pathway, which prevents the expression of genes required for the utilization of alternative carbon source. Central to this regulatory network is the yeast ortholog of the heterotrimeric 5'AMP-activated protein kinase (AMPK), which adjusts gene expression in response to glucose availability. The activity of the yeast AMPK complex is primarily regulated by the phosphorylation state of its catalytic subunit Snf1, a process orchestrated by a balance between upstream kinases and phosphatases. Among the latter, the Protein Phosphatase 1 (PP1) complex Reg1/Glc7 plays a critical role in repressing Snf1 activity under glucose-rich conditions. Despite its importance, the precise mechanism by which glucose availability leads to Snf1 inhibition remains incompletely understood. Evidence suggests that hexokinase 2 (Hxk2) participates in this pathway, potentially coupling the early steps of glucose metabolism to Snf1 signaling. Notably, the toxic glucose analog 2-deoxyglucose (2DG)- which is phosphorylated by Hxk2 but not further metabolized-mimics glucose in its ability to repress Snf1, implicating glucose or 2DG phosphorylation as a key regulatory signal. In this study, we explore the molecular basis of 2DG resistance in yeast through a large-scale genetic screen. The identified mutations confer resistance either by reducing 2DG phosphorylation (e.g., mutations in HXK2) or by enhancing constitutive Snf1 activity, via gain-of-function alleles in AMPK subunits or loss-of-function mutations in REG1 and GLC7. We describe a novel class of REG1 missense mutations, including reg1-W165G, that maintain normal basal Snf1 activity but fail to mediate 2DG-induced Snf1 inhibition, rendering cells effectively immune to 2DG toxicity. These findings position Reg1 as a central mediator in glucose sensing and further suggest that 2DG toxicity primarily results from aberrant Snf1 inactivation rather than a direct metabolic disturbance.

cell biology↗

bioRxiv2025

Likely circulation of a coronavirus in roe deer (Capreolus capreolus) populations suggested by SARS-CoV-2 serological investigation in France

The risk of viral transmissions from domestic/wild animals to humans is a major public health concern. Humans can also transmit viruses back to domestic and wild animals, acting as reservoir for virus maintenance and sources of epidemic diseases re-emergence. The SARS-CoV-2, causing COVID-19, likely originated from wildlife and has been evidenced to transmit from humans to captive, domestic and wild animals. White-tailed deer (Odocoileus virginianus) show high SARS-CoV-2 prevalence following human contamination, suggesting they could act as an emerging virus reservoir. We completed recent research on European cervid species by investigating whether SARS-CoV-2 had emerged in longitudinally-monitored European roe deer (Capreolus capreolus) populations in direct contact with humans in France. We performed indirect tests (serological ELISAs and seroneutralization) on sera collected before and after the virus emergence in humans, and direct RT-PCR tests on nasal swabs collected in 2022. We also investigated the virus exposure and prevalence in three other cervid species. ELISA tests were positive for 2.20% of sera, pre- and post-pandemic, but seroneutralization and PCR tests were negative. Although one population showed increased seroprevalence post-2020, results suggest that SARS-CoV-2 has not emerged in those populations, and that ELISA cross-reaction with one or several unidentified circulating coronaviruses is possible.

immunology↗

bioRxiv2025

Large extracellular vesicles containing mitochondria (EVMs) derived from Alzheimer disease cells harbor pathologic functional and molecular profiles and spread mitochondrial dysfunctions

In addition to small extracellular vesicles known as exosomes, cells release large extracellular vesicles containing mitochondria (EVMs). However, the molecular and functional characteristics of EVMs, as well as the impact of EVM secretion on the spreading of mitochondrial dysfunction between cells, remain unknown in the context of Alzheimers Disease (AD). Here, we provide an ultrastructural, biochemical, and functional characterization of EVMs isolated from cells expressing the amyloid precursor protein (APP) with the familial Swedish mutation (APPswe). We identified differential proteomic and lipidomic signatures in APPswe-derived EVMs compared to control EVMs and revealed a specific proteomic profile in EVMs isolated from conditioned media of fibroblasts from AD patients at the prodromal stage of the disease. Our findings show that APP-C terminal fragments (APP-CTFs) pathogenic accumulation in cells potentiates EVM secretion through the budding of the plasma membrane. We lastly demonstrated that APP-CTFs loaded EVMs are active carriers of dysfunctional mitochondria that transfer mitochondrial pathology to naive control recipient cells.

neuroscience↗

bioRxiv2024

A recombinant CHIKV-NLuc virus identifies chondrocytes as target of Chikungunya virus in a immunocompetent mouse model

First isolated in 1953 in Tanzania, the arthritogenic Chikungunya virus (CHIKV) re-emerged globally in 2005, leading to widespread outbreaks. Unlike other arboviruses, CHIKV predominantly induces symptomatic infections (72-96%), marked by fever, myalgia, polyarthralgia, and rash. Although rarely fatal, atypical forms such as encephalopathies can occur. Notably, 75.4% of patients experience persistent arthralgias for up to three years after the acute phase. Understanding CHIKVs pathophysiology in the joints is challenging due to the difficulties to obtain biological samples. The study employs a mouse model infected with a reporter virus expressing a Nano Luciferase to investigate the diseases transition to chronic arthritis. The murine model reveals viral replication in metatarsi joints, particularly in chondrocytes, confirmed in primary human chondrocytes undergoing viral-induced apoptosis. Ex vivo analysis confirmed viral replication in leg bones and articular cartilages, with histological evidence of focal erosive lesions and periarticular inflammation. The study further utilizes an in vivo imaging mouse model to monitor viral replication over time. Human chondrocytes prove susceptible to CHIKV infection, exhibiting active viral replication, bioluminescence activity, and increased viral production. CHIKV induced apoptosis, the upregulation of markers associated with cartilage remodeling and altered the cytokine production. This comprehensive study, utilizing advanced techniques and models, provides insights into CHIKVs ability to infect articular cartilages, shedding light on the mechanisms of chronic arthritis following infection.

microbiology↗

bioRxiv2023

Qualitative rather than quantitative phosphoregulation shapes the end of meiosis I in budding yeast

Exit from mitosis is brought about by dramatic changes in the phosphoproteome landscape. A drop in Cyclin-dependent kinase (Cdk), the master regulatory kinase, and activation of counteracting phosphatases such as Cdc14 in budding yeast, results in ordered substrate dephosphorylation, allowing entry into a new cell cycle and replication licensing. In meiosis however, two cell divisions have to be executed without intermediate DNA replication, implying that global phosphorylation and dephosphorylation have to be adapted to the challenges of meiosis. Using a global time-resolved phosphoproteomics approach in budding yeast, we compared the phosphoproteome landscape between mitotic exit and the transition from meiosis I to meiosis II. We found that unlike exit from mitosis, Cdk phosphomotifs remain mostly stably phosphorylated at the end of meiosis I, whereas a majority of Cdk-unrelated motifs are reset by dephosphorylation. However, inducing an artificial drop of Cdk at metaphase of meiosis I leads to ordered substrate dephosphorylation, comparable to mitosis, indicating that phosphoregulation of substrates at the end of meiosis I is thus mainly qualitatively rather than quantitatively ordered.

cell biology↗

bioRxiv2023

The nuclear PIWI-interacting protein Gtsf1 controls the selective degradation of small RNAs in Paramecium

Ciliates undergo developmentally programmed genome elimination, in which small RNAs direct the removal of target DNA segments, including transposable elements. At each sexual generation, the development of the macronucleus (MAC) requires massive and reproducible elimination of a large proportion of the germline micronuclear (MIC) genome, leading to a highly streamlined somatic MAC genome. 25-nt long scnRNAs are produced from the entire germline MIC genome during meiosis, and this initial complex small RNA population is then transported to the maternal MAC, where selection of scnRNAs corresponding to germline (MIC)-specific sequences is thought to take place. Selected scnRNAs, loaded onto the PIWI protein Ptiwi09, guide the deposition of histone H3 post-translational modifications (H3K9me3 and H3K27me3) onto transposable elements in the developing macronucleus, ultimately triggering their specific elimination. How germline-specific MIC scnRNAs are selected remains to be determined. Here, we provide important mechanistic insights into the scnRNA selection pathway by identifying a Paramecium homolog of Gametocyte specific factor 1 (Gtsf1) as essential for the selective degradation of scnRNAs corresponding to retained somatic MAC sequences. Consistently, we also show that Gtsf1 is exclusively localized in the maternal macronucleus and associates with the scnRNA-binding protein Ptiwi09. Furthermore, Gtsf1 is necessary for DNA elimination and correct H3K9me3 and H3K27me3 localization in the new developing macronucleus, demonstrating that the scnRNA selection process is important for genome elimination. We propose that Gtsf1 is required for the coordinated degradation of Ptiwi09-scnRNA complexes that pair with nascent RNA transcribed from the maternal MAC genome, similarly to the mechanism suggested for microRNA target-directed degradation in metazoans.

genetics↗

bioRxiv2023

Mechanisms of Nuclear Pore Complex disassembly by the mitotic Polo-Like Kinase 1 (PLK-1) in C. elegans embryos

The nuclear envelope, which protects and organizes the interphase genome, is dismantled during mitosis. In the C. elegans zygote, nuclear envelope breakdown (NEBD) of the parental pronuclei is spatially and temporally regulated during mitosis to promote the unification of the parental genomes. During NEBD, Nuclear Pore Complex (NPC) disassembly is critical for rupturing the nuclear permeability barrier and removing the NPCs from the membranes near the centrosomes and between the juxtaposed pronuclei. By combining live imaging, biochemistry, and phosphoproteomics, we characterized NPC disassembly and unveiled the exact role of the mitotic kinase PLK-1 in this process. We show that PLK-1 disassembles the NPC by targeting multiple NPC sub-complexes, including the cytoplasmic filaments, the central channel, and the inner ring. Notably, PLK-1 is recruited to and phosphorylates intrinsically disordered regions of several multivalent linker nucleoporins, a mechanism that appears to be an evolutionarily conserved driver of NPC disassembly during mitosis. (149/150 words) One-Sentence SummaryPLK-1 targets intrinsically disordered regions of multiple multivalent nucleoporins to dismantle the nuclear pore complexes in the C. elegans zygote.

cell biology↗

bioRxiv2022

A large-scale serological survey in pets from October 2020 through June 2021 in France shows significantly higher exposure to SARS-CoV-2 in cats

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect many animals, including pets such as dogs and cats. Many studies have documented infection in companion animals by bio-molecular and serological methods. However, only a few have compared seroprevalence in cats and dogs from the general population, and these studies were limited by small sample sizes and collections over short periods. Our goal was to obtain a more accurate evaluation of seroprevalence in companion animals in France and to determine whether cats and dogs differ in their exposure to SARS-CoV-2. For this purpose, we conducted an extensive SARS-CoV-2 cross-sectional serological survey of 2036 cats and 3577 dogs sampled by veterinarians during medical examinations in clinics throughout France. Sampling was carried out from October 2020 through June 2021, a period encompassing the second and third waves of SARS-CoV-2 infections in humans in the country. Using a microsphere immunoassay targeting the receptor binding domain and trimeric spike protein, we found 7.1% seroprevalence in pets. In a subset of 308 seropositive samples, 26.3% had neutralizing antibodies. We found that cats were significantly more likely to test positive than dogs, with seropositivity rates of 9.3% and 5.9% in cats and dogs, respectively. Finally, data for both species showed that seroprevalence was lower in older animals and was not associated with the date of sampling or the sex of the animal. Our results show that cats are significantly more sensitive to SARS-CoV-2 than dogs, in line with experimental studies. Our large sample size provides for a reliable, statistically robust estimate of the frequency of infection of pets from their owners and offers strong support for the notion that cats are more sensitive to SARS-CoV-2 than dogs. Our findings emphasise the importance of a One-Health approach to the SARS-CoV-2 pandemic and raise the question of whether companion animals in close contact with humans should be vaccinated.

microbiology↗

bioRxiv2021

New detection of SARS-CoV-2 in two cats height months after COVID-19 outbreak appearance in France

Although there are several reports in the literature of SARS-CoV-2 infection in cats, few SARS-CoV-2 sequences from infected cats have been published. In this report, SARS-CoV-2 infection was evaluated in two cats by clinical observation, molecular biology (qPCR and NGS), and serology (Microsphere immunoassay and seroneutralization). Following the observation of symptomatic SARS-CoV-2-infection in two cats, infection status was confirmed by RT-qPCR and, in one cat, serological analysis for antibodies against N-protein and S-protein, as well as neutralizing antibodies. Comparative analysis of five SARS-CoV-2 sequence-fragments obtained from one of the cats showed that this infection was not with one of the three recently emerged variants of SARS-CoV-2. This study provides additional information on the clinical, molecular, and serological aspects of SARS-CoV-2 infection in cats.

microbiology↗

bioRxiv2021

Myocarditis in naturally infected pets with the British variant of COVID-19

Domestic pets can contract SARS-CoV-2 infection but, based on the limited information available to date, it is unknown whether the new British B.1.1.7 variant can more easily infect certain animal species or increase the possibility of human-to-animal transmission. In this study, we report the first cases of infection of domestic cats and dogs by the British B.1.1.7 variant of SARS-CoV-2 diagnosed at a specialist veterinary hospital in the South-East of England. Furthermore, we discovered that many owners and handlers of these pets had developed Covid-19 respiratory symptoms 3-6 weeks before their pets became ill and had also tested PCR positive for Covid-19. Interestingly, all these B.1.1.7 infected pets developed atypical clinical manifestations, including severe cardiac abnormalities secondary to myocarditis and a profound impairment of the general health status of the patient but without any primary respiratory signs. Together, our findings demonstrate for the first time the ability for companion animals to be infected by the B.1.1.7 variant of SARS-CoV-2 and raise questions regarding its pathogenicity in these animals. Moreover, given the enhanced infectivity and transmissibility of B.1.1.7 variant for humans, these findings also highlights more than ever the risk that companion animals may potentially play a significant role in SARS-CoV-2 outbreak dynamics than previously appreciated.

microbiology↗

bioRxiv2020

High prevalence of SARS-CoV-2 antibodies in pets from COVID-19+ households

In a survey of household cats and dogs of laboratory-confirmed COVID-19 patients, we found a high seroprevalence of SARS-CoV-2 antibodies, ranging from 21% to 53%, depending on the positivity criteria chosen. Seropositivity was significantly greater among pets from COVID-19+ households compared to those with owners of unknown status. Our results highlight the potential role of pets in the spread of the epidemic.

microbiology↗

bioRxiv2020

The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a {beta}-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins. The involvement of each of these proteins and their interplays during the assembly process of this new virus are poorly-defined and are likely {beta}-coronavirus-type different. Therefore, we sought to investigate how SARS-CoV-2 behaves for its assembly by expression assays of S, in combination with E, M and/or N. By combining biochemical and imaging assays, we showed that E and M regulate intracellular trafficking of S and hence its furin-mediated processing. Indeed, our imaging data revealed that S remains at ERGIC or Golgi compartments upon expression of E or M, like for SARS-CoV-2 infected cells. By studying a mutant of S, we showed that its cytoplasmic tail, and more specifically, its C-terminal retrieval motif, is required for the M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlighted that E and M induce a specific maturation of S N-glycosylation, which is observed on particles and lysates from infected cells independently of its mechanisms of intracellular retention. Finally, we showed that both M, E and N are required for optimal production of virus-like-proteins. Altogether, our results indicated that E and M proteins influence the properties of S proteins to promote assembly of viral particles. Our results therefore highlight both similarities and dissimilarities in these events, as compared to other {beta}-coronaviruses. Author SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Its viral particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins, though their involvement in the virion assembly remain unknown for this particular coronavirus. Here we showed that presence of E and M influence the localization and maturation of S protein, in term of cleavage and N-glycosylation maturation. Indeed, E protein is able to slow down the cell secretory pathway whereas M-induced retention of S requires the retrieval motif in S C-terminus. We also highlighted that E and M might regulate the N glycosylation maturation of S independently of its intracellular retention mechanism. Finally, we showed that the four structural proteins are required for optimal formation of virus-like particles, highlighting the involvement of N, E and M in assembly of infectious particles. Altogether, our results highlight both similarities and dissimilarities in these events, as compared to other {beta}-coronaviruses.

microbiology↗