domain: sdv chim

text:
Van-Trang Dinh

Nadège Loaëc

Alicia Quillévéré

Ronan Le Sénéchal

Marc Keruzoré

Rodrigo Prado Martins

Anton Granzhan

Marc Blondel
email: marc.blondel@univ-brest.fr

The hide-and-seek game of the oncogenic Epstein-Barr virus-encoded EBNA1 protein with the immune system: An RNA G-quadruplex tale

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Although first observed at the beginning of the 20 th century [START_REF] Davies | Cancer in an African Community, 1897--1956. An Analysis of the Records of Mengo Hospital, Kampala, Uganda[END_REF], the Burkitt lymphoma (BL) was first described in 1958 as a prevalent lymphosarcoma affecting young children in sub-Saharan Africa [START_REF] Burkitt | A sarcoma involving the jaws in African children[END_REF]. It was then rapidly realized that the zone of incidence of BL coincided with the one of malaria, leading to the hypothesis that malaria could be responsible, or at least a cofactor for BL. In this context, Anthony Epstein and his student Yvonne Barr made the first observation, thanks to electron microscopy, of the so-called Epstein-Barr virus (EBV) in cultured lymphoblasts from Burkitt lymphoma ( [START_REF] Epstein | Virus Particles in Cultured Lymphoblasts from Burkitt's Lymphoma[END_REF][START_REF] Epstein | Cultivation in Vitro of Human Lymphoblasts from Burkitt's Malignant Lymphoma[END_REF]). A couple of years later, translocation and resulting activation of the c-MYC oncogene was detected in BL [START_REF] Klein | Specific chromosomal translocations and the genesis of B-cell-derived tumors in mice and men[END_REF][START_REF] Manolov | Marker band in one chromosome 14 from Burkitt lymphomas[END_REF].

Altogether these observations have paved the way for the current model that EBV, c-MYC and malaria collaborate to cause BL (reviewed in [START_REF] Thorley-Lawson | The curious case of the tumour virus: 50 years of Burkitt's lymphoma[END_REF]). Indeed, shortly after its discovery in 1964, the hypothesis that EBV may cause cancers has emerged, notably based on the observation that almost 100% of BL in tropical Africa were latently infected by the virus [START_REF] Magrath | The pathogenesis of Burkitt's lymphoma[END_REF] and carry the viral genome as a clonal extrachromosomal episome [START_REF] Lindahl | Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line[END_REF]. This way, EBV was the first oncogenic virus described in human and is still nowadays one of the most efficient means for transforming and immortalizing human B-cells. EBV infects more than 95% of the human population worldwide, but most EBV infections are asymptomatic even though, when EBV primary infection occurs during adolescence or young adulthood, it can lead to an acute infection known as infectious mononucleosis. After the primary infection, the virus persists lifelong in the memory B cells of the infected individuals. Under certain conditions the virus can cause several human cancers, that includes lymphoproliferative disorders such as Burkitt and Hodgkin lymphomas, non-lymphoid malignancies such as 100% of nasopharyngeal carcinoma, 10% of gastric cancers, rare T-and NK-cells lymphomas as well as leiomyosarcoma [START_REF] Daskalogianni | Epstein-Barr virus-encoded EBNA1 and ZEBRA: targets for therapeutic strategies against EBV-carrying cancers[END_REF][START_REF] Longnecker | Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis[END_REF][START_REF] Rezk | Epstein-Barr virus (EBV)-associated lymphoid proliferations, a 2018 update[END_REF] and, potentially, gliomas [START_REF] Akhtar | Epstein-Barr Virus in Gliomas: Cause, Association, or Artifact?[END_REF]. EBV is therefore found in a variety of human cancers whose localisation mostly overlaps with the known tissue tropism of the virus for B and epithelial cells. According to some estimations, about 200,000 EBV-related cancers emerge per year, hence accounting for at least 1% of cancer worldwide [START_REF] Cohen | Epstein-Barr virus: an important vaccine target for cancer prevention[END_REF]. In addition, a link between EBV primo-infection and multiple sclerosis has been suspected for a long time and firmly confirmed recently [START_REF] Bjornevik | Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis[END_REF]. For all these reasons, EBV became one of the most studied human viruses so far. As a latent virus, like all the gammaherpesviruses, EBV has evolved a strategy to escape the host immune system. This strategy is mainly based on the tight control of the expression of its Epstein-Barr nuclear antigen-1 (EBNA1) protein, the EBV-encoded genome maintenance protein of the virus [START_REF] Wilson | EBNA1: Oncogenic Activity, Immune Evasion and Biochemical Functions Provide Targets for Novel Therapeutic Strategies against Epstein-Barr Virus-Associated Cancers[END_REF]. Indeed, EBNA1 is considered as the Achilles heel of EBV due to the fact it is essential for viral genome replication and maintenance but, at the same time, it is also highly antigenic and T cells raised against EBNA1 exist in infected individuals [START_REF] Blake | Human CD8+ T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)containing protein requires exogenous processing[END_REF][START_REF] Lee | CD8 T cell recognition of endogenously expressed epstein-barr virus nuclear antigen 1[END_REF][START_REF] Leen | Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses[END_REF][START_REF] Munz | Epstein-barr virus nuclear antigen 1: from immunologically invisible to a promising T cell target[END_REF][START_REF] Munz | Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1[END_REF][START_REF] Voo | Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes[END_REF]. This review aims at recapitulating the knowledge about the EBNA1-based immune evasion of EBV and how the discovered mechanisms can define original therapeutic targets to unveil EBV-related cancers to the immune system.

EBNA1 is the Genome Maintenance Protein (GMP) of EBV

EBNA1 is a multifunctional EBV-encoded protein whose primary function is to ensure EBV genome replication and maintenance, hence being considered the genome maintenance protein (GMP) of the virus [START_REF] Blake | Immune evasion by gammaherpesvirus genome maintenance proteins[END_REF]. In addition, EBNA1 plays a role in transcriptional activation of the EBV latency genes in latent infection [START_REF] Gahn | An EBNA-1-dependent enhancer acts from a distance of 10 kilobase pairs to increase expression of the Epstein-Barr virus LMP gene[END_REF][START_REF] Sugden | A promoter of Epstein-Barr virus that can function during latent infection can be transactivated by EBNA-1, a viral protein required for viral DNA replication during latent infection[END_REF]. EBNA1 is a multi-domain protein (Figure 1) that contains, from residue 90 to 328 in its N-term part, a glycine alanine repeat (GAr) that consists of a stretch of single alanines separated by one, two or three glycines. This GAr domain is conserved across all EBV strains and plays a critical role in the ability of EBNA1, and thereby of EBV, to evade the immune system during the latent phase of viral infection (described in details later in this review). Importantly, a natural polymorphism in the length of GAr exists and the ability of GAr to allow immune evasion of EBNA1 is GAr-length-dependent: the longer GAr, the most efficient the immune evasion is. The GAr domain is flanked on each side by a glycine-arginine-rich (RGG) motif. RGG motifs (aka RGG boxes) are low complexity sequences that contain repetitions of the arginine-glycine-glycine tripeptide. RGG motifs can lead to intrinsically disordered regions [START_REF] Poornima | RGG-motif self-association regulates eIF4G-binding translation repressor protein Scd6[END_REF] and have been involved in interactions with nucleic acids and various proteins [START_REF] Thandapani | Defining the RGG/RG motif[END_REF]. The first RGG motif of EBNA1 is located from residue 33 to 64 whereas the second lies between residues 329 and 377 and both are responsible for EBNA1 association to host cell metaphase chromosomes, hence supporting EBV genome replication and maintenance [START_REF] Kang | Epstein-Barr virus latent genes[END_REF][START_REF] Mackey | The linking regions of EBNA1 are essential for its support of replication and transcription[END_REF][START_REF] Sears | The amino terminus of Epstein-Barr Virus (EBV) nuclear antigen 1 contains AT hooks that facilitate the replication and partitioning of latent EBV genomes by tethering them to cellular chromosomes[END_REF]. The second RGG motif of EBNA1 is followed by a monopartite nuclear localization signal NLS (KRPRSPSS, residues 379 to 386) and then by a C-terminal DNA-binding and dimerization domain (from residue 459 to 609), which is also responsible for the attachment of the viral genome to the cellular chromatin to ensure the viral replication [START_REF] Wilson | EBNA1: Oncogenic Activity, Immune Evasion and Biochemical Functions Provide Targets for Novel Therapeutic Strategies against Epstein-Barr Virus-Associated Cancers[END_REF][START_REF] Frappier | The Epstein-Barr Virus EBNA1 Protein[END_REF].

The function of EBNA1 in genome replication and maintenance also relies on its capacity to bind to multiple sites on the origin of DNA replication (OriP) of EBV genome. The EBV OriP contains two cisacting components, a dyad symmetry (DS) element, and a family of repeats (FR) [START_REF] Gahn | The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication[END_REF][START_REF] Reisman | A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components[END_REF]. The DS element is a repetition of four EBNA1 binding sites divided in two by a 9 bp sequence, it is responsible for DNA replication and it has also been shown to be sufficient for EBNA1-dependent EBV replication [START_REF] Frappier | The Epstein-Barr Virus EBNA1 Protein[END_REF][START_REF] Gahn | The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication[END_REF][START_REF] Wysokenski | Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus[END_REF]. The FR element consists of 20 tandem copies of a 30 bp sequence containing a 18 bp palindromic element on which EBNA1 binds, followed by 12 bps corresponding to an AT-rich element [START_REF] Rawlins | Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region[END_REF]. This FR element is not directly involved in DNA replication but, the mitotic segregation of the EBV episome requires binding of EBNA1 to its multiple recognition sites in the FR element for ensuring efficient attachment to host cell chromosomes [START_REF] Frappier | The Epstein-Barr Virus EBNA1 Protein[END_REF][START_REF] Hodin | Efficient replication of Epstein-Barr virus-derived plasmids requires tethering by EBNA1 to host chromosomes[END_REF].

In addition, thanks to a yeast-based two-hybrid screen, EBNA1 was shown to specifically interact also with a host cell nuclear protein, the so-called Epstein-Barr binding protein 2 (EBP2). This protein-protein interaction appears to be important for the role of EBNA1 in EBV episome stable segregation through association to host cell chromosomes in metaphase [START_REF] Frappier | The Epstein-Barr Virus EBNA1 Protein[END_REF][START_REF] Nayyar | Mitotic chromosome interactions of Epstein-Barr nuclear antigen 1 (EBNA1) and human EBNA1-binding protein 2 (EBP2)[END_REF][START_REF] Shire | EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance[END_REF]. EBNA1 region that binds to EBP2 has been mapped to the second RGG motif of EBNA1 (residues 329 to 377) whose deletion prevents interaction of EBNA1 with EBP2 and consequently affects EBV genome maintenance [START_REF] Shire | EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance[END_REF]. This EBNA1-EBP2 proteins interaction has been furthermore reconstituted in the budding yeast Saccharomyces cerevisiae. This way, EBP2 was demonstrated to be crucial for the maintenance of an EBV FR elementcontaining plasmid maintenance [START_REF] Kapoor | EBNA1 partitions Epstein-Barr virus plasmids in yeast cells by attaching to human EBNA1-binding protein 2 on mitotic chromosomes[END_REF][START_REF] Kapoor | Reconstitution of Epstein-Barr virus-based plasmid partitioning in budding yeast[END_REF][START_REF] Lista | The long-lasting love affair between the budding yeast Saccharomyces cerevisiae and the Epstein-Barr virus[END_REF]. EBNA1 is therefore a crucial protein for EBV DNA replication and episome maintenance, particularly in B cells which are actively dividing cells.

As stated above, EBNA1 is also implicated in transcriptional activation of the EBV latency genes in latent infection [START_REF] Gahn | An EBNA-1-dependent enhancer acts from a distance of 10 kilobase pairs to increase expression of the Epstein-Barr virus LMP gene[END_REF][START_REF] Sugden | A promoter of Epstein-Barr virus that can function during latent infection can be transactivated by EBNA-1, a viral protein required for viral DNA replication during latent infection[END_REF]. The 65-83 N-terminal EBNA1 residues [START_REF] Kennedy | EBNA-1, a bifunctional transcriptional activator[END_REF][START_REF] Wu | Separation of the DNA replication, segregation, and transcriptional activation functions of Epstein-Barr nuclear antigen 1[END_REF] as well as the second RGG motif (residues 329 to 377) [START_REF] Ceccarelli | Functional analyses of the EBNA1 origin DNA binding protein of Epstein-Barr virus[END_REF][START_REF] Van Scoy | Human p32: a coactivator for Epstein-Barr virus nuclear antigen-1-mediated transcriptional activation and possible role in viral latent cycle DNA replication[END_REF][START_REF] Wang | P32/TAP, a cellular protein that interacts with EBNA-1 of Epstein-Barr virus[END_REF] are required for transcriptional activation. EBNA1 requires both of these regions to activate transcription, as deletion of either one abrogates the transcriptional activation function of EBNA1 [START_REF] Wu | Separation of the DNA replication, segregation, and transcriptional activation functions of Epstein-Barr nuclear antigen 1[END_REF][START_REF] Ceccarelli | Functional analyses of the EBNA1 origin DNA binding protein of Epstein-Barr virus[END_REF]. Of note, a Δ61-83 EBNA1 mutant was found to be fully active for replication and segregation functions, indicating that transcriptional activation is a distinct EBNA1 function, separable from its GMP role [START_REF] Wu | Separation of the DNA replication, segregation, and transcriptional activation functions of Epstein-Barr nuclear antigen 1[END_REF].

EBNA1 is the Achilles heel of EBV

Like all the gamma herpesviruses, EBV is a latent virus that evades the host immune system. However, as said above, EBV possesses an Achilles heel: its virally-encoded genome maintenance protein EBNA1 (Epstein-Barr nuclear antigen 1). Indeed, being essential for replication and maintenance of the viral genome, EBNA1 is required for viral persistence but, at the same time, highly antigenic and T cells directed EBNA1 epitopes exist in nearly all EBV-infected individuals [START_REF] Blake | Human CD8+ T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)containing protein requires exogenous processing[END_REF][START_REF] Lee | CD8 T cell recognition of endogenously expressed epstein-barr virus nuclear antigen 1[END_REF][START_REF] Leen | Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses[END_REF][START_REF] Munz | Epstein-barr virus nuclear antigen 1: from immunologically invisible to a promising T cell target[END_REF][START_REF] Munz | Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1[END_REF][START_REF] Voo | Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes[END_REF]. Hence, EBV evolved a mechanism by which EBNA1 limits the presentation of EBNA1-derived antigenic peptides. This ability of EBNA1 to evade the immune system has been rapidly attributed to its GAr domain which prevents EBNA1-derived antigenic peptides to be presented to the MHC class I pathway [START_REF] Levitskaya | Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1[END_REF]. Importantly, this in cis effect of GAr is autonomous and transferable since, when fused to other proteins such as EBNA4, ovalbumin (OVA), or the herpes simplex virus thymidine-kinase, GAr also interfere with their antigenic presentation [START_REF] Levitskaya | Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1[END_REF][START_REF] Apcher | Epstein Barr virus-encoded EBNA1 interference with MHC class I antigen presentation reveals a close correlation between mRNA translation initiation and antigen presentation[END_REF][START_REF] Ossevoort | Characterization of an immuno 'stealth' derivative of the herpes simplex virus thymidine-kinase gene[END_REF][START_REF] Yin | Self-inhibition of synthesis and antigen presentation by Epstein-Barr virus-encoded EBNA1[END_REF]. Of note, the study by Maria Masucci and collaborators represents a milestone since, not only it demonstrated the crucial role of GAr in the immune evasion of EBNA1, but it also represented a proof of principle that interfering with GAr-based immune evasion of EBNA1 may represent an intervention point to unveil EBV-infected cells, in particular tumoral cells from EBVassociated cancers, to the immune system [START_REF] Munz | Epstein-barr virus nuclear antigen 1: from immunologically invisible to a promising T cell target[END_REF][START_REF] Levitskaya | Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1[END_REF].

A historical perspective on the role of GAr in EBNA1 immune evasion: the proteasome era followed by the ribosome era At the time the role of GAr in immune evasion of EBNA1, and thus of EBV, was discovered, the main dogma was that most antigenic peptides for the MHC class I pathway comes from 26S proteasomemediated degradation of full-length proteins (reviewed in [START_REF] Rock | Degradation of cell proteins and the generation of MHC class I-presented peptides[END_REF]). In this context, several teams have observed that the GAr domain is able to efficiently inhibit in cis protein degradation by the 26S proteasome. This has been shown by observing that the fusion of GAr to several short half-life proteins, that include p53 and Iκβα, interferes with their 26S proteasome-dependent degradation [START_REF] Daskalogianni | Gly-Ala repeats induce position-and substrate-specific regulation of 26 S proteasome-dependent partial processing[END_REF][START_REF] Levitskaya | Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1[END_REF][START_REF] Sharipo | A minimal glycine-alanine repeat prevents the interaction of ubiquitinated I kappaB alpha with the proteasome: a new mechanism for selective inhibition of proteolysis[END_REF] and this effect has been shown to be GAr-length-dependent [START_REF] Levitskaya | Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1[END_REF][START_REF] Dantuma | Inhibition of proteasomal degradation by the gly-Ala repeat of Epstein-Barr virus is influenced by the length of the repeat and the strength of the degradation signal[END_REF][START_REF] Heessen | Inhibition of ubiquitin/proteasome-dependent proteolysis in Saccharomyces cerevisiae by a Gly-Ala repeat[END_REF]. It was thus quite logical, on this ground, to infer that most of the capacity of GAr to inhibit antigen presentation was due to its ability to inhibit 26S-dependent proteolysis (Figure 2, left part). However, at the same time, some important problems associated to this model of antigenic presentation, in particular regarding the kinetics of antigenic presentation by the MHC class I pathway, have started to emerge [START_REF] Yewdell | DRiPs solidify: progress in understanding endogenous MHC class I antigen processing[END_REF][START_REF] Yewdell | Defective ribosomal products (DRiPs): a major source of antigenic peptides for MHC class I molecules?[END_REF][START_REF] Yewdell | The DRiP hypothesis decennial: support, controversy, refinement and extension[END_REF]. In line, as early as 1996, Jonathan Yewdell has proposed an alternative hypothesis in which the most important source of antigenic peptides would mainly originate from the so-called defective ribosomal products (DRiPs) [START_REF] Yewdell | Defective ribosomal products (DRiPs): a major source of antigenic peptides for MHC class I molecules?[END_REF][START_REF] Yewdell | The DRiP hypothesis decennial: support, controversy, refinement and extension[END_REF]. DRiPs correspond to abortive byproducts of the translation process that include defective, misfolded or prematurely terminated polypeptides. The first observation supporting this hypothesis is that most viral proteins are very stable, suggesting that the degradation of full-length proteins (the so-called "retirees") by the proteasome and the consecutive presentation of derived antigenic peptides by the MHC class I would take long time, from hours to days, which is not consistent with the experimental observations that the kinetic of presentation of antigenic peptides by the MHC class I pathway following viral infection is rather rapid (from a few minutes to a few hours) and may happen even before the synthesis of mature full-length proteins. In addition, there is a tight correlation between the onset of protein expression and epitope display for most antigens, which represents a strong indication that antigen presentation is largely linked to translation and not to degradation of mature full-length proteins (retirees) [START_REF] Yewdell | DRiPs solidify: progress in understanding endogenous MHC class I antigen processing[END_REF][START_REF] Croft | Kinetics of antigen expression and epitope presentation during virus infection[END_REF]. Indeed, from a number of T cell based-studies, it is clear that numerous antigenic peptides from multiple viruses are rapidly produced following infection [START_REF] Anton | Translating DRiPs: MHC class I immunosurveillance of pathogens and tumors[END_REF]. The tight kinetic link between viral gene translation and antigenic peptides generation has been extended to the proteomic level using mass spectrometry, for the vaccinia virus [START_REF] Croft | Kinetics of antigen expression and epitope presentation during virus infection[END_REF] and recently for the influenza A virus (IAV) [START_REF] Wu | Quantification of epitope abundance reveals the effect of direct and cross-presentation on influenza CTL responses[END_REF]. Hence, the idea that most viral antigenic peptides come from the degradation of full-length viral proteins did not seem anymore a viable hypothesis. Finally, the ribosome hypothesis was further supported by data showing that the inhibition of de novo protein synthesis rapidly reduces the antigenic presentation by MHC class I molecules [START_REF] Dolan | Distinct pathways generate peptides from defective ribosomal products for CD8+ T cell immunosurveillance[END_REF][START_REF] Khan | Cutting edge: neosynthesis is required for the presentation of a T cell epitope from a longlived viral protein[END_REF]. All these findings suggest that antigenic peptides have a main source from translation itself (Figure 2, right part), in particular from DRiPs [START_REF] Yewdell | DRiPs solidify: progress in understanding endogenous MHC class I antigen processing[END_REF][START_REF] Yewdell | DRiPs get molecular[END_REF]. Hence, the model in which the ability of the GAr domain of EBNA1 to enable its immune evasion, and therefore immune evasion of EBV, comes from its capacity to inhibit own degradation by the 26S proteasome started to be fragilized.

In addition, several studies have found that EBNA1 protein, with or without GAr, is a very stable protein [START_REF] Yin | Self-inhibition of synthesis and antigen presentation by Epstein-Barr virus-encoded EBNA1[END_REF][START_REF] Daskalogianni | Gly-Ala repeats induce position-and substrate-specific regulation of 26 S proteasome-dependent partial processing[END_REF][START_REF] Tellam | Endogenous presentation of CD8+ T cell epitopes from Epstein-Barr virus-encoded nuclear antigen 1[END_REF] like most viral proteins, although its stability may be tissue-dependent [START_REF] Tellam | Endogenous presentation of CD8+ T cell epitopes from Epstein-Barr virus-encoded nuclear antigen 1[END_REF]. Also, the steadystate level of EBNA1 protein was strongly increased in cells expressing a version of EBNA1 deleted for its GAr domain (EBNA1ΔGAr) as compared to cells expressing the wild-type EBNA1 (EBNA1wt), which was not expected when removing a domain that would inhibit proteasomal degradation [START_REF] Yin | Self-inhibition of synthesis and antigen presentation by Epstein-Barr virus-encoded EBNA1[END_REF][START_REF] Levitskaya | Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1[END_REF]. Finally, when expressing in the budding yeast Saccharomyces cerevisiae GAr domains of various lengths fused to destabilized versions of the green fluorescent protein (GFP), harboring N-end rule or ubiquitin fusion degradation signals, the length-dependent effect of the GAr domain on protein stability was confirmed.

However, the steady-state level of the various GAr-GFP fusions was inversely correlated to their degree of stabilization: the longer the GAr domain, the more the reporter protein was stable, but the smaller its steady-state level, which appears quite counterintuitive [START_REF] Heessen | Inhibition of ubiquitin/proteasome-dependent proteolysis in Saccharomyces cerevisiae by a Gly-Ala repeat[END_REF]. Altogether, all these indirect data suggested that the ability of GAr to promote immune evasion was not linked to its ability to inhibit the 26S proteasome-mediated proteolysis and, even if GAr is able to inhibit proteasomal degradation of unstable proteins to which it is fused, this effect is not operant in EBNA1 which is a stable protein.

The ability of GAr to interfere with EBNA1 immune evasion is linked to its capacity to interfere in cis with the translation of its own mRNA

In a seminal study published in 2003, the use of in vitro expression systems and cell-based metabolic labeling led to the demonstration that the increase in EBNA1ΔGAr, as compared to EBNA1wt, was due to an increase in the rate of EBNA1 synthesis [START_REF] Yin | Self-inhibition of synthesis and antigen presentation by Epstein-Barr virus-encoded EBNA1[END_REF]. Not only this striking result explained all the previously described results that did not fit with an effect of GAr on proteasomal degradation, but, more importantly, it was the starting point for other studies that unambiguously demonstrated that GAr does in fact inhibit in cis the translation of its own mRNA (EBNA1 mRNA) or of any messenger RNA (mRNA) to which it is fused, without affecting in trans the translation of other mRNA [START_REF] Apcher | Epstein Barr virus-encoded EBNA1 interference with MHC class I antigen presentation reveals a close correlation between mRNA translation initiation and antigen presentation[END_REF][START_REF] Apcher | mRNA translation regulation by the Gly-Ala repeat of Epstein-Barr virus nuclear antigen 1[END_REF]. This important demonstration was fully in line with the growing role of translation and of DRIPs in the genesis of antigenic peptides for the MHC class I pathway and readily explained the effect of GAr on immune evasion of EBNA1, and therefore of EBV [START_REF] Fahraeus | Do peptides control their own birth and death?[END_REF]. These important results have then been confirmed [START_REF] Tellam | Endogenous presentation of CD8+ T cell epitopes from Epstein-Barr virus-encoded nuclear antigen 1[END_REF] and converged to the model in which the endogenous presentation of EBNA1-derived antigenic peptides is dependent on the newly synthesized EBNA1 protein rather than on the long-lived stable EBNA1 protein, hence highlighting the inhibitory role of GAr on translation as the main mechanism explaining its inhibitory effect on antigen presentation (Figure 2b, right part). Importantly, the GAr-encoding sequence does not represent per se a hurdle to mRNA translation by the ribosome since the insertion of the c-Myc internal ribosome entry site (IRES) in the 5' untranslated transcribed region (UTR) of EBNA1 or GAr-OVA fusion-coding mRNA abolishes GAr-based inhibition of both translation and antigen presentation [START_REF] Apcher | Epstein Barr virus-encoded EBNA1 interference with MHC class I antigen presentation reveals a close correlation between mRNA translation initiation and antigen presentation[END_REF][START_REF] Apcher | mRNA translation regulation by the Gly-Ala repeat of Epstein-Barr virus nuclear antigen 1[END_REF]. This result also indicated that the GAr-inhibitory effect on translation requires cap-dependent translation initiation factors.

Tools to monitor the GAr inhibitory effect on translation: setup of a yeast-based assay amenable to high throughput drug and genetic screenings

Once the GAr inhibitory effect on translation firmly established, it rapidly appeared as a relevant intervention point to unveil EBV-infected cells, in particular tumor cells of cancers linked to EBV, to the immune system, hence the need for robust cell-based systems allowing both drug and genetic screenings. For various reasons, the budding yeast Saccharomyces cerevisiae has emerged as a relevant model system: notably, the study by the Masucci group already discussed above and based on the expression in yeast of destabilized GFP fused to GAr domains of various lengths indirectly suggested that the length-dependent inhibitory effect of GAr on translation is operant in yeast [START_REF] Heessen | Inhibition of ubiquitin/proteasome-dependent proteolysis in Saccharomyces cerevisiae by a Gly-Ala repeat[END_REF]. In addition, the budding yeast S. cerevisiae is an eukaryote as easy to handle as a prokaryote and is most certainly the model system for which the most molecular tools are available. Finally, for decades, yeast has been harnessed by biomedical research, notably on the Epstein-Barr virus itself (reviewed in [START_REF] Lista | The long-lasting love affair between the budding yeast Saccharomyces cerevisiae and the Epstein-Barr virus[END_REF]) and on virus immune evasion [START_REF] Angrand | Sneaking Out for Happy Hour: Yeast-Based Approaches to Explore and Modulate Immune Response and Immune Evasion[END_REF], and widely used for modeling various pathophysiological mechanisms responsible for a large variety of human disorders [START_REF] Bach | Budding yeast, a model and a tool... also for biomedical research[END_REF][START_REF] Kachroo | Humanized yeast to model human biology, disease and evolution[END_REF]. A number of these yeast-based models have been successfully used for performing large-scale pharmacological screening that aimed at identifying candidate drugs or chemical probes (for examples see [START_REF] Bach | Isolation of drugs active against mammalian prions using a yeast-based screening assay[END_REF] or [START_REF] Couplan | A yeast-based assay identifies drugs active against human mitochondrial disorders[END_REF]) as well as genetic screenings that focused on deciphering novel key factors involved in the considered diseases (for examples see [START_REF] Giorgini | A genomic screen in yeast implicates kynurenine 3-monooxygenase as a therapeutic target for Huntington disease[END_REF] or [START_REF] Lista | Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to Gquadruplexes of EBNA1 mRNA[END_REF]). Therefore, a yeast model for monitoring the GAr-based inhibition of the translation of its own mRNA in cis has been set up [START_REF] Voisset | A yeast-based assay identifies drugs that interfere with immune evasion of the Epstein-Barr virus[END_REF]. In this model (described in Figure 3a), the yeast protein Ade2 belonging to adenine biosynthesis pathway was used as a reporter protein since: (i) like EBNA1 it is a stable protein and (ii) a convenient colorimetric system allows to easily (without any treatment) determine its steady-state level in living yeast cells. Indeed, whereas yeast cells that express Ade2 at a functional level form white colonies, yeast cells that do not express Ade2 (for example cells deleted for the ADE2 gene -termed ade2Δ-) readily form red colonies on rich YPD medium and any intermediate level of Ade2 leads to the formation of pink colonies whose color intensity is inversely proportional to the level of Ade2 expressed. Hence, this simple reporter system allows to easily monitor subtle changes in the level of Ade2. For this reason, and based on the fact that GAr-based inhibition of translation is probably operant in yeast, fusions of GAr domains of various lengths [START_REF] Munz | Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1[END_REF][START_REF] Wu | Separation of the DNA replication, segregation, and transcriptional activation functions of Epstein-Barr nuclear antigen 1[END_REF] or 235 amino acids) to Ade2 were tested in yeast and led to the formal demonstration that GAr inhibits in cis and in a length-dependent manner the translation of its own mRNA in yeast, without any significant effect on mRNA level nor on protein stability (which was expected given that Ade2 is a very stable protein). Hence, the 43GAr-Ade2 fusion leads to pink yeasts, thereby allowing to easily monitor the effect of GAr and serving as the screening basis for identifying compounds or genes able to interfere with its in cis inhibitory effect on translation: modifiers that counteract the GAr effect on translation would lead to whiter yeast colonies, whereas those exacerbating the GAr effect would lead to redder yeast colonies. This yeast-based assay has first been used for drug screening (Figure 3b, left part) and led to the identification of doxorubicin as a compound specifically counteracting the inhibitory effect of GAr on the expression of GAr-Ade2. This effect was GAr-dependent since doxorubicin had no effect on the expression of Ade2. The yeast assay was then definitively validated when doxorubicin was not only shown to stimulate EBNA1 expression in a GAr-dependent manner in human cells, but also to overcome the GAr-dependent restriction of MHC class I antigen presentation [START_REF] Voisset | A yeast-based assay identifies drugs that interfere with immune evasion of the Epstein-Barr virus[END_REF]. These results also suggested that, should host cell factors involved in GAr-based translation inhibition exist, they would be functionally conserved from yeast to human. This conclusion was the ground for the use of the yeast-based assay to perform a genetic screening (Figure 3b, right part) that aimed at identifying those potential host factors. The same readout (changes in the pink color of the 43GAr-Ade2 yeast strain) was used and the effect of overexpressing every single yeast gene was thus tested and led to the identification of the yeast NSR1 gene [START_REF] Lista | Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to Gquadruplexes of EBNA1 mRNA[END_REF][START_REF] Lista | A yeast model for the mechanism of the Epstein-Barr virus immune evasion identifies a new therapeutic target to interfere with the virus stealthiness[END_REF] which encodes the Nsr1 protein, the yeast ortholog of the human nucleolin (NCL).

When overexpressed in yeast, Nsr1 or NCL led to a darker color of the 43GAr-Ade2 yeast strain due to the exacerbation of the GAr-based inhibition of translation, whereas the 43GAr-Ade2 yeast strain deleted for the NSR1 gene form white colonies that express a higher level of the 43GAr-Ade2 fusion protein. Similar results were obtained when overexpressing or downregulating human NCL in human cells expressing EBNA1 of GAr-OVA fusions [START_REF] Lista | Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to Gquadruplexes of EBNA1 mRNA[END_REF]. Altogether these results have highlighted nucleolin (NCL) as the first identified host cell factor required for GAr-based inhibition of translation, the mechanism at the basis of EBNA1 immune evasion. The question that came next was the precise role of NCL in GAr-based inhibition of translation.

Crucial role of RNA G-quadruplex (rG4) and of their interaction with NCL in GAr-based inhibition of translation and of antigen presentation

G-quadruplexes (G4) are non-canonical secondary structures that may assemble in guanine-rich DNA or RNA. G4 are formed by the stacking of at least two G-quartets which consist of a planar arrangement of four guanines connected by Hoogsteen hydrogen bonds and stabilized by a central cation, most often K + . G4 have been involved in gene regulation where they can affect transcription, splicing or translation [START_REF] Beaudoin | 5'-UTR G-quadruplex structures acting as translational repressors[END_REF][START_REF] Didiot | The G-quartet containing FMRP binding site in FMR1 mRNA is a potent exonic splicing enhancer[END_REF][START_REF] Gomez | Telomerase downregulation induced by the G-quadruplex ligand 12459 in A549 cells is mediated by hTERT RNA alternative splicing[END_REF][START_REF] Marcel | G-quadruplex structures in TP53 intron 3: role in alternative splicing and in production of p53 mRNA isoforms[END_REF][START_REF] Siddiqui-Jain | Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription[END_REF]. Importantly, the central regions of most gamma herpesviruses genome maintenance proteins are highly homologous in terms of nucleotide sequence and are purine enriched [START_REF] Cristillo | Double-stranded RNA as a not-self alarm signal: to evade, most viruses purine-load their RNAs, but some (HTLV-1, Epstein-Barr) pyrimidine-load[END_REF]. At about the time NCL was identified as the first host factor involved in GAr effect on translation, the ability of the GAr-encoding sequence of EBNA1 mRNA to form a cluster of up to thirteen RNA G-quadruplex (rG4) was demonstrated and the possibility that these rG4 could impede ribosome progression has been proposed [START_REF] Murat | G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation[END_REF]. In line, in the same study, it was shown that destabilization of these rG4 by antisense oligonucleotides enhances both EBNA1 translation and presentation of EBNA1-derived antigenic peptides. However, as stated above, the fact that driving the translation initiation of a GAr-OVA mRNA by the c-Myc internal ribosome entry site (IRES) abolishes GAr-based inhibition of both translation and antigen presentation suggests that the GAr-encoding sequence does not represent per se a hurdle to the ribosome progression [START_REF] Apcher | Epstein Barr virus-encoded EBNA1 interference with MHC class I antigen presentation reveals a close correlation between mRNA translation initiation and antigen presentation[END_REF][START_REF] Apcher | mRNA translation regulation by the Gly-Ala repeat of Epstein-Barr virus nuclear antigen 1[END_REF]. In addition, at a low concentration (less than 1 µM), EBNA1 mRNA was translated in vitro as efficiently as the EBNA1∆GAr mRNA [START_REF] Daskalogianni | Epstein-Barr virus-encoded EBNA1 and ZEBRA: targets for therapeutic strategies against EBV-carrying cancers[END_REF]. In line, GAr-based inhibition of translation is not operant in a yeast nsr1Δ strain deleted for nucleolin [START_REF] Lista | Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to Gquadruplexes of EBNA1 mRNA[END_REF][START_REF] Lista | A yeast model for the mechanism of the Epstein-Barr virus immune evasion identifies a new therapeutic target to interfere with the virus stealthiness[END_REF] also suggesting that the series of rG4 that may assemble in GAr-encoding RNA are not per se able to significantly impede ribosome progression. Importantly, human nucleolin (NCL) has long been known to interact with Gquadruplex [START_REF] Dempsey | G4 DNA binding by LR1 and its subunits, nucleolin and hnRNP D, A role for G-G pairing in immunoglobulin switch recombination[END_REF][START_REF] Hanakahi | High affinity interactions of nucleolin with G-G-paired rDNA[END_REF], hence opening the possibility that NCL interacts with rG4 of the EBNA1 mRNA and that this interaction is crucial for GAr-based inhibition of EBNA1 translation and antigenic peptides presentation. This potential NCL/rG4 of EBNA1 mRNA interaction has been evidenced using both RNA pulldown [START_REF] Lista | Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to Gquadruplexes of EBNA1 mRNA[END_REF][START_REF] Von Hacht | Identification and characterization of RNA guanine-quadruplex binding proteins[END_REF] and an adaptation of the proximity ligation assay (PLA) for monitoring RNA/protein interactions [START_REF] Lista | Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to Gquadruplexes of EBNA1 mRNA[END_REF][START_REF] Prado Martins | In Cellulo Protein-mRNA Interaction Assay to Determine the Action of G-Quadruplex-Binding Molecules[END_REF]. Following these various observations, the benchmark G4-ligand PhenDC3 was tested and shown to interfere with NCL binding to rG4 of EBNA1 mRNA, thereby relieving GAr-based inhibition of both translation and antigenic peptide presentation by the MHC class I [START_REF] Lista | Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to Gquadruplexes of EBNA1 mRNA[END_REF]. This result has motivated the screening of a series of 20 cationic bis(acylhydrazone) derivatives and led to the identification of two original G4-ligands, PyDH2 and PhenDH2, more active and less cytotoxic than PhenDC3 and interfering with EBV immune evasion also by disrupting the interaction between NCL and rG4 of EBNA1 mRNA [START_REF] Reznichenko | Novel cationic bis(acylhydrazones) as modulators of Epstein-Barr virus immune evasion acting through disruption of interaction between nucleolin and G-quadruplexes of EBNA1 mRNA[END_REF]. Of note, doxorubicin, the compound isolated in the yeast assay that models GAr-based inhibition of translation described above [START_REF] Voisset | A yeast-based assay identifies drugs that interfere with immune evasion of the Epstein-Barr virus[END_REF] was recently shown to be a G4-ligand [START_REF] Manet | Affinity of the anthracycline antitumor drugs Doxorubicin and Sabarubicin for human telomeric G-quadruplex structures[END_REF][START_REF] Scaglioni | Nemorubicin and doxorubicin bind the G-quadruplex sequences of the human telomeres and of the c-MYC promoter element Pu22[END_REF], thus giving a plausible explanation for its ability to interfere with EBNA1 immune evasion. Also, it has been recently observed that insertion of the c-Myc IRES to the 5' UTR of GAr-OVA RNA prevents the interaction between NCL and rG4 of the GAr-encoding RNA, thus readily explaining its ability to abolish GAr-based inhibition of both translation and antigen presentation [START_REF] Martins | Nuclear processing of nascent transcripts determines synthesis of full-length proteins and antigenic peptides[END_REF]. In addition, this result further confirms the importance of the NCL/rG4 of EBNA1 mRNA interaction for EBNA1 immune evasion.

Finally, the mRNA encoding LANA1, the genome maintenance protein of the Kaposi virus, another oncogenic herpesvirus that also evades the immune system, has been proposed to also form rG4 in a central region presenting sequence similarity with the one encoding GAr, readily suggesting rG4 could represent a common feature that may allow immune evasion of various stealthy oncoviruses [START_REF] Murat | G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation[END_REF][START_REF] Murat | Effects of messenger RNA structure and other translational control mechanisms on major histocompatibility complex-I mediated antigen presentation[END_REF].

The existence and the crucial role of the rG4 of LANA1 mRNA in immune evasion of the Kaposi virus have recently been shown [START_REF] Dabral | LANA and hnRNP A1 Regulate the Translation of LANA mRNA through G-Quadruplexes[END_REF][START_REF] Zheng | The different activities of RNA G-quadruplex structures are controlled by flanking sequences[END_REF].

The C-terminal RGG motif of NCL is crucial for its role in GAr-based inhibition of both translation and antigen presentation

The importance of the various domains of NCL protein for GAr-based inhibition of translation has been tested. NCL is composed of three domains: (i) an N-terminal domain that contains acidic stretches; (ii) a central region that contains four tandem RNA recognition motifs (RRM1 to RRM4); and (iii) a Cterminal RGG motif. Most nucleolin homologues known to date share the same organization, with some variation in the number of RRMs. Hence, the yeast nucleolin Nsr1 contains only two RRMs but possesses the acidic N-terminal domain as well as the C-terminal RGG motif. Importantly, and in contrast to NCL in human, Nsr1 is not essential in yeast as nsr1Δ strains, although growing rather poorly, are viable [START_REF] Lee | The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs[END_REF]. In addition, human nucleolin NCL can complement the effect of the loss of yeast Nsr1 on GAr-based inhibition of translation in yeast [START_REF] Lista | Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to Gquadruplexes of EBNA1 mRNA[END_REF][START_REF] Lista | A yeast model for the mechanism of the Epstein-Barr virus immune evasion identifies a new therapeutic target to interfere with the virus stealthiness[END_REF]. For these reasons, an approach combining the use of the yeast model for GAr-based inhibition of translation in which the NSR1 gene has been deleted (43GAr-ADE2 nsr1Δ strain) and the expression of either Nsr1 or NCL, or of parts of them, has been exploited to determine which domain of nucleolin is involved in this process and has identified the Cterminal RGG motif of both yeast and human nucleolin. Then, this RGG motif has been shown to interact with the rG4 of EBNA1 mRNA, thus being essential for EBNA1 ability to self-limit its expression [START_REF] Angrand | Type I arginine methyltransferases are intervention points to unveil the oncogenic Epstein-Barr virus to the immune system[END_REF].

One or two methyl groups can be attached to the nitrogen atoms of arginine, a process termed arginine methylation. This post-translational modification is conducted by protein arginine methyltransferases (PRMTs) [START_REF] Wu | Protein arginine methylation: from enigmatic functions to therapeutic targeting[END_REF]. These enzymes catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to the guanidino nitrogen atoms of arginine. There are nine PRMTs in human that are divided into three groups: type I, type II and type III which differ in the pattern of arginine methylation catalyzed [START_REF] Blanc | Arginine Methylation: The Coming of Age[END_REF]. Arginine methylation occurs in a variety of protein motifs, but the RGG motifs are the most commonly reported [100, 101] and, in budding yeast, it is estimated that the majority of arginine methylation occurs in RGG motifs [102]. For these reasons, the potential role of PRMTs in GArbased inhibition of translation and antigen presentation has been tested. Hence, using drug-based or siRNA-mediated inhibition of PRMTs, it has been shown that type I PRMTs are required for the crucial role of nucleolin in GAr-based inhibition of translation and antigen presentation [START_REF] Angrand | Type I arginine methyltransferases are intervention points to unveil the oncogenic Epstein-Barr virus to the immune system[END_REF]. Next, the role of methylation of the arginines of the RGG motif of both yeast and human nucleolins has been directly evaluated by changing the eight arginines of the RGG motif of Nsr1, or the ten arginines of the RGG motif of NCL, by either alanines (to prevent methylation), lysines (to prevent methylation while maintaining the positive charge), or phenylalanines as mimics of methylated arginines. Indeed, phenylalanine has been shown to mimic methylated arginine as it carries a bulky hydrophobic moiety [103,104]. Changing arginines into alanine or lysine suppress the interaction of NCL and Nsr1 with rG4 of EBNA1 mRNA, and thereby GAr-based inhibition of translation, whereas replacement of arginines by phenylalanines maintains both these phenomena [START_REF] Angrand | Type I arginine methyltransferases are intervention points to unveil the oncogenic Epstein-Barr virus to the immune system[END_REF]. Altogether, these results indicate that methylation of the arginines of the C-terminal RGG motif of nucleolin is necessary for its interaction with rG4 of the GAr-encoding sequence of EBNA1 mRNA, an interaction which is required for GAr-based inhibition of translation and, as a consequence, for EBNA1 immune evasion. Following these observations, two possible models for the role of methylation of the arginines of the C-terminal RGG motif of nucleolin could be envisioned: either this methylation directly promotes the interaction of NCL with rG4 of EBNA1 mRNA, or this methylation may interfere with the ability of the RGG motif of nucleolin to interact, and thus to be sequestered, by some of its partners (that can be proteins, DNA or RNA). In favor of this second possibility is the recent observation that Scd6, a yeast protein involved in translation inhibition, is able to self-associate via its RGG motif and that this self-association prevents its ability to inhibit translation and is negatively regulated by arginine methylation catalyzed by Hmt1, the main yeast type I PRMT [START_REF] Poornima | RGG-motif self-association regulates eIF4G-binding translation repressor protein Scd6[END_REF]. In vitro experiments based on arginine methylation of bacterially produced recombinant NCL, or on either methylated or non-methylated RGG peptides have shown that methylation of the arginines of the C-terminal RGG motif of NCL does not significantly change its ability to interact with rG4 of EBNA1 mRNA [START_REF] Angrand | Type I arginine methyltransferases are intervention points to unveil the oncogenic Epstein-Barr virus to the immune system[END_REF]. Hence, it seems that the RGG motif of NCL may be trapped by an unknown partner, thereby preventing its interaction with rG4 of EBNA1 mRNA and, upon type I PRMTs-mediated arginine methylation, the RGG motif of NCL is released and thus free to interact with rG4 of EBNA1 mRNA, hence leading to immune evasion of EBNA1 (Figure 4, left part). Of note, the unknown partner of the RGG motif of NCL could be L3, a ribosomal protein that has been shown to interact with the RGG motif of NCL [105].

Finally, all these results bring out an intriguing point regarding the role of NCL in GAr-based selflimitation of EBNA1 expression and antigenic presentation. Indeed, NCL has also been positively involved in EBV episome maintenance and transcription through a direct interaction with the EBNA1 protein [106]. Hence, NCL appears to positively control both EBV episome maintenance and transcription on the one hand, and the self-limitation of the EBV GMP (EBNA1) expression on the other.

As for EBV, one can consider it makes sense to have the same host cell protein regulating these two key aspects of EBV's latency. Indeed, if NCL level is low, then the maintenance and transcription of EBV episome should be compromised but, as a result of NCL role in GAr-based limitation of EBNA1 expression, EBNA1 mRNA should be more efficiently translated, which may compensate for its reduced level and favour the maintenance of EBV genome. On the contrary, if NCL level is high, then EBV episome will be efficiently maintained and transcribed, hence leading to a high level of EBNA1 mRNA, but then the increased NCL could further downregulate its translation, thereby limiting the level of EBNA1 protein and therefore its detection by the immune system. Importantly, as stated above, the role of NCL in EBNA1 immune evasion involves its ability to interact with G4 structures present in EBNA1 mRNA, whereas its role in episome maintenance and transcription involves its ability to interact with EBNA1's N-terminal 100 amino acids (hence upstream of the GAr domain of EBNA1 protein) [106].

Therefore, targeting the NCL-EBNA1 mRNA interaction should specifically affect EBNA1 immune evasion.

Therapeutic perspectives: potential intervention points and therapeutic avenues

Based on the current model of GAr-based inhibition of both EBNA1 expression and antigen presentation, two intervention points may be envisioned to unveil EBV-infected cells, in particular tumor cells of EBVrelated cancers, to the immune system: (i) the interaction between rG4 of EBNA1 mRNA and the Cterminal RGG motif of NCL, and (ii) type I PRMTs (Figure 5). Importantly, in both cases proof-ofprinciples exist as G4 ligands that interfere with the rG4/NCL interaction on the one hand, and drugs targeting type I PRMTs on the other, interfere with the GAr-based inhibition of translation and antigen presentation [START_REF] Lista | Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to Gquadruplexes of EBNA1 mRNA[END_REF][START_REF] Reznichenko | Novel cationic bis(acylhydrazones) as modulators of Epstein-Barr virus immune evasion acting through disruption of interaction between nucleolin and G-quadruplexes of EBNA1 mRNA[END_REF][START_REF] Angrand | Type I arginine methyltransferases are intervention points to unveil the oncogenic Epstein-Barr virus to the immune system[END_REF]. Of note, for both these therapeutic avenues, selectivity may be an issue (as for most of the drugs). Indeed, type I PRMTs may appear as relatively non-specific therapeutic targets as their inhibition could, in principle, affect the methylation of many proteins, similar to what has been initially considered for drugs inhibiting protein kinases or phosphatases. Of note, this potential lack of specificity has not prevented the development to the clinics of a number of kinases inhibitors [107], in particular in the field of cancer, and several inhibitors of PRMTs are currently being evaluated in clinical trials. Also, since RGG motifs are the main substrates of PRMTs and as, in various cell-based assays, type I inhibitors did not exhibit significant toxicity at concentration ranges in which they significantly affect both GAr-based translation inhibition and the interaction between NCL and rG4 of EBNA1 mRNA, the lack of specificity may not be an issue that could preclude their potential further developments to the clinics [START_REF] Angrand | Type I arginine methyltransferases are intervention points to unveil the oncogenic Epstein-Barr virus to the immune system[END_REF]. As for G4 ligands, an inherent limitation in their development to the clinics may also be their potential lack of specificity given that, in addition to the numerous rG4, there are hundreds of thousands potential DNA G4 (dG4) that may form in the human genome. Importantly however, G4 in the RNA context are often in a dynamic equilibrium with other structural isoforms, and even low doses of G4 ligands may have a profound impact on this equilibrium, preventing or leading to the recruitment of the corresponding structure-specific binding factors (nucleolin in the case of the rG4 of EBNA1 mRNA).

Another important point is that, besides the G-quartets, which are the constant elements constitutive of G4, there are other structural characteristics which vary from one sequence to another: the loops and the flanking regions. These elements ensure that each G4 is, in principle, unique within the whole genome or transcriptome and, as such, could be exploited as key targets to gain specificity through, for instance, the coupling of complementary oligonucleotides to G4-ligands [108]. Hence, it is not excluded that some specificity toward particular G4, notably rG4, may be reached and various methods to assess G4 ligands specificity have been described [109,110]. Finally, the combined use of G4-ligands and inhibitors of type I PRMTs may also help to solve the specificity issue by potentially allowing the use of much reduced doses of both types of drugs.

The citrullination of arginines of the RGG motif of NCL may also be involved in the GAr-based mechanism of inhibition of both EBNA1 translation and antigen presentation. Indeed, deamination, also called citrullination, is the process of conversion of arginine into citrulline through enzymatic activation of peptidyl arginine deiminases (PADIs), notably PADI4 (aka PAD4), the main peptidyl arginine deiminase in human, thus preventing their methylation by PRMTs [111,112]. Citrullination of RGG motifs was observed in several RGG-containing proteins, including NCL [113]. Interestingly, it has been reported that EBV infection activates several PRMTs, especially PRMT1, the main type I PRMTS, and at the same time inactivates PADI4 [114]. Hence, a possibility exists that EBV infection, by activating PRMT1 as well as inactivating PADI4, activates arginine methylation of the NCL C-terminal RGG motif, thus promoting the interaction of this protein with the G4s of EBNA1 mRNA, leading to limitation of its translation, and therefore to the evasion of EBNA1/EBV from the immune system (Figure 4).

Importantly, in addition to the GAr-encoding mRNA sequence, the GAr peptide could also participate to the inhibition of its own expression. For example, the addition of antibodies directed against the GAr domain can enhance the translation of GAr-carrying mRNAs, which supports the idea that the nascent GAr polypeptides could participate to the translation inhibition of its own mRNA [START_REF] Apcher | Epstein Barr virus-encoded EBNA1 interference with MHC class I antigen presentation reveals a close correlation between mRNA translation initiation and antigen presentation[END_REF][START_REF] Apcher | mRNA translation regulation by the Gly-Ala repeat of Epstein-Barr virus nuclear antigen 1[END_REF]. In addition, the nascent GAr peptides have recently been shown to interact with the nascent polypeptide-associated complex alpha (NACA) and displace it from the ribosome [115]. Endogenous NACA, in addition to bind to the GAr peptides, also interacts with GAr mRNA sequence as well as with NCL in the cell nucleus, thus facilitating NCL-EBNA1 mRNA interaction, thereby favoring mRNA translation inhibition in cis.

Importantly, siRNA-mediated silencing of NACA expression interferes with the interaction between NCL and the rG4 of GAr mRNA and thus on GAr-based inhibition of both translation and antigenic peptides production [115]. Also, as stated above, the EBNA1 protein itself contains two RGG motifs: one upstream and one downstream of the GAr domain, and it has been shown that EBNA1 could bind to its own mRNA through its two RGG motifs [116] and also that these two RGG motifs could bind to rG4 [117]. Moreover, these RGG domains are encoded by G-rich sequences in the EBNA1 mRNA that also have potential to form G-quadruplexes. These observations open two non-mutually exclusive possibilities that would implicate the RGG domains of EBNA1 in the mechanism of regulation of its expression. Firstly, like NCL, the EBNA1 protein could bind to the rG4 of its own mRNA through its two RGG motifs, and thus participate in the inhibition of its translation. The other possibility would be that, like the GAr-encoding sequence, the EBNA1 mRNA sequences encoding the two RGG motifs of EBNA1 would be capable of forming rG4 that may also recruit NCL and/or EBNA1 protein(s) and would therefore participate to the inhibition of EBNA1 translation as well as the production of EBNA1-derived antigenic peptides, leading thus to the immune evasion of EBNA1/EBV. Of note, in both cases, type I PRMTs and PADIs may play a regulatory role. Interestingly, NCL and EBNA1 proteins have been reported to interact

[106], hence opening the possibility of a cooperative binding on rG4 of EBNA1 mRNA, and thereby of a cooperative inhibitory effect of these two proteins on EBNA1 mRNA translation. All these possibilities clearly deserve detailed investigations.

Importantly, in addition to its crucial role in EBV genome replication and maintenance and in the virus immune evasion, EBNA1 has also been linked to cell transformation and, if the mechanism underlying its oncogenic activity is still unclear [START_REF] Wilson | EBNA1: Oncogenic Activity, Immune Evasion and Biochemical Functions Provide Targets for Novel Therapeutic Strategies against Epstein-Barr Virus-Associated Cancers[END_REF], it may involve the c-Myc oncogene. Indeed, the EµEBNA1 mice that express EBNA1 in their B-cells thanks to the immunoglobulin heavy chain intronic enhancer (Eµ) develop and succumb to B-cell lymphoma [118]. Importantly, the B cell tumours of these mice are monoclonal and remarkably similar to those induced by transgenic c-Myc expression (Eµmyc transgenic mice, [119,120]). However, contrary to the characteristic translocations observed in the c-MYC locus which are linked to the deregulation of its expression, in B cells tumours arising from EµEBNA1 transgenic mice the murine c-MYC locus was intact with no 5' rearrangements detected whereas the expression of c-Myc is also deregulated [118]. Hence, EBNA1 is able to induce overexpression of the c-MYC oncogene and, as such, may participate to the oncogenicity of EBV. Interestingly, recently a direct link between EBNA1 and C-Myc expression was revealed with the identification of a unique mechanism involving the GAr domain of EBNA1 [121]. In this model, the in cis GAr-mediated mRNA translation suppression causes a translational stress that activates the E2F1 transcription factor expression via phosphoinositide-3-kinase-δ-(PI3Kδ-) dependent signalling. This increase in the level of E2F1 causes the induction of c-Myc. In support of this model are the observations that drug-based or siRNa-mediated specific inhibition of PI3Kδ abrogate EBNA1-dependent induction of both E2F1 and c-Myc. Hence, in addition to be the GMP of EBV and to play a central role in the ability of the virus to evade the immune system, EBNA1 participate to the oncogenicity of EBV. Interestingly, both oncogenicity and immune evasion of EBV depend on the GAr domain of EBNA1. For this reason, pharmacological means that would result in exacerbating the GAr inhibitory effect on translation, by further decreasing EBNA1 level, may also be of therapeutic value for EBV-related cancers.

Concluding remark

Beyond the clear interest of all the studies on EBNA1 biological roles and immune evasion for defining intervention points and novel therapeutic avenues to treat EBV-related diseases, in particular EBVrelated malignancies, they also represent another illustration that "viruses are the best cell biologists" in that their study quite often leads to seminal discovery in cell biology.

EBNA1 immune evasion

On the left (Proteasome era) is represented the former model in which processing of the full-length EBNA1 protein by the 26S proteasome would be the main source of EBNA1-derived antigenic peptide.

On this ground, the ability of GAr to inhibit in cis 26S proteasome-mediated proteolysis gave a plausible explanation for its role in immune evasion of EBNA1.

On the right (Ribosome era) is represented the current model in which most of antigenic peptides would originate from byproducts of the translation (the so-called defective ribosomal products -DRIPs-) of EBNA1 mRNA. Hence, in this model, the ability of GAr to limit in cis the translation of its own mRNA fully explains its inhibitory effect on antigen presentation. In this model on the left, NCL would be trapped by an unknown factor (Protein X) which interacts with the unmethylated C-terminal RGG motif of NCL. Upon type I PRMTs-mediated methylation of the arginines of this RGG motif, the NCL/Protein X complex would be dissociated. Hence, the RGG motif of NCL would now be available to interact with the RNA G-quadruplexes (rG4) of EBNA1 mRNA, which is required for GAr-based limitation of translation, a phenomenon at the basis of EBNA1 immune evasion.

Citrullination (on the right), by preventing arginine methylation, would interfere with type I PRMTsmediated methylation of the C-terminal RGG motif of NCL, hence interfering with GAr-based inhibition of translation and presentation of EBNA1-derived antigenic peptides. Based on the model presented in Figure 4, at least to intervention points could be exploited to unveil EBV-infected cells (in particular tumour cells of EBV-related cancers) to the immune system: G4 ligands such as PhenDC3 that interfere with the binding of NCL on rG4 of EBNA1 mRNA (left) and compounds inhibiting type I PRMTs (right). By distinct modes of action, both types of drugs interfere with the binding of NCL on rG4 of EBNA1 mRNA, and thereby with the ability of GAr to self-limit the translation of its own mRNA. This, in turn, relieves the limitation of the production of EBNA1-derived antigenic peptides, ultimately interfering with immune evasion of EBNA1 and thus of EBV.

Finally, beyond

  cancer, EBV, and more particularly EBNA1, have been involved in other diseases, notably multiple sclerosis (MS). Indeed, the long-time suspected link between EBV and MS has recently been firmly confirmed in a large-scale study in which the authors analysed serum EBV antibodies from individuals who developed MS among a cohort of more than 10 million young adults in a more than 20 years follow-up, and the results indicated a 32-fold increase in the risk of developing MS after EBV infection [15]. In parallel, a role for EBNA1 in MS has been proposed, notably via molecular mimicry leading to the development of cross-reactive antibodies and T cells [122, 123]. In line, a vaccine against EBNA1 appears a relevant therapeutic option to treat EBV-related disorders, in particular EBV-related cancers and MS, and several experimental vaccines have been developed against EBV, including against EBNA1 [124-126]. Notably, vaccination against EBNA1 has been shown efficient in eliciting comprehensive CD4 + and CD8 + T cell responses which can translate into protection against EBV antigen expressing lymphomas [125, 127].

Figure 1 :

 1 Figure 1: Schematic representation of the EBV-encoded EBNA1 protein highlighting the essential role of the GAr domain in immune evasion of the virusThe first 32 amino acids of EBNA1 full length are not associated to a particular function. The two RGG motifs (Arginine-Glycine-Glycine) are highlighted in gold (from amino acids 33 to 64 and 329 to 377), the GAr domain (Glycine-Alanine repeat) is shown in orange (from amino acids 90 to 328), the nuclear localization signal (NLS) is located between amino acids 378 and 386 and colored in black. The DNAbinding and dimerization domain (amino acids 459 to 609) is colored in purple. As EBNA1 full length, EBNA1ΔGAr is able to fulfill its essential functions for EBV genome replication and maintenance.

Figure 2 :

 2 Figure 2: The proteasome and the ribosome era parallel with the two consecutive models for

Figure 3 :

 3 Figure 3: Rationale of the yeast-based assay for GAr-based inhibition of translation and use for both drug and genetic screenings a. Rationale of the use of the Ade2 reporter protein in yeast. When Ade2 is expressed at a functional level yeast cells are white. When not expressed (ade2Δ cells), yeast cells are red. Any intermediate level of Ade2 leads to pink colonies whose colour intensity is inversely proportional to the level of Ade2 expression. This allows to easily monitor the GAr-inhibitory effect on translation in yeast. b. Examples of use of the Ade2 reporter system for drug screening (left panel) aiming at identifying compounds that counteract the in cis inhibitory effect of GAr on translation, or genetic screening (right panel) to identify host genes involved in GAr-based inhibition of translation.

Figure 4 :

 4 Figure 4: Model for the role of NCL and of the arginine methylation and citrullination of its Cterminal RGG motif in EBNA1 immune evasion

Figure 5 :

 5 Figure 5: Possible intervention points to unveil EBNA1 to the immune system.

Figure 1

 1 Figure 1

Acknowledgments

This work was supported by the following grant agencies: 'La Ligue contre le cancer CSIRGO' and 'Fondation pour l'Avenir' to MB, 'Agence Nationale de la Recherche' (ANR-17-CE07-0004-01, to AG), French Ministry of Higher Education, Research and Innovation (PhD fellowships to RLS), 'La Ligue contre le cancer Bretagne' and 'Région Bretagne' (PhD fellowship to VTD), INCa PLBIO (PLBIO16-225) to MB and 'Fondation pour la Recherche Médicale' (DCM20181039571) to MB.

Nucleus

Ribosome GAr-encoding sequence

In cis translation Inhibition ?

PRMT I inhibitors


Julien Salama
email: julien.salama@sciencespo-lille.eu

) Financer

Financing the post-growth state

Introduction

The issue of financing the state in a post-growth economy combines several facets of the general post-growth challenge. Most notably, it concentrates some of the contradictions between the social and the ecological dimensions of the post-growth program. This financing problem feeds a common criticism addressed to post-growth proponents, and while it is often mentioned by the latter, it is seldom addressed in depth, as Daniel Bailey (2015a, p. 126) remarked:

The biggest unacknowledged consequence of the post-growth policy approach […] relates to the effects of the end of growth on the capitalist state.

Therefore, it seems that facing the challenge of financing the state in a post-growth economy is a crucial step in strengthening its credibility, both scientifically and politically. However, the research field in the state's fiscal dependency on growth is still emerging. [START_REF] Bailey | How sustainable is the welfare state in the context of an economic, fiscal and environmental crisis[END_REF][START_REF] Bailey | The Environmental Paradox of the Welfare State: The Dynamics of Sustainability[END_REF][START_REF] Bailey | Re-thinking the Fiscal and Monetary Political Economy of the Green State[END_REF] and Damien [START_REF] Demailly | Une société post-croissance pour le xxie siècle -Peut-on prospérer sans attendre le retour de la croissance ?[END_REF] have been the first authors to examine this looming problem in depth, later followed by Claudio Cattaneo and Aaron Vansintjan (2016), Timothée [START_REF] Parrique | The political economy of degrowth[END_REF], Ulrich [START_REF] Petschow | Social well-being within planetary boundaries: The precautionary post-growth approach[END_REF], Martin Fritz and Katharina [START_REF] Bohnenberger | Money, Vouchers, Public Infrastructures? A Framework for Sustainable Welfare Benefits[END_REF] and Christine [START_REF] Corlet-Walker | Welfare systems without economic growth: A review of the challenges and next steps for the field[END_REF]. Occupying a distinctive position in this field, Eloi Laurent (2019[START_REF] Laurent | Penser l'État-providence post-croissant[END_REF]Laurent ( , 2021a;;[START_REF] Laurent | From welfare to farewell : The European social-ecological state beyond economic growth[END_REF] seems to be the only author to contest the assertion that states may have a problematic dependence on growth. Due to space limitations, his arguments cannot be discussed here.

Finally, it is striking that all of these works, including Bailey's, focus on what post-growth would do specifically to the welfare state, rather than state institutions as a whole; thus the state seems to be a largely unthought issue within the field of post-growth research, as noted by Giacomo D'Alisa and Giorgos Kallis (2020).

A problematisation proposal: the state is trapped in a double social and ecological unsustainability

The problem can be divided into five steps.

First, it seems that absolute, continued, permanent and global decoupling between economic growth and ecological degradation is unlikely [START_REF] Ward | Is Decoupling GDP Growth from Environmental Impact Possible?[END_REF][START_REF] Parrique | Decoupling debunked, Evidence and arguments against green growth as a sole strategy for sustainability[END_REF][START_REF] Hickel | Is Green Growth Possible?[END_REF][START_REF] Haberl | A systematic review of the evidence on decoupling of GDP, resource use and GHG emissions, part II: synthesizing the insights[END_REF]. Besides these ecological limits, growth also faces social limits -i.e. it is no longer correlated with an increase in human well-being -as well as socioeconomic ones, which means that countries in the Global North may face economic stagnation in the years to come [START_REF] Kallis | Degrowth[END_REF][START_REF] Parrique | The political economy of degrowth[END_REF][START_REF] Corlet-Walker | Welfare systems without economic growth: A review of the challenges and next steps for the field[END_REF]. For these reasons, a post-growth economy seems necessary. Post-growth is understood here as an umbrella term that gathers proposals that share a common deprioritisation of growth in order to focus on human well-being. Christian [START_REF] Kerschner | Economic de-growth vs. Steady-state economy[END_REF], Dan O'Neill (2012), Milena Büchs andMax Koch (2017) and Brian Czech and Riccardo Mastini (2020) expound it as the degrowth process towards a stationary steady state. It is defined by equitable, democratic and selective downscaling of production and consumption levels and therefore energy and materials until reaching sustainable levels, while improving human well-being.

Secondly, the state is a crucial institution to drive this transformation towards a post-growth economy in an effective and equitable way. A Gramscian-inspired conception of the state could give the theoretical basis to back this argument. The state can be conceived as a social relation, i.e. a 'condensation' of a society's changing balances of social forces that aim to influence the orientation and the contents of policies [START_REF] Poulantzas | L'Etat, le pouvoir, le socialisme[END_REF][START_REF] Jessop | The State : Past, Present, Future[END_REF][START_REF] Koch | The state in the transformation to a sustainable postgrowth economy[END_REF][START_REF] D'alisa | Degrowth and the State[END_REF]. As a consequence, the state is the subject of a struggle from within and without, to determine the nature of its means and its ends. As argued by Peter [START_REF] Ferguson | Post-growth Politics. A Critical Theoretical and Policy Framework for Decarbonisation[END_REF], this dynamic conception of the state introduces the possibility of a disjunction between the state and capitalist interests. This could happen by building broad social coalitions along shared interests and a new, counter-hegemonic common sense, e.g. through coordinated grassroots initiatives, thus contributing to social change and paving the way towards a post-capitalist state (see for example D'Alisa & Kallis, 2020;Latour & Schulz, 2022; and the contributions in [START_REF] Barlow | Degrowth & Strategy: how to bring about social-ecological transformation[END_REF].

Moreover, if the state has to be the subject of such a transformation, it is because it is a crucial tool for the political regulation of conflicts and the maintenance of social cohesion in general, and for the ecological transformation in particular. Conceived as the coordinator of local, communal, international and private actors, it seems that it is the organisation that can implement 'macro-limits' on detrimental economic freedoms in a legitimate and effective way [START_REF] Eckersley | The Green State[END_REF]. The state, provided that it is transformed to meet these aims, may be our best readily available institution to plan the vast, costly and long-term transformation which cannot be achieved by near-sighted market forces or local uncoordinated initiatives. This argument could also be made for the equitable downscaling of energy and material throughput, as well as the just distribution of sufficiency efforts. Furthermore, the decommodification of economic relations, ensured by protection safety nets, would be of great importance in these turbulent times.

Third, beyond its ideological imperative to promote growth, the state also faces a 'functional' [START_REF] Eckersley | The Green State[END_REF] dependency on growth to finance itself. While this fact is well established [START_REF] Petschow | Social well-being within planetary boundaries: The precautionary post-growth approach[END_REF]Bohnenberger, 2021;[START_REF] Corlet-Walker | Welfare systems without economic growth: A review of the challenges and next steps for the field[END_REF], the post-growth literature often fails to emphasise that this is more fundamentally a dependency on capital accumulation, which is the economic phenomenon that generates growth [START_REF] Blauwhof | Overcoming accumulation : Is a capitalist steady-state economy possible?[END_REF][START_REF] Kallis | Degrowth[END_REF]. A state dependent on the taxation of private wealth and income is thus ultimately dependent on the ability of capital accumulation to generate profits and wages from which taxes are collected [START_REF] Harman | The state and capitalism today[END_REF][START_REF] Blauwhof | Overcoming accumulation : Is a capitalist steady-state economy possible?[END_REF][START_REF] Ferguson | Post-growth Politics. A Critical Theoretical and Policy Framework for Decarbonisation[END_REF]. Claus [START_REF] Offe | Contradictions of the welfare state[END_REF] thus makes 'taxation constraints' one of the constitutive elements of the capitalist state.

The state is therefore doubly challenged by the transition away from growth: because it is dependent on ecologically unsustainable growth, and because the exit from growth may dry up its sources of funding, while it may face rising expenditures as we will discuss later.

Fourth, an exit from growth that would weaken state fiscal resources would in turn threaten the social sustainability of the ecological transformation, and thus its effectiveness. As a matter of fact, departing from growth could affect the state's ability to steer, finance and regulate the transition and to ensure its fairness. In this case, a poorly financed and steered post-growth transition would rather mirror a recession, with its inherent devastating effects on subjective and objective dimensions of human well-being [START_REF] Büchs | Postgrowth and wellbeing. Challenges to sustainable welfare[END_REF]. Added to its potentially inegalitarian dynamics, such a transition would weaken social cohesion and, as a result, its own legitimacy [START_REF] Wilkinson | The Spirit Level. Why More Equal Societies Almost Always Do Better[END_REF][START_REF] Bailey | How sustainable is the welfare state in the context of an economic, fiscal and environmental crisis[END_REF]. This would be the case all the more if austerity policies were to emerge as a response to this financial destabilisation of the state: this is what Bailey (2015a) calls 'the unsustainability of welfare state retrenchment'.

Fifth, this tension can be summed up, after Bailey (2015a), as a double unsustainability: on the one hand, the current ecological unsustainability of state financing, which depends on ecologically problematic economic growth; on the other hand, the hypothetical socioeconomic unsustainability of a possible disengagement or weakening of the state resulting from the exit from growth. This disengagement could undermine the ecological transformation itself.

The state's vulnerability to a contraction of its tax base and an increase in its expenditures: what's exactly at stake?

First, one should underline the fact that the taxable base, for both the state and its welfare organisms, depends mainly on economic activity, although compulsory levies have their own sensitivity to its variations [START_REF] Büchs | Postgrowth and wellbeing. Challenges to sustainable welfare[END_REF][START_REF] Petschow | Social well-being within planetary boundaries: The precautionary post-growth approach[END_REF][START_REF] Bohnenberger | Making welfare resilient -Creating stable & sustainable welfare systems in times of declining economic growth[END_REF]. It should also be noted that the taxable base is partly determined by sociodemographic factors such as population growth and the health status of populations [START_REF] Laurent | From welfare to farewell : The European social-ecological state beyond economic growth[END_REF].

A transition to post-growth will probably lead to an overall contraction of that taxable base.

The post-growth imperative is to reduce the level of production, consumption of goods and services, rates of profit or hours of wage labour in order to bring energy and material throughput back to environmentally and socially sustainable levels via diverse measures (see e.g. [START_REF] Bailey | How sustainable is the welfare state in the context of an economic, fiscal and environmental crisis[END_REF][START_REF] Cattaneo | ALTERNATIVES TO GROWTH[END_REF][START_REF] Büchs | Postgrowth and wellbeing. Challenges to sustainable welfare[END_REF][START_REF] Parrique | The political economy of degrowth[END_REF][START_REF] Bohnenberger | Making welfare resilient -Creating stable & sustainable welfare systems in times of declining economic growth[END_REF][START_REF] Mastini | A Green New Deal without growth[END_REF][START_REF] Corlet-Walker | Welfare systems without economic growth: A review of the challenges and next steps for the field[END_REF]. This seems to be supported by two post-growth macroeconomic models that examined this issue. In the 'Sustainable Prosperity' scenario of the LowGrow model for the Canadian economy [START_REF] Jackson | LowGrow SFC: a stock-flow-consistent ecological macroeconomic model for Canada[END_REF], 2020) the debt to GDP ratio, which stood at 55% in 2017, could reach 80% in 2067 in this scenario, as opposed to 60% in the business-as-usual scenario. This is notably due to a decrease in revenues and an increase in the deficit. Eurogreen's 'De-growth' scenario [START_REF] D'alessandro | Feasible alternatives to green Growth[END_REF] reaches similar conclusions: modelling a contraction of GDP, it anticipates a deficit of 3% of GDP per year in 2035, which increases to 7% in 2050, compared to the 2% deficit in the 'Green Growth' scenario; part of the difference between these figures can be explained by the contraction of the denominator.

At the same time, the state will face additional expenditures, notably linked to rising human needs: the costs of the investments needed, those implied by the correction of the regressive effects of transition policies, those of adaptation policies, those linked to the consequences of ecological upheavals (extreme climatic events, migrations, collapse of biodiversity, etc.), ageing populations in the Global North and an increase in the relative costs of healthcare systems [START_REF] Bailey | How sustainable is the welfare state in the context of an economic, fiscal and environmental crisis[END_REF][START_REF] Gough | Climate Change, Capitalism and Sustainable Wellbeing[END_REF][START_REF] Petschow | Social well-being within planetary boundaries: The precautionary post-growth approach[END_REF][START_REF] Corlet-Walker | Welfare systems without economic growth: A review of the challenges and next steps for the field[END_REF].

Prospects for the post-growth state: how to finance it without growth?

According to the trends described above, if the financing framework of governments is not transformed, a transition out of growth would considerably increase public deficits and consequently debt ratios, in volume and in proportion to GDP. These ratios could be rendered unsustainable with the slowdown of growth itself. [START_REF] Bailey | How sustainable is the welfare state in the context of an economic, fiscal and environmental crisis[END_REF][START_REF] Bailey | Re-thinking the Fiscal and Monetary Political Economy of the Green State[END_REF] argues that even if current growth were to stabilise, current levels of public spending would already be fiscally unsustainable. In a post-growth future, economic growth will no longer be able to balance or mitigate deficits or ensure debt sustainability, while debt servicing will become increasingly difficult.

The challenge is therefore to ensure both efficient and ecologically responsible financing of the ecological transformation itself, as well as to find ways in which a state can finance itself in an economy that has reached a sustainable steady state. In other words, what are the mutations needed to overcome the 'double unsustainability' laid out earlier?

According to the simple typology used by [START_REF] Parrique | The political economy of degrowth[END_REF] and [START_REF] Mastini | A Green New Deal without growth[END_REF], three axes can be considered: transforming expenditures, the tax system and the monetary system.

Transforming public spending to meet the challenges of post-growth

Savings could be made from the elimination of ecologically unsustainable public expenditures, like fossil fuel subsidies (which account for an average of 55 billion euros per year in the EU, according to a 2022 European Court of Auditors report) as well as 'megaprojects such as highways, shipping ports, military bases, power plants and airports' (Cattaneo & Vansintjan, 2016, p. 21). The collective utility of spendings, in a context of scarcer fiscal resources, will need to be democratically deliberated.

There are also savings to be expected from the benefits of ecological transformation, and the induced suppression of the socio-environmental risks created and maintained by growth [START_REF] Büchs | Postgrowth and wellbeing. Challenges to sustainable welfare[END_REF]Laurent, 2019). [START_REF] Mastini | A Green New Deal without growth[END_REF] also argue that the post-growth transition will produce positive effects in terms of reducing 'public health costs, unemployment benefits, defence spending and adaptation to climate change'. As a matter of fact, the WHO estimates that 12% of all deaths in the OECD can be attributed to preventable environmental causes (Laurent, 2021). A preventive approach to welfare provision, rather than a curative one, could thus bring considerable benefits, by focusing public policies on the social, political and environmental determinants of health and needs [START_REF] Gough | The Political Economy of Prevention[END_REF][START_REF] Gough | Climate Change, Capitalism and Sustainable Wellbeing[END_REF][START_REF] Bohnenberger | Making welfare resilient -Creating stable & sustainable welfare systems in times of declining economic growth[END_REF][START_REF] Corlet-Walker | Welfare systems without economic growth: A review of the challenges and next steps for the field[END_REF]. It is nonetheless doubtful that these benefits could compensate for the massive increase in needs and expenditures that we have discussed previously.

A more fundamental transformation of public expenditure could consist in recalibrating the welfare state around the provision of basic human needs [START_REF] Gough | Climate Change, Capitalism and Sustainable Wellbeing[END_REF][START_REF] Büchs | Postgrowth and wellbeing. Challenges to sustainable welfare[END_REF]. Focusing the welfare state on effectively fulfilling basic human needs to all would therefore allow it to concentrate its expenditure on a number of essential objectives. On the one hand, this would help to get rid of insatiable or superfluous preferences; on the other hand, it would help prevent social risks and thus the increase in the 'demand' for social protection induced by economic growth (Büchs 2021a). Universal Basic Services and Universal Basic Vouchers could pave the way for meeting these objectives [START_REF] Gough | Universal Basic Services : A Theoretical and Moral Framework[END_REF]Büchs, 2021a;[START_REF] Bohnenberger | Money, Vouchers, Public Infrastructures? A Framework for Sustainable Welfare Benefits[END_REF].

A transformation of the state taxation architecture to mitigate the contraction of the tax base

Secondly, the recalibration of state and social protection budgets could also involve stronger, fairer and more progressive taxation on those that are already subject to it, as well as extended taxation, for example to areas of economic activity that are not currently subject to it. Income, property and inheritance could be more strongly taxed or even capped, and taxation could be extended to financial transactions, advertising, luxury consumptions, etc.

Proposals for an ecological tax reform often involve progressive taxes on pollution as well as carbon emissions, natural capital depletion and energy consumption [START_REF] Daly | Ecological Economics. Principles and Applications[END_REF][START_REF] Cattaneo | ALTERNATIVES TO GROWTH[END_REF][START_REF] Büchs | Postgrowth and wellbeing. Challenges to sustainable welfare[END_REF][START_REF] Gough | Climate Change, Capitalism and Sustainable Wellbeing[END_REF][START_REF] Parrique | The political economy of degrowth[END_REF].

However, as [START_REF] Demailly | Une société post-croissance pour le xxie siècle -Peut-on prospérer sans attendre le retour de la croissance ?[END_REF], [START_REF] Bailey | How sustainable is the welfare state in the context of an economic, fiscal and environmental crisis[END_REF] and [START_REF] Cattaneo | ALTERNATIVES TO GROWTH[END_REF] point out, behavioural and ecological taxes would fade with the progressive disappearance of the degradations they disincentivize. It should also be noted that eco-taxes could have regressive distributional effects, particularly on the most vulnerable, thus requiring corrective measures that mobilise part of the resources that they themselves have generated. Moreover, the tax reforms detailed above aimed at increasing public revenues may not compensate for the fiscal losses caused by the absence or contraction of GDP: the very purpose of a post-growth transition is to generate an economy in which the levels of production and consumption have been brought down to, and stabilised at, environmentally sustainable levels. These reforms thus aim to curb and mitigate this contraction by maximising the revenues from a smaller taxable base. Eventually, the tax reforms described above refer to the more fundamental balance of power that determines the level of socialisation of wealth that a society wants to achieve.

Achieving the decorrelation of the state budget from capitalist growth through an overhauling of the monetary system

A first step could be to call into question one of the main characteristics of modern money, namely its general purpose, by creating special-purpose monies like local and complementary currencies [START_REF] Parrique | The political economy of degrowth[END_REF]. One of their crucial benefits is their 'non-dissipative' function, as Parrique puts it: they do not require wealth to be created in the first place to provide tax revenues and eventually be reinvested in public goods and services. Instead, the provision of these is done at a 'pre-monetary' stage, focusing directly on the flows of time, energy and matter. Such local currencies would save the interest that would otherwise have to be paid by taking out a loan and make possible repayment in kind over time. They would allow a number of goods and services usually provided by public authorities to be removed from the scope of public expenditure in a non-dissipative way, through their decentralisation, decommodification and diversification. However, as Christian Arnsperger (2020) points out, these currencies cannot finance heavy, complex and unprofitable investments or programmes involving large numbers of people, goods and services. And as they are complementary, they are not intended to replace the state and national mechanisms of solidarity and economic exchange.

A second step lies in the transformation of money creation: this is the aim of the 'sovereign money' proposals (Positive Money, 2016;[START_REF] Huber | Sovereign Money. Beyond Reserve Banking[END_REF][START_REF] Grandjean | Une monnaie écologique[END_REF]. They propose the nationalisation of the money creation process by issuing debt-free money managed by a democratised Central Bank in order to decommodify it, to democratise its creation and distribution, and to put it at the service of the common good. Some of these proposals also advocate the removal of the quasi-monopoly of money creation held by commercial banks, which would result in an abolition of profit-driven money creation (e.g. [START_REF] Mellor | Debt Or Democracy : Public Money for Sustainability and Social Justice[END_REF][START_REF] Parrique | The political economy of degrowth[END_REF]. This would allow the state to finance its expenditures, for example costly, long-term and non-profitable investments, or to offset all or part of its deficit.

A monetary sovereign state -that is yet to be achieved in the eurozone and in many countries of the Global South -would then only be limited by the availability of real resources (labour, natural resources and productive capacity) for its spending. Inflation is also one of these limits, but may not be an immediate threat if money is well injected, managed and destroyed [START_REF] Kelton | The Deficit Myth, Public Affairs[END_REF][START_REF] Grandjean | Une monnaie écologique[END_REF].

A third alternative and radical step could be the adoption of the Modern Monetary Theory (MMT) paradigm (e.g. Randall [START_REF] Wray | Modern Money Theory. A Primer on Macroeconomics for Sovereign Monetary Systems[END_REF][START_REF] Kelton | The Deficit Myth, Public Affairs[END_REF]. According to MMT, which aims to be a descriptive rather than a prescriptive approach, if a state is monetarily sovereign, it does not have to balance its budget like a household, notably because it does not have to borrow or tax what it spends. Indeed, according to the MMT, a monetarily sovereign state spends not from its tax revenues, but from the issuance of its own currency: being the sole issuer of this currency, it therefore has no constraint on its capacity to spend. Thus, taxation is not used to finance spending: the government spends, then collects taxes afterwards as a compensation.

The deficit is therefore a 'myth' [START_REF] Kelton | The Deficit Myth, Public Affairs[END_REF], and state budgetary constraints are mainly ideological. This reversal of perspective may help to address part of the problem of financing the state without growth [START_REF] Hickel | The Virtuous Circle of Sustainable Welfare as a Transformative Policy Idea[END_REF][START_REF] Bailey | Re-thinking the Fiscal and Monetary Political Economy of the Green State[END_REF][START_REF] Williams | MMT, Post-growth economics, and avoiding collapse[END_REF][START_REF] Helker-Nygren | Degrowth & Modern monetary theory: building bridges for socio-ecological sustainability and justice[END_REF], provided it is adopted as the main monetary paradigm. The transformation of state expenditures would rather serve to limit the need for monetary creation and taxation would regulate the excess amount of money in circulation. This is also generally the case for sovereign money proposals, that share a lot with MMT. But these two paradigms, which would complete a decoupling of public spending from capitalist accumulation through public monetary financing, seem to envisage it in different ways. Sovereign money initiatives seem to conceive tax revenues as the main means of financing state expenditure, and here it has been embedded in a more global transformation, along with a recalibration of expenditures and taxation, to adapt the state to the post-growth economic downscaling. Meanwhile, MMT asserts that there is no direct link between public spending and taxation: however, there is still a need to confront MMT more directly with the post-growth challenge, as Ellen Helker-Nygren (2022) has started to do.

These two alternatives, at times complementary 'packages' (recalibration of expenditures, taxation and money creation on the one hand, and MMT on the other hand), have the potential to lead a major opportunity to decorrelate the state from capital accumulation and its subsequent growth imperative. This could pave the way to the rise of a post-growth and therefore post-capitalist state, decoupled from capital accumulation. The question of the nature of post-growth economic activity remains open.

Fig. 1 :

 1 Fig. 1: The double unsustainability of the post-growth state.

Degrowth Journal Volume 1 (2023) 00036

Acknowledgements

I would like to thank S. Rousseau, my master thesis supervisor, as well as the Veblen Institute that helped me writing this master thesis, and especially L. Hallak.

Funding

The authors did receive any funding for this research.

Conflict of interest

The author has no conflict of interest to disclose.