Innocent J Macha
email: imacha@udsm.ac.tz

Besim Ben-Nissan

Jerran Santos

Sophie Cazalbou

Artemis Stamboulis

David Grossin

Gerard Giordano

Biocompatibility of a new biodegradable polymer-hydroxyapatite composite for biomedical applications

Keywords:

The rise in the number of musculoskeletal disorders (MSDs) due to an increasingly aging population has led to a growing demand for medication to prevent and treat these diseases. An increased interest in the development of new drugs to allow treatment of these diseases in their very early stages is currently observed. The current approach on local direct delivery of medication and key minerals to support bone repair and regeneration at the defect site, from flexible degradable devices, seems to be an effective strategy. Polylactic acid (PLA) and microspheres of hydrothermally converted coralline hydroxyapatite (cHAp) were used to develop PLA thin film composites as drug delivery systems. The PLA provided flexibility and biodegradability of the systems, while coralline hydroxyapatite provided the required calcium and phosphate ions for bone regeneration. These coralline hydroxyapatite microspheres have a unique architecture of interconnected porosity, are bioactive in nature and suitable for drug loading and controlled slow drug release. The cell attachment and morphology of the PLA thin film composites were evaluated in vitro using cell cultures of human adipose derived stem cells (hADSC). It was shown that hADSC cells exhibited a strong attachment and proliferation on PLA thin film cHAp composites, signi fying high biocompatibility and a potential for osteointegration due to the presence of HAp.

Introduction

Different types of cells have been used in tissue engineering and in therapeutic strategies in cell therapy, including stem cell trans plantation. Stem cells are primarily used to understand the mech anisms by which natural or synthetic biomaterials are able to elicit a cellular response when implanted in vivo. In the early days, adult stem cells were used in this process but proved difficult to grow in culture because they were fully developed [1]. Attention was later focused onto the use of fetal cells, which are not stem cells but behaved similarly and better than adult cells when tested in conjunction with biomaterials [1,2].

Stem cells are capable of continuous self renewal for an extended period of time with minimal phenotypic changes. They are also unspecialized and can be differentiated into mature func tional cells in any of the dermal linages. The two most widely uti lized adult stem cells are derived from bone marrow extracts and adipose lipoaspirates and are termed as bone marrow stem cells (BMSCs) and adipose derived stem cells (hADSCs), respectively. BMSCs are involved in repair mechanisms during injury and have been extensively investigated, specifically in orthopaedic applications.

Biocompatibility of biomaterials involves three important stages: adhesion of cells on the surface, proliferation and finally, differentiation. When a biomaterial is implanted into a living body, the biomaterial is rapidly coated with a protein layer before the cells' attachment. Serum protein and various extracellular matrices (ECM) are involved, such as fibronectin, fibrinogen, albumin and vitronectin [3]. The protein adsorption and conformation is mainly influenced by the morphology of the biomaterial surface, which also influences the cell adhesion and proliferation process [4]. It has been reported that micro/nano surface topography has a direct impact on cell adhesion and proliferation, with a micro and nano textured surface favouring adhesion.

Apart from the morphology, the surface charge and chemical composition of the biomaterial surface has been suggested to play a significant role in the adhesion and proliferation of cells. Kese lowsky et al. [START_REF] Keselowsky | 66 Fig. 5. SEM micrographs of cell cultured samples coated with poly-L-lysine a) PLA b) PLAGM[END_REF] showed that cell adhesion through the integrin group of cell surface receptors, depends on the conformation of adsorbed fibronectin. In their demonstration, they used a self assembly monolayer of different functional groups such as OH, COOH, NH 2 and CH 3 termini, to create surfaces with different chemistry that were hydrophilic and neutrally charged; hydrophilic and acidic; hydrophilic and basic; and hydrophobic, respectively. Their findings suggested that the adhesion strength of cell binding (as determined by a centrifugation assay) followed the trend: OH > COOH > NH 2 > CH 3 . They also found that the specific gene expression of cells such as osteoblast, alkaline phosphatase enzyme activity and matrix mineralization, showed dependence on surface chemistry in which OH and NH 2 terminated surfaces were more advantageous, compared with COOH and CH 3 . Hydrophobic sur faces seem to cause denaturation of proteins and prevent the sur face exposure to cell binding groups responsible to cell adhesion. On the other hand, hydrophilic and neutrally charged surfaces induce the least extent of unfolding or denaturation, leading to a good cell adhesion on the fibronectin [6].

It has been reported that calcium phosphate bone substitutes derived from mixed hydroxyapatite (HAp) and b tricalcium phos phate (b TCP) are one of the most promising materials for bone drug delivery systems. Major attention has been focused on the delivery of antibiotics, due to their wide areas of application as prevention against infection during surgical interventions, or in general in the treatment of bone related infections [7].

For slow drug delivery applications, ceramics and other mate rials such as polymers and biocomposites have been proposed in the past, but they are difficult to form into an appropriate shape with adequate interconnected micro porosity in order to be fitted into any type and size of bone defect. The treatment of bone infection remains difficult because of problems with the local penetration of systemically administered antibiotics. Recently, Chu et al. demonstrated that marine shells with specific micro spherical design offer desired functions for the delivery of bisphosphonate (paminodrate) and an antibiotic (gentamicin) [8]. This has been possible by virtue of their unique structure and the architecture of the foraminifera shells, which are extraordinarily difficult to manufacture currently [9,10].

In this paper, polylactic acid (PLA) and microspheres of hydro thermally converted coralline hydroxyapatite (cHAp) were used to develop PLA thin film composites as drug delivery systems. PLA provided flexibility and biodegradability; whereas cHAp provided bioactivity and osteoconductivity. The cell attachment and cell morphology on PLA thin film composites were evaluated in vitro using cultures of human adipose derived stem cells (hADSC).

Materials and methods

Coral skeleton samples were obtained from the Great Barrier Reef, QLD Australia; gentamicin sulfate, clodronate (dichlor omethylenediphosphonic acid disodium salt), chloroform dia mmonium hydrogen phosphate ((NH 4 ) 2 HPO 4 , 98%), and sodium hypochlorite (NaClO) were obtained from Sigma Aldrich, Castle Hill, Australia. The PLA was purchased from NatureWorks Australia.

Porosity measurement

Porosity measurements were carried out with a mercury intrusion porosimeter (Autopore III, Micromeritics Instruments Inc., Norcross, GA, USA) with a 5 cm3 powder penetrometer.

PLA thin film composites and drug loading

Hydrothermally converted coralline hydroxyapatite particles were loaded with 10% w/w of gentamicin as described in previous works [7,11]. The required amount of gentamicin was dissolved in polished 18 MU water (MilliQ, Millipore, Victoria, Australia), mixed with HAp, sonicated and then dried using a Rotavapor R 210 coupled with a V 850, BUCHI (In vitro Technologies, Australia, vacuum controller) at 60 C. Gentamicin loaded cHAp micro spheres were then dried in vacuumed desiccators and used to produce PLA thin film composites. Drug loading of the PLA films was performed as described by Macha et al. [12] by adding the required amount of gentamicin or gentamicin loaded cHAp into PLA chloroform solution to give 10% (w/w) drug in the composites under magnetic stirring, followed by sonication to break down the agglomerations of particles, cast in Petri dishes and then dried in a desiccator under vacuum to produce transparent pore free thin films. The films thickness were measured to be in the range 0.1e0.2 mm.

The samples for the stem cell experiments were cast into ster ilized glass Petri dishes 60 Â 15 mm. Before cell seeding, the samples were sterilized in UV for 40 min.

Antibacterial efficacy test was performed and presented in our previously work [13]. Briefly, an antimicrobial efficacy test was conducted in Tryptone Soya Broth (TSB). Inoculum density equiv alent to 0.5 McFarland standards (1 Â 108 CFU/ml) from overnight culture, with a 50 ml TSB sub culture was used. PLA film compos ites loaded with gentamicin were either introduced in the begin ning or after 90 min of bacterial growth in a dry incubator at 37 ± 0.1 C, with shaking at 220 rpm. The optical density of the culture was monitored by spectrophotometer at 595 nm from the experiment at time 0, which was immediately after introduction of gentamicin loaded PLA films, and every half an hour for the first 6e6.5 h. When OD reaches about 1, samples were diluted to 1:10 before measurement. An additional time point was obtained after 24 h to check for recovery. Replicates of all experiments were done in different days. cultures of human adipose derived stem cells (hADSC) at passage 5 in biological triplicates. Approximately 5 Â 10 4 cells/ml were seeded on glass Petri dishes with the cast samples in DMEM/F12 Glutmax (Gibco) 10% fetal bovine serum (Gibco) and incubated at 37 C, 5% CO 2 for 7 days. Briefly, the PLA and PLAGM samples were coated with poly L lysine at room temperature for 30 min and rinsed twice with phosphate buffered saline (pH 7.4) before cell culture. Samples were washed with PBS (pH 7.4), fixed in 4% formaldehyde (Sigma) and then dehydrated in an ethanol gradient from 10% to 100% at 10% increments at 10 min each, before the cells were observed in SEM (ZEISS Supra55VP, Zeiss, Germany) operated at 5 kV accelerated voltage, using a back scatter electron detector.

Results and discussions

Fig. 1 shows the morphology of coralline materials hydrother mally converted to hydroxyapatite in a phosphate solution. Fig. 1a suggests that HAp retained the morphology of the original coral after conversion.

Fig. 1d also shows coral after conversion, revealing the hy droxyapatite platelet/needle like morphology. After ball milling coralline particles still contain meso and nanopores for drug loading and release. The porosity analysis of coral powder (Fig. 2), shows the pore sizes in the range of 3 nme~350 mm. It was also indicated that most of the crushed and ground particles have pore sizes around 4.5 mm with a surface area determined to be 3.23 m 2 /g. The porosity of coral particles was determined to be 58% in the size range considered (3 nm 350 nm). The results are consistent with results reported previously, using Nuclear Magnetic Resonance (NMR) [14].

Scanning electron microscopy (SEM) was used to confirm the physical presence of loaded drugs in HAp particles as shown in Fig. 3. FTIR was used in previous work to evaluate any alteration of drugs loaded in a PLA matrix [7]. Fig. 3 shows micrographs of HAp before (Fig. 3a) and after loading with gentamicin (Fig. 3b). It is evident that converted coral particles are coated with the drug on the surface and into the micro, meso and nanopores, as the drug solution could easily penetrate into these pores.

Synthetic polymeric biomaterials can only support cell adhesion and proliferation to a limited extent due to the lack of functional groups necessary for cell interaction [15]. Fig. 4 presents SEM im ages showing the morphology and attachment of hADSC seeded on PLA thin film composites. The results show abundant cell attach ment on PLAHAp and PLAHApGM samples, but no cell attachment on PLA and PLAGM.

PLA has an alkyl pendant group (CH 3 ) in its backbone, which makes the polymer more hydrophobic and tends to denaturalize proteins responsible for cell binding and adhesion. Gentamicin has NH 2 groups, which with the CH 3 groups on the polymer back bone, reduce any chance for protein binding on the surface. This was evident because PLA and PLGM samples do not show any cells on their surfaces. The addition of hydroxyapatite (HAp) in the matrix results in an improvement of bioactivity of composite films by changing the surface chemistry from hydrophobic to hydro philic. The presence of eOH groups due to HAp on the other hand, favours the binding of adhesive proteins such as vitronectin and fibronectin and subsequently cellular interaction [16]. There are several methods currently used to treat biomaterial surfaces in order to improve their biocompatibility, including physical, chem ical and mechanical treatments. Surface treatments such as expo sure to plasma, formation of coatings, corona discharge, implantation of ions and ultraviolet (UV) ozone treatments can enhance cell attachment. Specific functional groups can be also covalently attached to biomaterial surfaces using chemical modi fication methods, to attach for example biomolecules [3].

In order to confirm the lack of protein adsorption on the surface of the PLA and PLAGM samples, they were coated with poly L lysine as an attachment factor to enhance the electrostatic interactions between negatively charged ions of the cell membrane and posi tively charged ions of the culture surface, by increasing the number of positively charged sites available for cell binding. The results suggest that cells were able to attach on the PLA and PLAGM sur faces after coating with poly L lysine in less than 24 h as shown in Fig. 5.

Conclusions

The study of PLAecalcium phosphate thin film composites re veals possible potential applications of these devices as drug de livery systems in the biomedical field. The flexibility they provide allows them to conform to any desired clinical shape and size. Incorporation of hydroxyapatite in the matrix has the added advantage of improving bioactivity and the continuous supply of calcium Ca 2þ and phosphate PO 4 3À ions which can assist in bone regeneration and repair. On one hand, PLAHAp and PLAHApGM display a strong affinity to hADSC, due to the presence of HAp in the polymeric matrix. On the other hand, a lack of cell attachment on the PLA and PLAGM could be observed. Coating PLA and PLAGM surfaces with poly L lysine reveals the possibility of surface treat ments that can enhance cell attachment, which could be of medical importance. Although, not carried out here, possibly other surface methods like plasma treatments could also be employed to render the surfaces suitable for protein binding. The use of PLA HAp composites as drug release systems with excellent biocompati bility could pave the way for a new generation of drug delivery systems for bone regeneration.

Fig. 1 .

 1 Fig. 1. SEM picture of (a) coral solid piece showing the retained porous morphology after conversion, b) coral solid piece before conversion for comparison, c) coralline piece after conversion to hydroxyapatite, d) converted coral piece showing platelets/needle-like morphology of hydroxyapatite.

2. 3 .Fig. 2 .

 32 Fig. 2. Porosity within (a) coral powder; pore size and pore size distribution and b) coral; evidence of pre-existing nanopores in a solid piece of coral.

Fig. 3 .

 3 Fig. 3. Hydrothermally coral-converted HAp (a) before gentamicin loading and (b) after gentamicin loading.

Fig. 4 .

 4 Fig. 4. SEM image of hADSCs cultured PLA thin film composites for 10 days, showing attachment and morphology of cells. The controls are presented on left hand column of media and films of PLA, PLAGM, PLAHAp and PLAHApGM with no cells. These are clear and devoid of cells. The column on the right are the films PLA, PLAGM, PLAHAp and PLAHApGM incubated with the cells. The image results show no cellular attachment of hADSCs to PLA or PLAGM with some precipitate forming on the latter. The final two images exhibit a highly confluent layer of attached homogenous hADSCs are present across the films PLAHAp and PLAHApGM.

Acknowledgement

We are grateful for the financial support from the Australian Academy of Science and the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska Curie grant agreement No 645749.

Author contributions

IJM, BB, and JS conceived and designed the experiments. IJM and JS performed the experiments. SC and AS analysed the data. All author contributed to the manuscript preparation. BB, AS and DG review the manuscript.

Conflicts of interest

The authors declare no conflict of interest.


Mariama Akodad

J Fauconnier

Pierre Sicard

Fabien Huet

Florence Blandel

Annick Bourret

Pascal De

Santa Barbara

Sylvain Aguilhon

Marion Le Gall

Gerald Hugon

Interest of colchicine in the treatment of acute myocardial infarct responsible for heart failure in a mouse model

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


Emilie M Bonnaud

Marion Szelechowski

Alexandre Bétourné

Charlotte Foret

Anne Thouard

Daniel Gonzalez- Dunia

Cécile E Malnou
email: cecile.malnou@inserm.fr

Borna disease virus phosphoprotein modulates epigenetic signaling in neurons to control viral replication

Understanding the modalities of interaction of neurotropic viruses with their target cells represents a major challenge that may improve our knowledge of many human neurological disorders for which viral origin is suspected. Borna disease virus (BDV) represents an ideal model to analyze the molecular mechanisms of viral persistence in neurons and its consequences for neuronal homeostasis. It is now established that BDV ensures its longterm maintenance in infected cells through a stable interaction of viral components with the host cell chromatin, in particular with core histones. This has led to our hypothesis that such an interaction may trigger epigenetic changes in the host cell. Here, we focused on histone acetylation, which play key roles in epigenetic regulation of gene expression, notably for neurons. We performed a comparative analysis of histone acetylation patterns of neurons infected or not by BDV, which revealed that infection decreases histone acetylation on selected lysine residues. We showed that the BDV phosphoprotein (P) is responsible for these perturbations, even when expressed alone independently of the viral context, and that this action depends on its phosphorylation by protein kinase C. We also demonstrated that BDV P inhibits cellular histone acetyl transferase activities. Finally, by pharmacologically manipulating cellular acetylation levels, we observed that inhibiting cellular acetyl transferases reduces viral replication in cell culture. Our findings reveal that manipulation of cellular epigenetics by BDV could be a mean to modulate viral replication and thus illustrate a fascinating example of virus/host cell interaction.

Importance

Persistent DNA viruses often subvert the mechanisms that regulate cellular chromatin dynamics, thereby benefitting from the resulting epigenetic changes to create a favorable milieu for their latent/persistent states. Here, we reasoned that Borna Disease Virus (BDV), the only RNA virus known to durably persist in the nucleus of infected cells, notably neurons, might employ a similar mechanism. In this study, we uncover a novel modality of virus/cell interaction in which BDV phosphoprotein inhibits cellular histone acetylation by interfering with histone acetyl transferase activities. Manipulation of cellular histone acetylation is accompanied by a modulation of viral replication, revealing the perfect adaptation of this "ancient" virus to its host that may favor neuronal persistence and limit cellular damage.

Introduction

Long-term persistence in the host cell is a real challenge for viruses, since it requires a tight control of viral replication in order to limit cytopathic effects. This is notably the case for viruses infecting cells with poor renewal capacities, such as neurons. Thus, neurotropic viruses provide a unique opportunity to decipher the molecular mechanisms underlying virus-cell interactions during persistence. Moreover, a better understanding of the physiological consequences of viral persistence in the central nervous system (CNS) may also help to clarify some unknown aspects of neuronal physiology, in normal and pathological conditions [START_REF] Matsumoto | Bornavirus closely associates and segregates with host chromosomes to ensure persistent intranuclear infection[END_REF][START_REF] Lipkin | Borna disease virus -fact and fantasy[END_REF].

Borna disease virus (BDV) represents an ideal paradigm for investigating the modalities of persistence of a non-cytolytic virus in the CNS. BDV exhibits a selective tropism for neurons of the limbic system, in particular the cortex and hippocampus, two structures that govern many cognitive and behavioral functions [START_REF] Gonzalez-Dunia | Borna disease virus interference with neuronal plasticity[END_REF]. Although BDV is highly neurotropic, it can also replicate in other cells of the CNS, as well as in many established cell lines in vitro.

BDV infects a wide variety of mammals, leading to a large spectrum of neurological disorders, ranging from acute encephalitis to behavioral alterations without inflammation [START_REF] Pletnikov | Borna disease virus infection of the neonatal rat: developmental brain injury model of autism spectrum disorders[END_REF][START_REF] Carbone | Borna disease: virusinduced neurobehavioral disease pathogenesis[END_REF]. Despite this wide host range, there is still controversy regarding whether BDV can infect humans and be associated with neurological diseases [START_REF] Lipkin | Borna disease virus -fact and fantasy[END_REF]. The recent detection of endogenized BDV-like sequences in a wide array of species, including primates, has however established that ancient bornaviruses actually infected our ancestors nearly 40 million years ago, thus revealing the long co-evolution between bornaviruses and animal species [START_REF] Horie | Endogenous non-retroviral RNA virus elements in mammalian genomes[END_REF].

BDV is an enveloped virus, with a non-segmented, negative strand RNA genome of 8.9 kb, the smallest of the Mononegavirales order [START_REF] De La Torre | Molecular biology of Borna disease virus and persistence[END_REF]. Its compact genome encodes six proteins, namely, the nucleoprotein (N), phosphoprotein (P), protein X, matrix protein (M), glycoprotein (G), and polymerase (L). Whereas M and G are involved in particle formation, P, N, and L are components of the ribonucleoprotein complex (RNP). One striking feature of BDV infection resides in the location of RNP into the nucleus, where viral transcriptions and replication take place [START_REF] Matsumoto | Bornavirus closely associates and segregates with host chromosomes to ensure persistent intranuclear infection[END_REF]. This is an exception for vertebrate RNA viruses, which usually replicate in the cytoplasm of infected cells. The only other family of RNA viruses that replicates in the nucleus, the Orthomyxoviridae, are cytolytic [START_REF] Gonzalez-Dunia | Borna disease virus interference with neuronal plasticity[END_REF]. Hence, BDV is the only RNA virus that durably persists in the nucleus of infected cells.

To achieve this efficient intra-nuclear persistence, BDV closely associates with chromatin and forms viral factories, designated as vSPOTs (viral SPeckles Of Transcripts), which contain all viral components necessary for viral transcriptions. It was shown that vSPOTs interact in a dynamic manner with host chromosomes, using High Mobility protein Group B-1 (HMGB-1) as a scaffold protein between RNP and chromatin [START_REF] Matsumoto | Bornavirus closely associates and segregates with host chromosomes to ensure persistent intranuclear infection[END_REF][START_REF] Charlier | Analysis of borna disease virus trafficking in live infected cells by using a virus encoding a tetracysteine-tagged p protein[END_REF]. Based on these findings, it was tempting to hypothesize that BDV may modify the host cell chromatin, or interact with factors controlling chromatin dynamics. Moreover, in a previous proteomic analysis using primary neuronal culture protein extracts, we observed that acetylation of histone H2B was changed upon infection, suggesting that epigenetic signaling could indeed be affected by BDV [START_REF] Suberbielle | Proteomic analysis reveals selective impediment of neuronal remodeling upon Borna disease virus infection[END_REF].

Amongst the multiple modes of virus-mediated hijacking of host cell functions, manipulation of epigenetic signaling appears particularly well suited for persistent viruses, especially for DNA viruses. This allows them to benefit from epigenetic changes they induce in the nucleus of target cells, thereby producing a favorable environment for their latent/persistent states or for reactivation [START_REF] Horwitz | Adenovirus small e1a alters global patterns of histone modification[END_REF][START_REF] Paschos | Epigenetic reprogramming of host genes in viral and microbial pathogenesis[END_REF][START_REF] Knipe | Snapshots: chromatin control of viral infection[END_REF][START_REF] Gomez-Diaz | Epigenetics of hostpathogen interactions: the road ahead and the road behind[END_REF]. This has been well described for herpesviruses, such as Kaposi's sarcoma-associated herpesvirus, for which the latencyassociated nuclear antigen has been shown to interact with host chromatin modifiers and induce epigenetic reprogramming [START_REF] Kim | Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen regulates the KSHV epigenome by association with the histone demethylase KDM3A[END_REF][START_REF] Hu | LANA binds to multiple active viral and cellular promoters and associates with the H3K4methyltransferase hSET1 complex[END_REF]. Such a scenario appeared however less likely for RNA viruses. The long lasting nuclear persistence of BDV, together with its close association to chromatin, led us to hypothesize that, similarly to large DNA viruses, BDV infection could be accompanied by virus-mediated changes in the host cell epigenome.

As histone acetylation is a crucial modification allowing a fine control of neuronal gene expression and is often perturbed in numerous neurological pathologies [START_REF] Peixoto | The role of histone acetylation in memory formation and cognitive impairments[END_REF][START_REF] Tsankova | Epigenetic regulation in psychiatric disorders[END_REF], we sought to analyze the impact of BDV infection on epigenetic signaling, with a particular focus on histone acetylation patterns. Using both primary neuronal cultures and persistently infected cell lines, we demonstrate that BDV infection leads to a decrease of histone acetylation levels, restricted to specific H2B and H4 lysine residues. We also identify the BDV P as the viral determinant responsible for this inhibition and show that it needs to be phosphorylated by the protein kinase C (PKC) to exert this effect. Finally, we show that decreased histone acetylation is due to inhibition of cellular histone acetyl transferases (HATs) and that pharmacological manipulation of cellular acetylation modulates viral replication. This suggests that inhibition of histone acetylation may serve to control viral replication during persistence.

Materials and methods

Ethics statement. Animal handling and care for the preparation of primary neuronal cultures from rat embryos was performed in accordance with the European Union Council Directive 86/609/EEC and experiments were performed following the French national chart for ethics of animal experiments (articles R 214-87 to 90 of the "Code rural"). Our protocol received approval from the local committee on the ethics of animal experiments (permit number: 04-U1043-DG-06). Pregnant rats were deeply anesthetized with CO2 before euthanasia to minimize suffering.

Primary neuronal cultures, virus infection and plasmid transfection. Primary cortical

neurons were prepared from Sprague-Dawley rat embryos at gestational day 17 by the papain dissociation method as described previously [START_REF] Chevalier | Neurons are MHC class Idependent targets for CD8 T cells upon neurotropic viral infection[END_REF]. At the end of the procedure, neurons were either directly plated on variable supports (glass coverslips or culture plates) depending on the experiments, or used for plasmid transfection before plating. Before seeding, supports were coated with 500 µg/ml poly-ornithine (Sigma), followed by 5 µg/ml laminin (Roche). Neurons were maintained in Neurobasal medium (Invitrogen) supplemented with 100 µg/ml penicillin/streptomycin, 0.5 mM glutamine and 2 % B-27 supplement (Invitrogen). Half of each culture was infected 4 h after plating with cell-free BDV. Cell-released virus stocks were prepared as described using Vero cells persistently infected by wild-type BDV (Giessen strain He/80) or different recombinant BDV (rBDV) [START_REF] Schmid | Protein kinase C-dependent phosphorylation of Borna disease virus P protein is required for efficient viral spread[END_REF]. By 14 days post-infection, the efficacy of viral spread into the culture (which routinely attained >95 %) was systematically assessed by indirect immunofluorescence for the detection of BDV antigens.

To achieve efficient expression of isolated BDV proteins, neurons were transfected using Amaxa technology and the rat neuron nucleofector kit (Lonza) following the manufacturer instructions (5.10 6 neurons with 3 µg of plasmid DNA, using the G-13 program), allowing routine transfection efficiencies of at least 75 % for primary neurons.

Neurons were plated for 14 days, a time at which transfection efficiency was systematically verified by indirect immunofluorescence before proceeding to histone preparation. Plasmids used for transfection contained genes encoding BDV N or BDV P proteins under the control of the CMV early enhancer/chiken b actin (CAG) promoter, or an empty vector as a control.

In addition, two plasmids encoding different mutants of BDV P were used, one in which the glutamate residue in position 84 was replaced by an asparagine (PE84N) to abolish interaction with HMGB1 [START_REF] Schmid | Protein kinase C-dependent phosphorylation of Borna disease virus P protein is required for efficient viral spread[END_REF], and another for which serine residues at positions 26 and 28 were replaced by alanine residues (PAA) to mutate the PKC phosphorylation site of P [START_REF] Schmid | Protein kinase C-dependent phosphorylation of Borna disease virus P protein is required for efficient viral spread[END_REF]. All plasmids were kind gifts from Pr. Schwemmle. Plasmid maps are available on request.

Cell lines, production of lentiviral vectors and transduction experiments. Vero and HEK-293T cells were obtained from the American Type Culture Collection. We also used Vero cells persistently infected with either wild-type BDV (Giessen strain He/80), recombinant BDV in which the two serine residues in position 26 and 28 of BDV-P were replaced by alanine residues (rBDV-PAA) or recombinant wild-type BDV (rBDV-Pwt), composed of the canonical sequence of wild-type BDV strain He/80 [START_REF] Schmid | Protein kinase C-dependent phosphorylation of Borna disease virus P protein is required for efficient viral spread[END_REF]. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10 % fetal bovine serum and 2 mM L-glutamine. For HEK-293T cells, 2 mg/ml geneticin was added to the medium. For the establishment of Vero cell lines stably expressing individual BDV proteins, Vero cells were transduced by lentiviral vectors at a multiplicity of transduction of 10. A lentiviral vector encoding green fluorescent protein (GFP) was used to establish the control cell line. For each cell line, transduction efficacy was assessed by immunofluorescence, to ensure that at least 95 % of the cells expressed the protein of interest.

Histone preparation and western blot analysis. The procedure for histone preparation was adapted from Shechter et al. [START_REF] Shechter | Extraction, purification and analysis of histones[END_REF]. Nuclei were isolated upon cell lysis in a hypotonic buffer containing 0.2 % Triton-X100, 20 mM b-glycerophosphate, 10 mM Hepes pH 7.6, 1.5 mM MgCl2, 10 mM KCl, 10 mM sodium butyrate, 1 mM DTT and protease inhibitors (Complete Mini, Roche). After centrifugation and washing, nuclei were incubated in 0.4 N HCl during 1 h at 4°C to allow histone solubilization. After removal of the nuclear debris by centrifugation, histones were precipitated from supernatant by the addition of 4 volumes of acetone and incubation overnight at -20°C. They were then centrifuged at 15,000 X g during 20 min and histone-enriched pellets were resuspended in Laemmli buffer for western blot analysis. Western blots were performed as previously described [START_REF] Schmid | Protein kinase C-dependent phosphorylation of Borna disease virus P protein is required for efficient viral spread[END_REF] using either histone preparations or whole cell extracts and the following primary antibodies: anti total H2B, H2B acetyl K5, H2B acetyl K12, H2B acetyl K15, H2B acetyl K20, H3 pan acetyl, total H3 and H4 acetyl K8 from Epitomics (rabbit monoclonal antibodies); anti total H4, H4 tetraacetyl, H4 acetyl K5, H4 acetyl K12 and H4 acetyl K16 from Active Motif (rabbit antibody); rabbit antisera anti BDV N and P proteins [START_REF] Volmer | Borna disease virus blocks potentiation of synaptic activity through inhibition of protein kinase C signaling[END_REF] and mouse monoclonal anti actin (Sigma). The secondary antibodies used were anti-rabbit and anti-mouse antibodies coupled to 680 nm and 770 nm infrared dyes (Biotium). Laser scanning of blots and quantitative analyses were performed using the Odyssey Infrared Imaging System (Li-Cor).

Quantification of histone acetylation level was carried out by measuring the intensity of the signal corresponding to the acetylated histone normalized by the corresponding signal for total histone. Results were expressed as variation of acetylation compared to the mean level of control cells (uninfected or mock transfected/transduced), which was set to 1. Data are presented as means ± SEM. Statistical significance was determined using paired t-test.

Indirect immunofluorescence analysis. Cells were fixed with 2 % paraformaldehyde at room temperature for 10 min followed by 4 % paraformaldehyde for 10 min. They were washed 3 times in PBS (Invitrogen), and permeabilized with PBS 0.1 % Triton-X100 during 4 min. Cells were washed twice in PBS, blocked in 2.5 % normal goat serum (Invitrogen) during at least 1 h. Primary antibodies (the same used for western blot analyses described above) were diluted in PBS containing 2.5 % normal goat serum and incubated 1 h at room temperature. For controlling infection, transduction or transfection efficiencies, we used rabbit antisera raised against BDV N or BDV P proteins [START_REF] Volmer | Borna disease virus blocks potentiation of synaptic activity through inhibition of protein kinase C signaling[END_REF]. To assess neuronal integrity, we used a mouse anti bIII-Tubulin antibody (Sigma). Upon incubation with primary antibodies, cells were washed 3 times with PBS 0.1 % Triton-X100 and incubated with secondary goat anti rabbit antibody coupled to Alexa Fluor 488 or goat anti mouse antibody coupled to Alexa Fluor 594, during 1 h at room temperature. After 3 washes, coverslips were mounted onto microscope slides using ProLong Gold (Invitrogen) containing DAPI.

To measure histone acetylation levels, images were acquired on a LSM 510 confocal microscope (Zeiss) using fixed acquisition parameters. The Imaris software (Bitplane AG, Switzerland) was used to automatically calculate the mean density of fluorescence signals for individual cells, after background subtraction and thresholding. The same parameters were used for the quantification of each picture. To assess BDV N and P protein expression, images were acquired on a Zeiss Apotome microscope.

Analysis of HAT and HDAC activities.

Nuclear extracts were prepared using a commercially available kit (Active Motif) and protein concentrations were determined by Bradford assays. Equal amounts of each extracts were then used to measure HAT and HDAC enzymatic activities, by using the HAT Assay Kit and the HDAC Assay Kit (Active Motif). For each reaction, 2 µg of nuclear extracts were used for HAT assays and 4 µg of nuclear extracts for HDAC assays. Reactions were systematically carried out in duplicate for each extract. Revelation of the assay was done by fluorescence quantification using a Varioskan Flash scanner (Thermo Scientific).

Analysis of BDV multiplication and protein expression by flow cytometry. 4.10 5

Vero cells were plated and infected with cell-free BDV at a multiplicity of infection of 0.5. They were treated with 1 mM sodium butyrate (Sigma) or 10 µM anacardic acid (Tocris bioscience) the same day of infection and again 48 h later. After 4 days of incubation, cells were dissociated with trypsin, harvested, and fixed in 2 % paraformaldehyde for 15 min at room temperature. After fixation, cells were permeabilized during 20 min with FACS buffer containing 0.5 % saponin, 1 % bovine serum albumin and 3 mM EDTA. Cells were then incubated in blocking buffer (3 % pre-immune rabbit serum diluted in FACS buffer) during 1 h before addition of 5 µg/mL of antibodies for 1 h at 4 °C. Anti BDV P and isotype control antibodies were purified from rabbit sera (Abselect Serum Antibody Purification System, Innova Biosciences) and pre-conjugated with an Atto-488 fluorescent label (Lightning-Link Conjugation System, Innova Biosciences). After extensive washes, flow cytometry data were collected on a FACSCalibur (BD Biosciences) and analyzed using FlowJo software (TreeStar,version 8.8.6).

Reverse-transcription and quantitative PCR for measurement of vRNA. 10 5 Vero cells were plated and infected with cell-free BDV at a multiplicity of infection of 0.5. At different times post infection, total RNA were prepared from cells using the Nucleospin kit (Macherey-Nagel) following the manufacturer's instructions. One µg of RNA was reversetranscribed using SuperScript III Reverse Transcriptase (Life Technologies) with a primer specific for genomic viral RNA (primer sequence: 5'GACACTACGACGGGAACGAT3').

The resulting cDNAs were used for quantitative PCR (qPCR) experiments with primers specific for BDV genome (forward primer: 5'CGGTAGACCAGCTCCTGAAG3', reverse primer: 5'GAGGTCCACCTTCTCCATCA3'). In parallel, one µg of RNA was reversetranscribed using random hexamers to amplify total cDNAs. These cDNAs were used for qPCR using primers specific for glyceraldehyde 3 phosphate deshydrogenase (GAPDH) for normalization (forward primer: 5'CAATGACCCCTTCATTGACC3', reverse primer: 5'GACAAGCTTCCCGTTCTCAG3'). qPCR reactions were prepared with LightCycler 480 DNA SYBR Green I Master reaction mix (Roche Diagnostics) and run in duplicate on a LightCycler 480 instrument (Roche Diagnostics). Viral genome quantification was expressed in arbitrary units according to the following formula: amount of vRNA = 2 -(Ct BDV - Ct GAPDH) .

Results

BDV infection decreases histone acetylation of specific lysine residues. To assess the impact of BDV infection on histone acetylation patterns, primary neuronal cultures were infected or not by BDV. Neurons were cultured for 14 days, a time sufficient to allow BDV spreading to all neurons, as verified by indirect immunofluorescence analysis (data not shown) and in agreement with our previous proteomics findings [START_REF] Volmer | Borna disease virus blocks potentiation of synaptic activity through inhibition of protein kinase C signaling[END_REF][START_REF] Prat | Mutation of the protein kinase C site in Borna disease virus phosphoprotein abrogates viral interference with neuronal signaling and restores normal synaptic activity[END_REF]. There was no overt impact of infection on the morphology or viability of the cultures, consistent with the non-cytolytic replication strategy of BDV. From these neurons, we prepared histone fractions [START_REF] Shechter | Extraction, purification and analysis of histones[END_REF], which were analyzed by western blot using antibodies targeting each known acetylable lysine of H2B (Figure 1A, left panel). We observed that BDV infection led to a significant decrease of H2B acetylation on K5 and K20 (33 and 26 % decrease, respectively, Figure 1A), consistent with our previous findings [START_REF] Suberbielle | Proteomic analysis reveals selective impediment of neuronal remodeling upon Borna disease virus infection[END_REF]. In addition, infection also induced a decrease of acetylation on K15, but not on K12. We next asked whether this acetylation defect was restricted to H2B or could be extended to two other core histones, H3 and H4, for which acetylation modifications have been extensively described [START_REF] Kouzarides | Chromatin modifications and their function[END_REF]. While global levels of H3 acetylation were unchanged by infection, a significant decrease of the global acetylation level of H4 was observed in infected neurons (Figure 1B), which was attributable to decreased acetylation of K5, K8 and K16 but not of K12 (Figure 1C).

As BDV is able to infect a wide variety of cell types in vitro, even from non-neuronal origin, we next asked whether this histone acetylation decrease was also observed in other cell types. To this aim, histone acetylation levels were evaluated in Vero cells persistently infected by BDV (Vero-BV ( 24)). We observed that histone acetylation levels were also decreased in Vero-BV cells and that, remarkably, it occurred exactly on the same lysine residues than in primary neuronal cultures (Figure 2). Interestingly, changes were even more pronounced, reaching around 40 to 50 % decrease depending on the lysine, presumably resulting from the fact that Vero-BV cells had been infected for numerous passages. As for neurons, there was no change in acetylation of H2B K12 or H4 K12, neither in global H3 acetylation, suggesting that BDV may impact a general process conserved in several cell types that selectively targets the acetylation status of specific lysine residues of H2B and H4.

To confirm our findings concerning the impact of BDV infection on histone acetylation using a different approach, we performed indirect immunofluorescence analysis of histone acetylation using Vero and Vero-BV cells. As shown in Figure 3, this analysis revealed a general decrease of fluorescence levels in infected cells when we assessed H2B acetylation on K5, K15 and K20, but not for K12, in perfect agreement with our western blot results. This experiment also revealed that the decrease of acetylation was similar in all cells (Figure 3, right panels) and thus was a global effect resulting from BDV infection.

BDV P is responsible for histone acetylation decrease. We next assessed which of the BDV-encoded proteins could be responsible for this acetylation decrease. Among these, BDV N and BDV P represent potential candidates for decreasing histone acetylation. Indeed, they belong to the RNP complex and are abundant in the nucleus of infected cells, where they locate in close association with chromatin (1). To identify which of these two proteins could account for decreased histone acetylation, plasmids encoding BDV N or BDV P were transfected using the Amaxa technology that allows a high rate of transfection for primary neuronal cultures without altering viability. Fourteen days after transfection, we verified that neuronal integrity was indeed preserved using immunofluorescence analysis for ßIII-Tubulin. Moreover, at least 75 % of neurons expressed the transfected BDV protein (Figure 4A). We also observed that both BDV N and BDV P were expressed predominantly in the nucleus of transfected neurons, consistent with the presence of nuclear localization signals in both proteins [START_REF] Shoya | Two proline-rich nuclear localization signals in the amino-and carboxyl-terminal regions of the Borna disease virus phosphoprotein[END_REF][START_REF] Kobayashi | Nuclear targeting activity associated with the amino terminal region of the Borna disease virus nucleoprotein[END_REF] (Figure 4A). Analysis of histone acetylation levels in transfected neurons was compared to levels in neurons transfected with an empty plasmid. As shown in Figure 4B for the acetylation of H2B K20, we observed that the singled-out expression of BDV P into primary neurons led to decreased histone acetylation levels, a result that was not observed for BDV N-expressing neurons (Figures 4B and4C). This experiment was reproduced for each lysine residue analyzed previously and results are summarized in Table 1. Strikingly, the isolated expression of BDV P in neurons had the same impact on histone acetylation than BDV infection, with the same lysine specificity and at the same level of decrease. In order to verify if the same findings were observed in non-neuronal cells, we generated stable Vero cell lines expressing each of the viral candidate proteins, or GFP as a control, and analyzed histone acetylation (Table 2). Acetylation of the different lysine residues of H2B and H4 was drastically and significantly reduced only in the cell line expressing BDV P, confirming that BDV P is responsible for decreased histone acetylation in a non-neuronal cell line. These data indicate that BDV P is the key viral determinant responsible for the decrease of histone acetylation during BDV infection.

BDV P binding to HMGB-1 is not required for decreased histone acetylation. One feature of BDV P is its ability to bind chromatin via its interaction with HMGB-1, a nonhistone architectural protein of the chromatin [START_REF] Matsumoto | Bornavirus closely associates and segregates with host chromosomes to ensure persistent intranuclear infection[END_REF][START_REF] Kamitani | Borna disease virus phosphoprotein binds a neurite outgrowth factor, amphoterin/HMG-1[END_REF]. Given the important roles of HMGB-1 on chromatin dynamics [START_REF] Bustin | High-mobility-group chromosomal proteins: architectural components that facilitate chromatin function[END_REF], we next reasoned that the impact of BDV P on histone acetylation might be dependent on its interaction with HMGB-1. To test this hypothesis, we used a mutant form of BDV P, BDV PE84N, which no longer binds to HMGB-1 due to a mutation in its interaction domain (1). As described above, primary neuronal cultures were transfected with plasmids encoding wild-type BDV P, the BDV PE84N mutant, or an empty plasmid, and we next analyzed histone acetylation levels (Figure 5). Indirect immunofluorescence analysis indicated that the transfection efficiency was around 80 % and that the mutant BDV PE84N still localized into the nucleus (Figure 5A). When using H2B K20 acetylation as a read-out, we observed that acetylation was still inhibited in neurons expressing the BDV PE84N mutant (Figure 5B). Thus, interaction of BDV P with chromatin via its binding to HMGB-1 appears dispensable for the protein to exert its action on histone acetylation.

BDV P phosphorylation by PKC is essential for its action on histone acetylation.

Similarly to other non-segmented negative-strand RNA viruses, BDV P is a subunit of the viral polymerase complex and its activity is regulated upon phosphorylation by host kinases.

BDV P is preferentially phosphorylated at serine residues 26 and 28 by PKC and, to a lesser extent, at serine residues 70 and 86 by casein kinase II [START_REF] Schmid | Functional characterization of the major and minor phosphorylation sites of the P protein of Borna disease virus[END_REF][START_REF] Schwemmle | Borna disease virus P-protein is phosphorylated by protein kinase c-epsilon and casein kinase II[END_REF]. PKC phosphorylation is of particular interest for BDV pathogenesis, since we previously showed that BDV P acts as a PKC decoy substrate and impairs synaptic plasticity by diverting PKC from its natural targets in neurons [START_REF] Volmer | Borna disease virus blocks potentiation of synaptic activity through inhibition of protein kinase C signaling[END_REF][START_REF] Prat | Mutation of the protein kinase C site in Borna disease virus phosphoprotein abrogates viral interference with neuronal signaling and restores normal synaptic activity[END_REF]. Interestingly, PKC also regulates histone acetylation levels, notably by controlling activity of cellular histone acetyl transferases (HATs) [START_REF] Wang | CBP histone acetyltransferase activity regulates embryonic neural differentiation in the normal and Rubinstein-Taybi syndrome brain[END_REF][START_REF] Clarke | PKCbetaII augments NF-kappaB-dependent transcription at the CCL11 promoter via p300/CBP-associated factor recruitment and histone H4 acetylation[END_REF]. Thus, we next asked whether BDV P phosphorylation by PKC was required for its impact on histone acetylation.

To this aim, we used the PKC non-phosphorylable BDV PAA mutant, in which the two serine residues at positions 26 and 28 of BDV P had been replaced by alanine (A) residues [START_REF] Prat | Mutation of the protein kinase C site in Borna disease virus phosphoprotein abrogates viral interference with neuronal signaling and restores normal synaptic activity[END_REF]. We used transfected primary neuronal cultures (Figure 6A), as well as Vero cell lines stably expressing wild type or mutant BDV P (Figure 6B). In addition, we also used Vero cells persistently infected by recombinant BDV (rBDV) expressing wild type or mutant PAA (Figure 6C) [START_REF] Prat | Mutation of the protein kinase C site in Borna disease virus phosphoprotein abrogates viral interference with neuronal signaling and restores normal synaptic activity[END_REF]. In each case, BDV PAA subcellular localization was assessed by indirect immunofluorescence and was similar to that of the wild type protein (Figure 6, left panels).

Histone preparations from these different cells were analyzed for acetylation levels by western blot (Figure 6, right panels for H2B K20 acetylation). In all experimental models, the BDV PAA mutant had lost the ability to inhibit histone acetylation. Moreover, results were similar when measurement of acetylation was done with anti acetylated H2B K5 and anti pan acetylated H4 antibodies (data not shown). Therefore, phosphorylation of BDV P by PKC is mandatory to mediate decrease of histone acetylation.

Impaired histone acetylation due to BDV infection results from inhibition of neuronal

HAT activities. Histone acetylation is a dynamic, enzyme-regulated process, which results from the balance between activities of HATs and histone deacetylases (HDACs), two wide families of enzymes acting in concert [START_REF] Struhl | Histone acetylation and transcriptional regulatory mechanisms[END_REF][START_REF] Grunstein | Histone acetylation in chromatin structure and transcription[END_REF]. To examine whether BDV infection perturbs the equilibrium between these two families, enzymatic assays were performed to evaluate cellular HAT and HDAC activities, using nuclear extracts prepared from non-infected or infected primary neuronal cultures. Levels of HDAC activities were similar in nuclear preparations from infected or non-infected neurons (Figure 7A). In contrast, HAT activities were significantly decreased in nuclear extracts from BDV-infected neurons, reaching a level of about 25 % of decrease (Figure 7B). The reduced histone acetylation levels consecutive to BDV infection are thus due to inhibition of acetylation rather than increased deacetylation. We next assessed whether the BDV P expressed alone in primary cortical neurons could also inhibit the cellular HAT enzymatic activities. Nuclear extracts prepared from mock or BDV P transfected neurons were used to perform HAT enzymatic assays as described above (Figure 7C). Isolated expression of wild-type BDV P in neurons led to an inhibition of HAT activities at similar level than infection (around 25%). This effect was partly reverted, although not entirely, in neurons expressing the non-phosphorylable BDV PAA mutant (Figure 7C). Thus, the BDV P protein mediates inhibition of cellular HAT enzymatic activities and its phosphorylation by PKC may be implicated in this inhibition.

HAT and HDAC inhibitors modulate BDV replication. Having shown that BDV infection modifies the histone acetylation pattern by inhibiting cellular HAT activities, we

next investigated whether such modifications would help to create a more favorable milieu for viral replication. To examine the interplay between cellular acetylation levels and BDV replication, HAT and HDAC activities were pharmacologically modulated and the resulting consequences on BDV replication were followed at different times post-infection. We used sodium butyrate, an inhibitor of HDAC activities [START_REF] Candido | Sodium butyrate inhibits histone deacetylation in cultured cells[END_REF] and anacardic acid, an inhibitor of HAT activities [START_REF] Balasubramanyam | Small molecule modulators of histone acetyltransferase p300[END_REF] to treat Vero cells that were concomitantly infected by BDV. We first checked that the two drugs did not affect cell viability at the concentrations used during the experiments (data not shown). We also verified that both inhibitors indeed modulated histone acetylation levels in non-infected cells when compared to untreated cells (Figure 8A). Four days after onset of treatment, anacardic acid induced a 50 % decrease of H2B K20 acetylation compared to control cells (Figure 8A), a level similar to that observed during BDV infection (Figure 2). In contrast, sodium butyrate treatment led to a large increase (seven-fold) of H2B K20 acetylation.

To measure the impact of drug treatment on viral replication, Vero cells were harvested four days after infection and percentages of infected cells were determined by flow cytometry upon cell staining with an anti BDV P antibody. The data presented in

Discussion

The objective of this study was to gain insight on the outcome of the intra-nuclear persistence of a RNA virus, such as BDV, on the host cell chromatin dynamics. Here, we demonstrate that BDV infection leads to a significant decrease of histone acetylation on selected lysine residues. We also show that this decrease is due to inhibition of cellular HAT activities, mediated by the viral phosphoprotein, and that P phosphorylation by PKC plays a role in its effect. Finally we show that manipulation of host cell epigenetics leads to modulation of viral replication (Figure 9). These data illustrate a novel and fascinating example of virus/host cell interaction, which could be a strategy to favor long lasting BDV persistence. This strategy, different from classical latency, would rely on "self-restriction" of replication. This would prevent exhaustion of the host cell consecutive to an excessive recruitment of host factors, thus limiting cellular damage. This acetylation decrease, which could seem moderate at first, was however consistently observed both in primary neurons and established cell lines. The observed levels of reduction (around 30 %) are actually close to levels observed in neurons from mice knockout for the CREB Binding Protein (CBP) HAT, which display clear memory defects [START_REF] Barrett | Hippocampal focal knockout of CBP affects specific histone modifications, long-term potentiation, and long-term memory[END_REF][START_REF] Chen | CREB binding protein is required for both short-term and long-term memory formation[END_REF].

Interestingly, our observation that the exact same lysine residues were affected in all cell types argues for a conserved mechanism of inhibition in neuronal and non-neuronal cells.

Interestingly, decreases of histone acetylation were more important in persistently infected Vero cells than in neurons, infected for 14 days, suggesting that BDV persistence into the host cell may potentiate the inhibition of histone acetylation. A recent SILAC-based analysis of the impact of BDV infection on the proteome of human oligodendroglial cells also revealed changes in histone lysine acetylation patterns [START_REF] Liu | Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells[END_REF]. This study reported similar decreased values for certain lysine residues, but also many differences, in particular for H2B K15 for which an increase of acetylation was observed. It would be interesting to determine whether such differences are due to divergences in cell types or viral strains.

In our study, the decrease of histone acetylation due to BDV infection appeared to be restricted to specific H2B and H4 lysine residues. It would be interesting in the future to determine their genome-wide distribution, similar to what has been reported for adenovirus E1a protein [START_REF] Ferrari | Reorganization of the host epigenome by a viral oncogene[END_REF]. In this case, ChIP-sequencing experiments revealed that the global decrease of acetylation on H3 K18 observed during infection was correlated to a redistribution of acetylation at the genome scale, with enrichment in promoter regions of genes needed for cell replication.

Our results unambiguously demonstrated that BDV P, by inhibiting cellular HAT activities, was responsible for decreased histone acetylation, even when expressed alone, thereby pointing to a new role for this multifunctional protein that lies at the crossroads between BDV and numerous cellular pathways [START_REF] Prat | Mutation of the protein kinase C site in Borna disease virus phosphoprotein abrogates viral interference with neuronal signaling and restores normal synaptic activity[END_REF][START_REF] Planz | Borna disease virus: a unique pathogen and its interaction with intracellular signalling pathways[END_REF][START_REF] Peng | Borna disease virus p protein affects neural transmission through interactions with gammaaminobutyric acid receptor-associated protein[END_REF]. Interestingly, the impact of BDV P on histone acetylation appears to be dissociated from its interaction with chromatin, in particular from its binding to HMGB-1, as revealed by the use of the BDV PE84N mutant, although we did not formally test that this was true for all lysine residues affected by BDV and only used H2B K20 as a read-out. This fact is consistent with our recent results using real-time live cell imaging, which showed that BDV P is actually highly mobile in vSPOTs, suggesting a labile and transitory interaction with chromatin that may not be sufficient to directly interfere with chromatin dynamics [START_REF] Charlier | Analysis of borna disease virus trafficking in live infected cells by using a virus encoding a tetracysteine-tagged p protein[END_REF].

In contrast, invalidation of the PKC phosphorylation sites on BDV P completely abrogated its impact on histone acetylation and, at least partially, on HAT activities. This may be due to a direct interference of phosphorylated P on HAT activities. Alternatively, BDV P could serve as a PKC decoy substrate, in agreement with our previous findings [START_REF] Volmer | Borna disease virus blocks potentiation of synaptic activity through inhibition of protein kinase C signaling[END_REF][START_REF] Prat | Mutation of the protein kinase C site in Borna disease virus phosphoprotein abrogates viral interference with neuronal signaling and restores normal synaptic activity[END_REF] and divert PKC from one or several HATs. Indeed it is known that the enzymatic activities of several HATs can be positively or negatively regulated by PKC [START_REF] Wang | CBP histone acetyltransferase activity regulates embryonic neural differentiation in the normal and Rubinstein-Taybi syndrome brain[END_REF][START_REF] He | Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein[END_REF][START_REF] Wang | Metformin activates an atypical PKC-CBP pathway to promote neurogenesis and enhance spatial memory formation[END_REF][START_REF] Yuan | Inhibition of histone acetyltransferase function of p300 by PKCdelta[END_REF][START_REF] Jin | alpha-Synuclein negatively regulates protein kinase Cdelta expression to suppress apoptosis in dopaminergic neurons by reducing p300 histone acetyltransferase activity[END_REF]. Such a mechanism would differ from what has been described for other viruses such as adenovirus, for which the viral protein E1a directly binds the CBP/p300 HAT [START_REF] Horwitz | Adenovirus small e1a alters global patterns of histone modification[END_REF]. Another example is influenza virus, for which it was shown that the non-structural protein-1 possesses a histone-like sequence that serves as mimic substrate for the Tip60 HAT [START_REF] Marazzi | Suppression of the antiviral response by an influenza histone mimic[END_REF].

Since restoration of HAT activities were not complete with the PAA mutant, we thus cannot formally exclude the possibility that other functions controlled by P may also play a role.

The characterization of which HAT(s) are actually impacted by BDV P will be challenging. Only little is known concerning the post-translational modifications of most HATs, which are often very large proteins with many potentially modifiable residues [START_REF] Legube | Regulating histone acetyltransferases and deacetylases[END_REF].

Moreover, although PKC can phosphorylate several HATs [START_REF] Wang | Metformin activates an atypical PKC-CBP pathway to promote neurogenesis and enhance spatial memory formation[END_REF][START_REF] Yuan | Inhibition of histone acetyltransferase function of p300 by PKCdelta[END_REF], the resulting impact on their activities, combined with other post-translational modifications, is poorly understood.

The fact that the pattern of impaired histone lysine acetylation was remarkably similar amongst cell types could provide some clues for a future identification of the HATs, although most of them present a broad specificity for their substrates and could thus virtually acetylate the vast majority of histone lysine residues [START_REF] Kouzarides | Chromatin modifications and their function[END_REF].

The role of BDV P phosphorylation in viral replication is still incompletely understood.

Phosphorylation mutants of BDV P display increased polymerase activity in minireplicon assays, whereas they induce cell-type dependent impaired viral spread, leading to the hypothesis that P might play multiple and contrasted roles in the viral cycle [START_REF] Schmid | Protein kinase C-dependent phosphorylation of Borna disease virus P protein is required for efficient viral spread[END_REF][START_REF] Schmid | Functional characterization of the major and minor phosphorylation sites of the P protein of Borna disease virus[END_REF].

Similarly, it has been shown that phosphorylation of Ebolavirus VP30 protein plays opposite roles in viral replication and transcription [START_REF] Biedenkopf | Phosphorylation of Ebola virus VP30 influences the composition of the viral nucleocapsid complex: impact on viral transcription and replication[END_REF]. Here, we demonstrate that the PAA mutant protein has lost the capacity to reduce histone acetylation, whereas it has been shown to increase viral RNA polymerase activity [START_REF] Schmid | Protein kinase C-dependent phosphorylation of Borna disease virus P protein is required for efficient viral spread[END_REF][START_REF] Schmid | Functional characterization of the major and minor phosphorylation sites of the P protein of Borna disease virus[END_REF]. These results are thus in agreement with our findings that BDV replication is favored when level of cellular histone acetylation is higher.

Finally, epigenetic modifications, in particular histone acetylation, have now emerged as central regulators of cognitive processes during learning and memory [START_REF] Peixoto | The role of histone acetylation in memory formation and cognitive impairments[END_REF][START_REF] Borrelli | Decoding the epigenetic language of neuronal plasticity[END_REF]. Hence, impaired histone acetylation occurring in the brain of BDV-infected animals may participate to behavioral perturbations observed in BDV-infected hosts (4). We are currently assessing the impact on cognition of the isolated expression of BDV P in neurons in order to expand results obtained in transgenic mice expressing BDV P in astrocytes [START_REF] Kamitani | Glial expression of Borna disease virus phosphoprotein induces behavioral and neurological abnormalities in transgenic mice[END_REF]. These data may reveal for the first time that an animal virus, or a singled-out viral protein, can perturb cognitive functions by interfering with PKC-dependent and neuronal epigenetic signaling pathways. Merged images are shown on the right. Bar = 50 µm. Right panels: comparative analysis of H2B K20 acetylation levels in cells expressing wild-type BDV P or the BDV PAA mutant. In all cases, acetylation levels were normalized to levels obtained for control conditions, which were set to 1 and which are indicated by a dashed line on the graphs. Data are expressed as means ± SEM from at least four independent experiments (i.e., independent sets of cultures of transfected neurons or cultures of Vero cells that were independently grown after the original split during 5 to 20 passages). **, P £ 0.01 by paired t test.

  Lentiviral vector plasmids allowing expression of BDV N or wild type or mutant BDV P under the control of the CAG promoter were engineered by insertion of viral gene sequences into BamHI/XhoI restriction sites of the pTrip-CAG plasmid (kind gift of Dr Charneau). Non-replicative vector particles were produced by transient transfection of HEK-293T cells with each vector plasmid, the second generation psPAX2 packaging plasmid (Addgene plasmid 12260, provided by D. Trono) and pMD2.G plasmid encoding the vesicular stomatitis virus envelope glycoprotein (VSV-G, Addgene plasmid 12259, provided by D. Trono). Particles were concentrated from cell culture supernatants by ultracentrifugation. Gene transfer titers were determined on transduced HEK-293T cells by indirect immunofluorescence using anti-BDV N or P protein sera.

Figure 8B ,

 8B Figure 8B, representative for three independent experiments, indicate that without drug treatment, 75 % of cells were positive for BDV P (Figure 8B, up panel). The percentage of infected cells dropped at 62 % upon treatment with anacardic acid, indicating that viral dissemination into the culture was reduced compared to control cells (Figure 8B, down panel). Moreover, the mean fluorescence intensity value (MFI), reflecting the amount of

Figure 1 .

 1 Figure 1. BDV infection decreases histone acetylation levels in primary neuronal cultures. A) Western blot analysis of H2B acetylation levels. Left panel: histone fractions were prepared from parallel non-infected (NI) or infected (BDV) neuronal cultures and analyzed by western blot with the indicated antibodies. Total H2B levels were assessed for each sample and used to normalize acetylation signals. Right panel: Quantification of H2B acetylation. Acetylation values (normalized for total H2B) obtained for infected neurons were normalized to values obtained for non-infected neurons, which was set to 1 and which is represented by the dashed line on the graph. Data are expressed as means ± SEM from at least nine independent sets of neuronal cultures. *, P £ 0.05; **, P £ 0.01; ***, P £ 0.001; ns = non significant, by paired t test. B) Analysis of global H3 and H4 acetylation by western blot, done as in A). C) Analysis of acetylation on each H4 lysine residues by western blot, done as in A).

Figure 2 .

 2 Figure 2. BDV infection decreases histone acetylation levels in persistently infected Vero cells. A) Analysis of H2B acetylation. Left panel: histone fractions were prepared from parallel non-infected (NI) or persistently infected (BDV) Vero cells and analyzed by western blot with the indicated antibodies. Total H2B levels were assessed for each sample and used to normalize acetylation signals. Right panel: Quantification of H2B acetylation. Acetylation values (normalized for total H2B) obtained for infected cells were normalized to values obtained for non-infected cells, which was set to 1 and which is represented by the dashed line on the graph. Data are expressed as means ± SEM from at least seven independent experiments, i.e., cultures of Vero cells that were independently grown after the original split during 5 to 20 passages. *, P £ 0.05; **, P £ 0.01; ***, P £ 0.001; ns = non significant, by paired t test. B) Analysis of H3 and H4 global acetylation by western blot, done as in A). C) Western blot analysis of acetylation on each H4 lysine residues, done as in A).

Figure 3 .

 3 Figure 3. Histone acetylation decrease due to BDV affects all infected cells.Immunofluorescence analysis was performed on non-infected (NI, left column) or persistently infected (BDV, right column) Vero cells, in order to visualize acetylation on

Figure 4 .

 4 Figure 4. The BDV P protein is responsible for decreased histone acetylation levels. A) Indirect immunofluorescence analysis of primary neurons 14 days post-transfection. Neurons were stained for DAPI, the viral BDV P or N proteins (green) and the neuronal marker bIII-tubulin (red). Merged images are shown on the right panels. Bar = 50 µm. B)Western blot analysis, performed on nuclear extracts to visualize expression levels of BDV P or N proteins (top panel; Empty = neurons transfected with an empty vector), and on histone extracts to assess the level of K20 acetylated and total H2B (lower panels). C) Quantification of H2B K20 acetylation levels in transfected neurons. Acetylation levels (relative to total H2B) were normalized to values obtained for neurons transfected by an empty plasmid, which was set to 1. Data are expressed as means ± SEM obtained from at least five independent sets of transfected neuronal cultures. **, P £ 0.01, by paired t test.

Figure 5 .

 5 Figure 5. BDV P binding to HMGB-1 is dispensable for its impact on histone acetylation. A) Indirect immunofluorescence analysis of primary neurons 14 days posttransfection. Neurons were stained for DAPI, the wild-type BDV P or PE84N mutant protein (green) and the neuronal marker bIII-tubulin (red). Merged images are shown on the right panel. Bar = 50 µm. B) Quantification of H2B K20 acetylation levels. Acetylation levels (relative to total H2B) were normalized to values obtained for neurons transfected by an empty plasmid, which was set to 1. Data are expressed as means ± SEM from six independent sets of transfected neuronal cultures. *, P£ 0.05; **, P £ 0.01, by paired t test.

Figure 6 .

 6 Figure 6. Mutation of the PKC phosphorylation sites of BDV P abrogates its action on histone acetylation. This analysis was performed in: A) transfected primary neuronal cultures (control: empty vector), B) Vero cells stably expressing isolated proteins (control: cell line expressing GFP) and C) Vero cells persistently infected by a recombinant BDV (rBDV) wild type or encoding the mutant PAA protein (control: non infected cells). Left

Figure 7 .

 7 Figure 7. BDV infection and BDV P inhibit neuronal HAT activities. Enzymatic assays for dosing HDAC (A) and HAT (B and C) activities were performed using nuclear extracts prepared from: non-infected (NI) or infected (BDV) primary neuronal cultures (A and B); neurons transfected with an empty plasmid or plasmids encoding wild type BDV P or BDV PAA mutant (C). Enzymatic activities measured as described in the methods are expressed as arbitrary units (A. U.). Data are the means ± SEM from at least four independent sets of neuronal cultures. **, P £ 0.01; *, P £ 0.05, ns = non significant, by paired t test.

Figure 8 :

 8 Figure 8: HAT and HDAC inhibitors interfere with BDV replication. Vero cells wereinfected by BDV at a multiplicity of infection of 0.5, treated with 1 mM sodium butyrate (NaBu) or 10 µM anacardic acid and followed daily for four days. Six independent sets of de novo infection of Vero cells with cell-free BDV were performed. Out of these, three were used for flow cytometry analysis and three to prepare protein/RNA extracts for western blots and RT-qPCR. A) Impact of a 4-day treatment on H2B K20 acetylation levels using noninfected cells. Acetylation levels (relative to total H2B) measured in treated cells were normalized to values obtained for non-treated cells, which was set to 1. B) Flow cytometrybased analysis of BDV spread in cell culture. Cells were stained with either control rabbit IgG (black line) or rabbit anti-BDV P polyclonal IgG antibodies (grey full histogram).Percentages of BDV P-positive cells and mean fluorescence intensities of this population(MFI) are reported on the top and in the right bottom corner of the graphs, respectively. One representative experiment out of three giving similar results is shown. C) Time-course western-blot analysis (from day 1 (d1) to day 4 (d4)) of viral protein levels in cells that were either left non-treated (NT), or treated with NaBu or anacardic acid (AA). The different

Figure 9 :

 9 Figure 9: A hypothetical model of BDV P-mediated manipulation of epigenetic signaling. During infection, BDV P decreases cellular HAT activities, partly upon its phosphorylation by PKC, thereby resulting in lowered histone acetylation levels. Such reduced histone acetylation levels would, in turn, lead to a reduced viral replication.

Acknowledgments

We thank the staff of the different facilities of our research center (imaging and flow cytometry platforms) for excellent assistance that was instrumental for the proper realization of this project. We thank D. Trono and P. Charneau for lentiviral vector plasmids. We are grateful to Stéphane Chavanas, Roland Liblau and Abdelhadi Saoudi for critical reading of the manuscript and insightful comments.

Funding

This work was supported by grants from ANR (ANR-10-Blanc-1322, to DGD), the Fondation pour la Recherche Médicale (Equipe FRM 2009, to DGD), a grant from the Toulouse III University research council (to CEM) and institutional grants from Inserm and CNRS (to DGD). This work is part of the Ph.D. thesis work of EMB, who was supported by a doctoral fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche.

Author Contributions