Sophie Sagnier

Coralie F Daussy

Sophie Borel

Véronique Robert-Hebmann

Mathias Faure

Fabien P Blanchet

Bruno Beaumelle

Martine Biard-Piechaczyk

Lucile Espert
email: lucile.espert@cpbs.cnrs.fr

Autophagy restricts HIV-1 infection by selectively degrading Tat in CD4 + T lymphocytes Running title: HIV-1 Tat is selectively degraded by autophagy

Autophagy is an ubiquitous mechanism involved in the lysosomal-mediated degradation of cellular components when engulfed in vacuoles called autophagosomes. Autophagy is also recognized as an important regulator of the innate and adaptive immune responses against numerous pathogens, which have, therefore, developed strategies to block or use the autophagy machinery to their own benefit.

Upon Human Immunodeficiency Virus-1 (HIV-1) infection, viral envelope glycoproteins (Env) induce autophagy-dependent apoptosis of uninfected bystander CD4 + T lymphocytes, a mechanism likely contributing to the loss of CD4 + T cells. In contrast, in productively infected CD4 + T cells, HIV-1 is able to block Env-induced autophagy in order to avoid its antiviral effect. To date, nothing is known about how autophagy restricts HIV-1 infection in CD4 + T lymphocytes. Here we report that autophagy selectively degrades the HIV-1 transactivator Tat, a protein essential for viral transcription and virion production. We demonstrated that this selective autophagy-mediated degradation of Tat relies on its ubiquitin-independent interaction with the p62/SQSTM1 adaptor. Taken together, our results provide evidence that the anti-HIV effect of autophagy is specifically due to the degradation of the viral transactivator Tat, but this process is rapidly counteracted by the virus to favor its replication and spread. autophagy restricts HIV-1 infection remain to be elucidated. Here, we report that the HIV-1 transactivator Tat, a protein essential for viral replication, is specifically degraded by autophagy in CD4 + T lymphocytes. Both Tat present in infected cells and incoming Tat secreted from infected cells are targeted for autophagy degradation through an ubiquitinindependent interaction with the autophagy receptor p62/SQSTM1. This study is the first to demonstrate that selective autophagy can be an anti-viral process by degrading a viral transactivator. In addition, the results could help in the design of new therapies against HIV-1 by specifically targeting this mechanism.

IMPORTANCE

Autophagy is recognized as one of the most ancient and conserved mechanism of cellular defense against invading pathogens. Crosstalks between HIV-1 and autophagy have been demonstrated depending on the virally challenged cell type, and HIV-1 has evolved strategies to block this process to replicate efficiently. However, the mechanisms by which

INTRODUCTION

Macroautophagy, herein referred to as autophagy, is a major cellular catabolic pathway highly regulated in eukaryotes. It is involved in the degradation of cytoplasmic material after its sequestration in vacuoles called autophagosomes. The autophagosomes fuse with lysosomes to form autolysosomes in which the sequestered material is degraded and then recycled [START_REF] Tanida | Autophagy basics[END_REF]. Since the discovery of the Atg genes that regulate this process, autophagy has been involved in a number of important cellular functions including cellular homeostasis, development, aging or innate and adaptive immune responses [START_REF] Suzuki | Interrelationships among Atg proteins during autophagy in Saccharomyces cerevisiae[END_REF][START_REF] Uchiyama | Autophagyphysiology and pathophysiology[END_REF]. Autophagy is believed to be one of the most ancient defense processes against invading pathogens. Its antiviral effect has been described in many studies through different mechanisms including a direct degradation of cytoplasmic viral components as for example shown for the Sindbis virus (SIN) capsids that are specifically targeted to autophagy upon interaction with p62 [START_REF] Orvedahl | Autophagy protects against Sindbis virus infection of the central nervous system[END_REF][START_REF] Sumpter | Selective autophagy and viruses[END_REF].

Importantly, pathogens have evolved different means to inhibit or use autophagy to their own profit [START_REF] Deretic | Autophagy in infection, inflammation and immunity[END_REF].

At the molecular level, two signaling complexes are involved in the induction, the elongation and the closure steps of autophagy, leading to the formation of autophagosomes. Briefly, the class III PI3Kinase, associated with p150 and Beclin 1, is responsible for the formation of the phagophore. Two ubiquitination-related conjugation systems, leading to the formation of the Atg12-Atg5-Atg16L complex and the Atg8-Phosphatidylethanolamine (PE) complex, are required for the elongation and closure of the autophagosome. These two conjugates are formed upon the action of a unique E1-activating enzyme called Atg7. ATG8-PE is inserted in the autophagic vacuole membranes and is present all along the pathway, a characteristic that makes it an autophagosomal marker. As autophagy proceeds, ATG8-PE is finally degraded in autolysosomes. Six orthologs of ATG8 exist in mammals, three microtubule-associated protein 1 light chain 3 (LC3) proteins (LC3A, B, and C), one gammaaminobutyrate receptor-associated protein (GABARAP) and two GABARAP-like proteins (GABARAPL1 and GATE16/GABARAPL2). All these proteins are synthesized as precursors that are rapidly processed at their C-terminus, leading to the exposure of a glycine residue that can be conjugated to PE [START_REF] Pyo | Molecules and their functions in autophagy[END_REF][START_REF] Weidberg | Mammalian Atg8s: one is simply not enough[END_REF][START_REF] Weidberg | LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis[END_REF]. LC3B is still the most extensively studied ATG8 protein and will be hereafter referred to as LC3.

Autophagy can be a very selective process by the action of adaptor proteins behaving as autophagy cargo receptors, themselves degraded by autophagy due to their interaction with LC3 [START_REF] Johansen | Selective autophagy mediated by autophagic adapter proteins[END_REF]. Autophagy cargo receptors share at least one domain, the LIR domain (LC3-Interacting Region), allowing interaction with ATG8 family members and thus targeting the cargos to autophagosomes [START_REF] Birgisdottir | The LIR motif -crucial for selective autophagy[END_REF]. p62/SQSTM1 (sequestosome 1, hereafter called p62) is a typical autophagy receptor that interacts with ubiquitinated substrates via its UBA domain (UBiquitin Associated domain) and that is able to multimerize via its PB1 domain (NH 2terminal Phox and Bem1p domain). These two motifs are important for selective autophagic degradation of ubiquitinated substrates [START_REF] Johansen | Selective autophagy mediated by autophagic adapter proteins[END_REF][START_REF] Bjorkoy | p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death[END_REF][START_REF] Bjorkoy | p62/SQSTM1: a missing link between protein aggregates and the autophagy machinery[END_REF][START_REF] Pankiv | p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[END_REF]. Of note, p62 has been involved in the lysosomal-mediated degradation of incoming pathogens, a process termed "xenophagy" [START_REF] Ishimura | Dissection of the role of p62/Sqstm1 in activation of Nrf2 during xenophagy[END_REF][START_REF] Knodler | Eating the strangers within: host control of intracellular bacteria via xenophagy[END_REF].

Type 1 Human Immunodeficiency Virus (HIV-1) infects immune cells, mainly CD4 + T lymphocytes and macrophages. In most instances, viral entry is mediated by the interaction of the viral envelope glycoproteins (gp120 and gp41, called Env) with CD4 and a co-receptor, mainly CCR5 or CXCR4, expressed at the surface of the target cells, to initiate a membrane fusion event. The gp120 interacts first with CD4, triggering conformational changes leading to increased exposure of gp120 regions able to bind to the co-receptor. This interaction induces a structural rearrangement in gp41 and the insertion of its N-terminus fusion peptide into the target cell membrane, leading to the fusion between viral and cell membranes and thus to viral entry [START_REF] Arrildt | The HIV-1 env protein: a coat of many colors[END_REF]. The RNA viral genome is then retro-transcribed in DNA by the viral reverse transcriptase and is transported to the nucleus where it integrates the genome of the host cell by the action of the viral integrase. After the integration step, the viral transactivator Tat enables the transcription of the viral DNA, leading to the production of the viral components necessary to synthesize new infectious particles that are released from the infected cells. The HIV-1 replication cycle can thus be divided in two phases: the early phase, from viral entry to provirus integration, and the late phase, from transcription of viral genes to the release of new viral particles [START_REF] Freed | The cell biology of HIV-1 and other retroviruses[END_REF][START_REF] Mohammadi | 24 hours in the life of HIV-1 in a T cell line[END_REF].

Tat is a small HIV-1 protein of 80 to 103 amino acids, depending on the viral strain, necessary for the transcription of all the viral genes after its binding to the TAR RNA loop present in the HIV-1 Long Terminal Repeat (LTR) promoter [START_REF] Ott | The control of HIV transcription: keeping RNA polymerase II on track[END_REF]. In addition to its role in infected cells, Tat can be secreted and captured by bystander cells where it can induce apoptosis or modulate the expression of cellular proteins, in particular pro-inflammatory cytokines [START_REF] Debaisieux | The ins and outs of HIV-1 Tat[END_REF][START_REF] Huigen | Multiple effects of HIV-1 trans-activator protein on the pathogenesis of HIV-1 infection[END_REF][START_REF] Rayne | The ability of chloroquine to prevent tat-induced cytokine secretion by monocytes is implicated in its in vivo anti-human immunodeficiency virus type 1 activity[END_REF][START_REF] Vendeville | HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses[END_REF].

Complex links exist between HIV-1 and autophagy, depending on the cell types and on the infectious status of the target cell type, i.e. if the target cell is productively infected or not [START_REF] Biard-Piechaczyk | HIV-1, ubiquitin and ubiquitin-like proteins: the dialectic interactions of a virus with a sophisticated network of post-translational modifications[END_REF][START_REF] Borel | Macroautophagy Regulation during HIV-1 Infection of CD4+ T Cells and Macrophages[END_REF][START_REF] Espert | Autophagy in HIV-induced T cell death[END_REF]. In dendritic cells, HIV-1 is able to block autophagy, impeding viral antigen presentation [START_REF] Blanchet | Human immunodeficiency virus-1 inhibition of immunoamphisomes in dendritic cells impairs early innate and adaptive immune responses[END_REF][START_REF] Blanchet | Immunoamphisomes in dendritic cells amplify TLR signaling and enhance exogenous antigen presentation on MHC-II[END_REF]. In CD4 + T lymphocytes, we previously demonstrated that autophagy is induced by the gp41 fusogenic function and that this process leads to apoptosis of uninfected bystander CD4 + T cells, i.e. cells that have been in contact with infected cells without being productively infected [START_REF] Denizot | HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells[END_REF][START_REF] Espert | Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4[END_REF]. In contrast, autophagy is blocked in productively infected CD4 + T cells [START_REF] Espert | Differential role of autophagy in CD4 T cells and macrophages during X4 and R5 HIV-1 infection[END_REF].

In macrophages that have been challenged by the virus but not productively infected, autophagy is inhibited via the activation of Stat3 and Src-Akt by Tat and IL10 [START_REF] Van Grol | HIV-1 inhibits autophagy in bystander macrophage/monocytic cells through Src-Akt and STAT3[END_REF]. HIV-1 Tat also suppresses STAT1 activation triggered by IFN in monocytes and IFN-induced autophagy in primary macrophages, suggesting that inhibition of autophagy could impair the immune defenses by blocking the antigen processing required for the recognition and killing of intracellular pathogens [START_REF] Li | HIV-1 trans-activator protein dysregulates IFN-gamma signaling and contributes to the suppression of autophagy induction[END_REF]. These results are consistent with the fact that macrophages do not undergo Env-mediated apoptosis and are not subjected to depletion during HIV-1 infection.

The mechanism by which autophagy exerts its restriction activity against HIV-1 infection in CD4 + T cells remains to be fully characterized. We demonstrate here that p62 interacts with Tat and targets it to lysosomal-mediated degradation via selective autophagy. This interaction is not mediated by a conventional domain of p62 since the UBA or the PB1 domains of this protein are not involved. Interestingly, both neo-synthesized Tat in infected cells and exogenous Tat internalized by uninfected cells are sensitive to autophagic degradation, indicating that all the functions of Tat could be counteracted by autophagy. In conclusion, we propose that degradation of the viral transactivator Tat by selective autophagy is a not yet appreciated potent antiviral cellular mechanism against HIV-1 infection of CD4 + T lymphocytes. Plasmids. The pNL4-3 molecular clone has been obtained from the NIH AIDS Reagent Program. The different p62 constructs, cloned in a pENTR plasmid, were a kind gift from Dr Terje Johansen (University of Tromsø, Tromsø, Norway). After a LR clonase reaction, we have cloned the different p62 cDNA in pDEST vectors in order to obtain GST-fused proteins, using the Gateway® Cloning Technology (Life Technologies). The LC3B cDNA was first cloned in a pENTR vector and then cloned in a pDEST vector using the Gateway® Cloning Technology (Life Technologies). The GST-LC3F52A mutant was obtained by mutagenesis using the Quickchange kit from Stratagene. The FLAG-tagged Tat (86 residues; BH10 isolate) has been cloned in the pBi-GL expression vector under the control of a tetracyclin response element (TRE) (The FLAG is placed at the C-terminal part of Tat). To express the gene of interest, the plasmid needs to be co-transfected with a pUHD-neo vector responsible for the expression of the tTA regulatory proteins needed to transactivate the TRE. The pBi-Tat K50-51A mutant, expressing the FLAG-TatK50-51A mutant, was generated from the pBi-Tat using the Quickchange kit from Stratagene [START_REF] Rayne | Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF]. The FLAG-Tat K71R expression vector was a kind gift from Monsef Benkirane (as for pBi-GL-Tat, the FLAG tag is located at the C-terminus of Tat) [START_REF] Bres | A non-proteolytic role for ubiquitin in Tat-mediated transactivation of the HIV-1 promoter[END_REF].

MATERIALS AND METHODS

Ethics

The different constructs expressing Vif, Nef and Vpr were cloned in a pENTR plasmid and transfered in a pCI-neo3xFLAG expression vector after a LR clonase reaction using the Gateway® Cloning Technology (Life Technologies). The expression vector coding for GFP-LC3 was kindly provided by T. Yoshimori (Osaka University, Japan) and R. Willey (NIAID, NIH). The expression vector coding for Gag was kindly provided by Nathalie Chazal (CPBS, Montpellier) [START_REF] Schneider | Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev-independent expression of Gag and Gag/protease and particle formation[END_REF]. TEM analysis. Cells were fixed in situ with 2.5% glutaraldehyde in cacodylate buffer (pH 7.4) for 60 min at 4°C, post-fixed with 2% osmium tetroxide, then washed in cacodylate buffer containing 0.5% tanic acid. After extensive washes in 0.1 M Sorensen phosphate buffer (pH 7.2), cells were included in a fibrin clot as described by Charret and Fauré-Fremiet (38). Cells were then post-fixed with 2% osmium tetroxide and 0.5% tannic acid, dehydrated, and embedded in epon (Embed-812, Electron Microscopy Sciences Inc.). Sections were counterstained with uranyl acetate and lead citrate and examined with a Hitachi H7100 TEM.

HIV-1 infection and production. To produce HIV virions, HEK cells were transfected with pNL4-3 and supernatants were collected 2 days post-transfection. Viral particles were concentrated by ultracentrifugation. Jurkat cells were then challenged for 24h with dilutions of the concentrated viruses. Flow cytometry analysis of Gag + cells was performed in order to quantify the multiplicity of infection (MOI). Quantification of the viral particles production was performed with a p24 Enzyme-linked immunosorbent assay from "Innogenetics" (Innotest® HIV antigen mAb) according to the manufacturer's instructions. CD4 + T cells were infected with concentrated viruses at a MOI comprised between 0.1 and 1. Co-culture experiments were done with chronically HIV-infected (MOLT-X4) or uninfected T cells (MOLT) co-incubated for indicated times with HEK/CD4/CXCR4 or MAGIC5B cells.

Quantification of HIV infection of target cells was measured by flow cytometry (for HEK cells) or β-galactosidase activity assay (for MAGIC5B cells).

GST pull-down experiments. Constructs expressing GST proteins fused to wild type LC3

(GST-LC3), mutated LC3 (GST-LC3 F52A), wild type p62 (GST-p62) or mutated p62 (GST-p62 ΔLIR, GST-p62 ΔUBA and GST-p62 ΔPB1) were co-transfected in HEK cells with the pBi-FLAG -Tat and pUHD vectors. Corresponding empty vectors were used as controls. Anti-proteases (E64d + Pepstatin A) were added to the transfected cells in order to optimize the expression of Tat. Cell lysis was performed 24h post-transfection in 150 mM NaCl, 20 mM Tris pH 7 and 0.5% NP40 at 4°C. After centrifugation for 20 min at 4°C, the lysates were incubated with glutathione-conjugated sepharose beads for 2h at 4°C. Unbound proteins were removed by 4 washes with cold lysis buffer. Bound proteins were then eluted by boiling for 10 min in Laemmli buffer and separated by gel electrophoresis followed by immunoblotting with specific antibodies.

Flow cytometry

The percentage of HIV-1-infected cells was determined by quantifying the cellular levels of p24 gag by flow cytometry using the HIV KC57 FITC kit according to the manufacturer's instructions (Beckman Coulter). Briefly, cells are fixed, permealized and the anti-p24 antibody is added to the cells. After staining for 30 minutes, cells were washed with PBS, and fluorescence intensity was measured on a COULTER EPICS XL Flow Cytometer (Beckman Coulter).

β-galactosidase assay. Chemiluminescent reporter assay system "Galacto-Star™ β-Galactosidase Reporter Gene Assay System for Mammalian Cells", from Life Technologies, was used to quantitate ß-galactosidase activity according to manufacturer's instruction. Briefly, 0.5x10 6 MAGIC5B cells were lysed in 200µl of lysis buffer provided by the manufacturer. 50µl of cell lysate was used in each assay, in duplicates. The results were normalized by quantifying the total protein concentrations using the Bradford assay from Sigma-Aldrich.

Transfections. 0.5x10 6 HEK or HEK/CD4/CXCR4 cells were cultured in 6-well plates 24h prior to transfection. Plasmids were then transfected using the TurboFect reagent (Fermentas) according to the manufacturer's instructions. Smartpool p62 siRNA and control siRNA were from ABgene. Beclin 1 siRNA and Atg7 siRNA were synthetized and annealed by Eurogentec.

The mRNA sequences to be targeted for Beclin 1 and Atg7 siRNA were 5'-CAGUUUGGCACAAUCAAUA-3' and 5'-GCAUCAUCUUCGAAGUGAA-3', respectively.

Transfection of siRNA into HEK/CD4/CXCR4 or MAGIC5B cells (0.5.10 6 cells maintained in 6well plates) was performed with the LipoRNAiMax reagent (Invitrogen) according to the manufacturer's instructions. In the case of siRNA and plasmid co-transfections, 0.25x10 6 HEK cells were first transfected with siRNA using the LipoRNAiMax reagent and, 24h later, siRNAtransfected cells were transfected with the plasmid using the TurboFect reagent.

Western blot. Cell lysates were loaded in a 12% Prosieve™ 50 gels (Lonza) and transferred to PVDF membranes. After a blocking step for 1h at room temperature in PBS containing 0.5% casein, membranes were incubated overnight at 4°C with the primary antibody (Ab) in the blocking buffer. After 3 washes with PBS supplemented with 0.05% Tween, the membranes were incubated for 1h at room temperature with peroxidase-coupled secondary Ab. Upon extensive washes, membranes were incubated with the Luminata Western HRP Substrate (Millipore). Proteins were detected by chemiluminescence and imaged with a G-box camera (Syngene imaging system). The expression level of GAPDH was used as loading control in all the western blots and for quantification. Quantification of protein expression was done with the Genesys software (Syngene).

The LC3-conjugated form to PE, named LC3-II, presents a higher electrophoretic mobility in gels and was, therefore, used to quantify autophagy.

Fluorescence analysis of autophagy. HEK/CD4/CXCR4 cells were cultured in six-well plates and transfected with pEGFP-LC3 using the TurboFect reagent (Fermentas) according to the manufacturer's instructions. Transfected cells were then left to adhere on coverslips before co-culture with MOLT cells (negative control) or chronically infected cells (MOLT-X4) for indicated times (from 2h to 48h). Human primary CD4 + T cells were transfected with pEGFP-LC3 using the Nucleofector apparatus (Amaxa Biosystems) according to the manufacturer's instructions. Transfected primary T cells were then co-cultured on coverslips for 4h with effector cells expressing or not the viral envelope at their surface (HEK.Env or HEK, respectively). Coverslips were washed with PBS and cells were fixed in a 3.7% paraformaldehyde/PBS solution for 10 min at room temperature and analysed by epifluorescence using a Leica microscope. Autophagic cells were defined as cells containing at least 5 GFP-LC3 puncta. More than 100 transfected cells were analysed by two different investigators. Results are from at least three independent experiments.

Statistics.

Analysis of the variance of the results was performed after arc sine transformation of the data when percentages were compared [START_REF] Zar | Biostatistical analysis[END_REF]. Differences were considered significant at *p < 0.05, **p < 0.01, and ***p < 0.001.

RESULTS

Induction of autophagy decreases viral production in HIV-1-infected CD4 + T lymphocytes.

We, and others, have previously showed that autophagy is blocked in productively HIV-1infected CD4 + T lymphocytes, suggesting that autophagy is an anti-HIV process counteracted by this virus [START_REF] Espert | Differential role of autophagy in CD4 T cells and macrophages during X4 and R5 HIV-1 infection[END_REF][START_REF] Zhou | Human immunodeficiency virus type-1 infection inhibits autophagy[END_REF]. To examine this hypothesis, activated primary CD4 + T cells were infected by HIV-1 for 3 days (~20% infection). Then, autophagy was induced by adding Torin 1, an inhibitor of the mTOR kinase, for 24h. The level of viral particle production was measured by quantifying the viral capsid protein p24 by ELISA. As expected, we detected a significant decrease of HIV-1 p24 levels in the supernatants of cells treated by Torin 1 (Fig. 1A). The same results were obtained when Torin 1 was added to infected Jurkat cells (Fig. 1B) or chronically infected CD4 + T cells (MOLT-X4) (Fig. 1C). Thus, addition of Torin 1 decreases viral production, supporting the anti-HIV role of autophagy.

Tat interacts with p62 and is specifically degraded by Torin 1-induced autophagy. We performed a two-hybrid screen to analyze the interactions between several HIV-1 proteins (Tat, Vif, Vpr and Nef) and a bank of ATG proteins. Among the viral proteins tested, the transactivator Tat was the only viral protein able to bind p62 (data not shown), highly suggesting a role for selective autophagy. This interaction was further confirmed in vitro using a GST pull down assay in which Tat was found to interact with GST-p62 but not GST alone (Fig. 2A). As p62 is an autophagy receptor involved in the specific degradation of proteins or organelles by autophagy, we then analyzed whether induction of autophagy could affect the expression level of Tat. To this aim, we transfected human embryonic kidney (HEK) cells with a plasmid expressing a FLAG-Tat construct and autophagy was induced upon addition of Torin 1 for 3h. We observed a significant decrease in Tat levels upon Torin 1 treatment (Fig. 2B). Importantly, this effect was reversed in cells pre-treated with a cocktail of anti-proteases (E64d + Pepstatin A, AP) previously described to block lysosomal-mediated degradation (Fig. 2B). Furthermore, inhibition of lysosomal proteases, in absence of autophagy induction, showed increased levels of Tat expression in cells, suggesting that the viral transactivator can be degraded by lysosomes (Fig. 2B and2D). The levels of the ectopically expressed viral proteins Gag, Vif or Nef was not affected by the addition of Torin 1 (Fig. 2C). These data strongly suggest that the Torin 1-induced decrease of Tat level is selective and rely on an autophagy-dependent lysosomal-mediated degradation. The results were confirmed in a context of viral infection using a HIV-1 chronically infected T cells (MOLT-X4 cells) which constitutively produce the viral Tat protein. As shown in figure 2D, Tat levels were also decreased in MOLT-X4 cells upon Torin 1 treatment for 3h, and the autophagy-inducing drug effect could be reversed by AP treatment. Since Tat is essential for HIV-1 replication cycle, these results extend further those presented in figure 1 in which drug-mediated induction of autophagy strongly decreased viral release.

To confirm that the autophagic process is responsible for the lysosomal-mediated degradation of Tat, we down-regulated the expression of the essential autophagy-related protein ATG7 using siRNA. As shown in figure 2E, while the control siRNA did not prevent Torin-1-induced Tat degradation, the viral transactivator expression levels were significantly increased in cells for which ATG7 expression was reduced. Outstandingly, even if siRNAmediated ATG7 decreased expression was of 30% only, Tat levels became totally insensitive to Torin 1 treatment, confirming the contribution of the autophagy pathway in Tat degradation. This result also indicates that this process is finely tuned and relies on a fully functional autophagy process.

Since we observed that Tat was interacting with p62 (see Fig. 2A), we aimed at analyzing Tat levels when p62 expression was decreased. Remarkably, we noted that the siRNA-mediated decrease of p62 expression correlated with a significant increase in Tat protein signal compared to levels obtained in lysates from siCT-treated cells (Fig. 2F). Overall, these results demonstrated that the autophagy pathway significantly contributes to Tat degradation and the autophagy receptor p62, shown to interact with Tat, could confer selectivity in this process.

The interaction between Tat and p62 is ubiquitin-independent. As we demonstrated that

Tat interacts with p62 and since p62 is known to induce the selective autophagic degradation of substrates by interacting with LC3, we further analyzed whether Tat could be present in complexes with LC3. We thus performed GST pull-down experiments after cotransfection of plasmids expressing GST-LC3 and FLAG-Tat in HEK cells. As shown in figure 3A, Tat co-precipitates with GST-LC3. To assess whether this interaction was p62-dependent, we co-transfected cells with constructs expressing FLAG-Tat and a GST-tagged mutant of LC3 unable to interact with p62 (GST-LC3F52A) [START_REF] Bres | A non-proteolytic role for ubiquitin in Tat-mediated transactivation of the HIV-1 promoter[END_REF][START_REF] Shvets | The N-terminus and Phe52 residue of LC3 recruit p62/SQSTM1 into autophagosomes[END_REF]. We observed that Tat did not coprecipitate with LC3F52A (Fig. 3A), indicating that the interaction between Tat and LC3 was indirect and seemingly regulated by p62. To confirm this hypothesis, we tested the interaction between Tat and a mutated form of p62 that does not interact with LC3 (GST-p62LIR). As expected, this mutant still interacted with Tat hence with an apparently increased efficacy, which could be explained by the lack of targeting to autophagy-mediated degradation and thus a prolonged protein complex stability (Fig. 3A, lane 6).

The autophagy receptor p62 has been reported to selectively address ubiquitinated proteins or ubiquitinated organelles to autophagic degradation via specific recognition of ubiquitin moieties through its UBA domain, and the PB1 domain contributes to this process. We thus analyzed the interaction between Tat and mutated forms of p62 for which the UBA (GST-p62 ΔUBA) or the PB1 (GST-p62 ΔPB1) domains were deleted. As shown in figure 3B (lanes 3 and 4), Tat still interacted with these two p62 mutants, indicating that ubiquitination might not influence the interaction between p62 and Tat in vitro. In accordance with this result, Tat mutants on known ubiquitinated lysines, previously reported to regulate Tat activity and stability (K71 and K50-51, respectively) [START_REF] Bres | A non-proteolytic role for ubiquitin in Tat-mediated transactivation of the HIV-1 promoter[END_REF][START_REF] Johri | Tits and bits of HIV Tat protein[END_REF][START_REF] Kiernan | HIV-1 tat transcriptional activity is regulated by acetylation[END_REF] are still degraded upon induction of autophagy by Torin 1 (Figure 3C).

Autophagy is induced by HIV-1 envelope glycoproteins (Env) at the very first steps of HIV-1 infection.

To further decipher whether Tat degradation by autophagy could be relevant in vivo, we analyzed its occurrence in the context of HIV-1 infection. Indeed, we already demonstrated that autophagy is induced in uninfected bystander CD4 + T lymphocytes after 48h of co-culture with Env-expressing cells [START_REF] Espert | Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4[END_REF]. We extended this finding with experiments showing that the fusogenic function of gp41 is responsible for the observed Env-induced autophagy [START_REF] Denizot | HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells[END_REF]. Given that the gp41-mediated fusion process is an early HIV-1 entry event, we investigated whether autophagy could be induced very rapidly after contact of Env with its receptors on target cells, before productive infection. To this aim, HEK cells expressing HIV receptors (HEK/CD4/CXCR4) and transfected with a plasmid expressing GFP-LC3, were co-cultured with chronically infected T cells (MOLT-X4), or uninfected T cells (MOLT) as a negative control. The level of autophagy and the percentage of infected cells were analyzed at different time points of co-culture. As shown in figure 4A, autophagy was rapidly induced in read-out cells upon co-culture with MOLT-X4, with a peak around 4h corresponding to the early phase of HIV-1 infection [START_REF] Mohammadi | 24 hours in the life of HIV-1 in a T cell line[END_REF]. Autophagy was then negatively regulated and finally completely inhibited after 24h of co-culture, a step corresponding to the late phases of HIV-1 infection. Importantly, induction of autophagy is inversely correlated with the level of infection. Of note, the same pattern was obtained when GFP-LC3 transfected cells were challenged with cell free HIV-1 (data not shown). We next analyzed whether the viral envelope was contributing to this early autophagy induction during early events of HIV-1 infection of CD4 + T cells. Primary CD4 + T cells, expressing GFP-LC3, were co-cultured with effector cells stably expressing Env, or not, at their surface (HEK.Env and HEK cells, respectively). As shown in figure 4B, Env alone was able to rapidly and significantly induce autophagy in target cells. To confirm and reinforce these data, we took advantage of another autophagy read-out by analyzing the phenotype of primary CD4 + T cells by transmission electron microscopy upon 4h of co-culture with HEK cells stably expressing Env or not. We detected the appearance of numerous autophagic vacuoles in the cytoplasm of target CD4 + T cells only in the presence of Env (Fig. 4C). Thus, Env is able to induce autophagy in target cells upon early contact with infected cells. It is interesting to note that, as shown for Env-mediated autophagy in longer kinetics of co-culture [START_REF] Denizot | HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells[END_REF], this early autophagy induction was dependent on the gp41 fusogenic function. Indeed, the addition of T20 to the co-culture, a gp41-mediated fusion peptide inhibitor, completely abolished the Env-induced autophagy (Figure 4D).

We then analyzed whether later steps of autophagy could be induced or blocked when cellular lysosomal proteases were inhibited by AP. As evidenced in figure 4E, the increased level of LC3-II obtained upon 4h of contact with Env-expressing HEK cells was further enhanced in lysates from cells also pre-treated with AP, confirming that the induced autophagy flux was not blocked during the early time points of contact between the viral envelope and CD4 + T cells.

To confirm that Tat could be a selective target for autophagic degradation in the context of productive infection, we analyzed its expression levels upon Env-induced autophagy during HIV-1 infection. For this purpose, we transfected a plasmid expressing FLAG-Tat in HEK/CD4/CXCR4 and co-cultured these cells with MOLT-X4 during 4h, a time previously shown to correspond to the peak of Env-mediated autophagy induction, or 48h, a time for which autophagy was seemingly inhibited (Fig. 4A). As expected, the level of LC3-II was increased up to 70% after 4h of co-culture while being decreased by 40% upon 48h of coculture with MOLT-X4 cells, strongly supporting an early induction of autophagy followed by its blockade during HIV-1 infection. Remarkably, Tat expression levels were inversely correlated to those of LC3-II, meaning that Tat is depleted when autophagy is induced while this effect was not observed under conditions where autophagy is inhibited by the virus (Fig. 4F).

Env-induced autophagy triggers Tat degradation and consequently represses HIV-1 LTR

activation. In order to assess the contribution of Env-mediated autophagy induction in Tat degradation, we transfected a plasmid expressing FLAG-Tat in HEK/CD4/CXCR4 and treated these cells with the autophagy initiation inhibitor 3-methyladenine (3-MA) before co-culture for 4h with uninfected or chronically HIV-infected T cells. As shown in figure 5A, Tat levels markedly decreased upon co-culture with MOLT-X4 cells, while they were fully rescued in the presence of 3-MA. This result confirms that Env-mediated autophagy flux induction is responsible for Tat degradation. We reproduced the experiments using 8.E5 cells as donor cells. These CD4 + T cells are productively infected, thus expressing Env at the surface but, because they do not express the reverse transcriptase, they do not produce infectious viral particles. With this model, the Env-mediated autophagy induction was even more pronounced as evidenced by the increased and sustained Tat degradation observed in target cells (Fig. 5B).

While viral challenge could rapidly and transiently induce autophagy, productive infection appeared to block this defense mechanism, thus potentially protecting the viral transactivator Tat from degradation. Interestingly, Tat can exert multiple effects on bystander cells when secreted from productively infected cells. For instance, secreted Tat was reported to transactivate the expression of genes under control of the HIV-LTR in bystander cells. To examine whether incoming Tat could be degraded by autophagy, we used HeLa cells expressing CD4 and CXCR4 together with the ß-galactosidase under control of the HIV-LTR promoter (MAGIC5B cells) [START_REF] Kimpton | Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene[END_REF]. MAGIC5B cells were co-cultured during 4h and 8h with MOLT-X4 cells in the presence or absence of 3-MA and ß-galactosidase activity was measured. An enzymatic activity could be detected in reporter target cells supporting the bystander effect of secreted Tat. Transactivation was significantly increased in the presence of 3-MA, strongly suggesting that autophagy controls Tat-mediated transactivation of HIV-LTR (Fig. 5C). Because 3-MA could have some side effects possibly affecting Tat secretion from infected cells, we aimed at confirming these results by directly downregulating the expression of essential ATG proteins in target cells. Therefore, MAGIC5B cells were transfected with siRNA directed against Beclin 1, or Atg7, or unrelated siRNA (unr) before co-culture with MOLT-X4 cells. As expected, the level of ß-galactosidase activity was increased at both time points of co-culture when Beclin 1 or Atg7 expression were silenced (Fig. 5D). Inversely, autophagy induction should inhibit HIV-LTR transactivation due to Tat degradation (see Fig. 2). Indeed, when MAGIC5B cells were co-cultured with MOLT-X4 cells in the presence of Torin 1, we observed a significant decrease of ß-galactosidase activity, inversely correlated with the increased enzymatic activity obtained upon autophagy flux blockade with AP (Fig. 5E). Hence, the effect of drug-mediated autophagy induction on transactivation by Tat appeared to be sustained as evidenced by the decreased transactivation upon 16h of co-culture (Fig. 5E). These data thus show that the autophagy pathway can be a major regulator of Tat-mediated transactivation and viral replication. This conclusion is valid whether Tat is produced intracellularly (i.e. by infected cells) or delivered from the outside (i.e. endocytosed). Indeed, to confirm that the detected viral promoter transactivation was exclusively Tatdependent and controlled by autophagy, we took advantage of a previously described Tat mutant that lacks its single tryptophan residue (Tat-W11Y). This mutant retains transactivation activity but is unable to enter bystander cells (35) (Figure 5F). MAGIC5B cells were transfected with a Beclin 1-specific siRNA or a control siRNA, then treated with 200nM of recombinant Tat-WT or Tat-W11Y and ß-galactosidase activity was measured after 48h.

We observed that, contrary to cells treated with Tat-WT, transactivation was not significant when Tat-W11Y was used (Fig. 5G). These results confirm that transactivation by incoming Tat is targeted by selective autophagy.

DISCUSSION

HIV-1, like other viruses, has to deal with cellular defenses in order to replicate efficiently.

Although several cellular HIV-1 restriction factors have been identified, most of them were shown to be affected by viral countermeasures [START_REF] Strebel | HIV accessory proteins versus host restriction factors[END_REF]. We previously demonstrated that HIV-1 Env induces autophagy in uninfected CD4 + T lymphocytes via the fusogenic function of gp41, leading to their apoptotic cell death [START_REF] Denizot | HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells[END_REF][START_REF] Espert | Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4[END_REF]. In this study, we demonstrate that induction of autophagy is readily detectable shortly upon viral challenge in an Env-dependent manner, while being compromised later in time. Autophagic activity is inversely correlated with HIV-1 production. We further provide evidence that autophagy, when pharmacologically induced in productively infected CD4 + T cells, leads to a significant decrease in viral production thus identifying this cellular pathway as an HIV-1 restriction mechanism. This observation led us to decipher the molecular mechanism of the autophagy-mediated antiviral effect. A twohybrid screen revealed a strong and specific interaction between p62 (also called SQSTM1), an autophagy receptor, and the viral transactivator Tat. We first confirmed the interaction between both proteins at the cellular level in pull-down assays and confirmed that Tat expression levels were modulated in cells upon autophagy activation or inhibition. We then demonstrated that Tat expression was significantly increased in p62-deficient cells, suggesting that p62 could be a mediator of Tat degradation by selective autophagy. We therefore analyzed the domains of p62 possibly involved in the interaction with Tat. Surprisingly, the interaction between Tat and p62 was not relying on a functional UBA domain of p62, suggesting that ubiquitination would not regulate p62-mediated Tat degradation, in contrast to the general paradigm reported for other substrates [START_REF] Rogov | Interactions between autophagy receptors and ubiquitin-like proteins form the molecular basis for selective autophagy[END_REF]. Consistently, Tat mutations at the level of lysines which were previously shown to regulate Tat stability and activity did not prevent its degradation upon autophagy induction. Although we cannot exclude modification of other residues or the involvement of unidentified cellular proteins belonging to the complex Tat/p62, our data represent the first demonstration of selective autophagic degradation of a viral transactivator. Indeed, in a context of viral infection, a selective p62-mediated degradation has only been demonstrated for two structural viral proteins, the capsid protein of the chikungunya virus (CHIKV) and the capsid protein of the Sindbis virus (SIN) [START_REF] Orvedahl | Autophagy protects against Sindbis virus infection of the central nervous system[END_REF][START_REF] Judith | Species-specific impact of the autophagy machinery on Chikungunya virus infection[END_REF]. In the case of CHIKV, p62 binds to ubiquitinated capsid and targets it to autophagic degradation while in the case of the SIN, and as reminiscent of our data for Tat, the interaction between p62 and the SIN capsid does not require the p62 UBA domain, highlighting a potential original mechanism by which p62 recognizes autophagic substrates. As p62 could frequently interacts with viral proteins [START_REF] Gregoire | Autophagy and RNA virus interactomes reveal IRGM as a common target[END_REF], it would be interesting to decipher the molecular mechanism behind this ubiquitinindependent p62-mediated autophagic degradation, with a particular emphasis on the identification of putative other cellular partners linking p62 to its substrates. One potential candidate is the histone deacetylase HDAC6 whose deacetylase activity was shown to be regulated upon interaction with p62 (49). Furthermore, HDAC6 was also reported to deacetylate Tat, leading to a decrease in Tat transactivation activity [START_REF] Huo | Regulation of Tat acetylation and transactivation activity by the microtubule-associated deacetylase HDAC6[END_REF].

Although cellular factors are involved in HIV-1 transcription, Tat is absolutely required for HIV-1 replication since it is needed for the elongation of viral transcripts [START_REF] Ott | The control of HIV transcription: keeping RNA polymerase II on track[END_REF]. Tat is thus a perfect target to shut down viral production. Interestingly, Tat was shown to be actively secreted from infected cells and captured by several cell types [START_REF] Debaisieux | The ins and outs of HIV-1 Tat[END_REF][START_REF] Johri | Tits and bits of HIV Tat protein[END_REF]. Our results suggest that incoming Tat is also a target for autophagic degradation following internalization by bystander cells. The consequences of this degradation on the different known effects of Tat, and in particular on the homeostasis of target cells, warrants further investigation.

We uncovered that autophagy is induced by HIV-1 Env at the very first steps of infection. This Env-induced autophagy is timely regulated and initially triggers the degradation of any incoming Tat, avoiding the death of the target cells long enough to allow the virus to initiate its replication. If productive infection of the target cells proceeds, the virus blocks autophagy, and Tat, thereby protected from degradation, can fulfill its transcriptional role, enabling robust viral production.

The regulation of intracellular Tat concentration is tightly regulated. Indeed, we demonstrated here that Tat can be degraded by basal constitutive autophagy and upon Envinduced autophagy. Tat was also reported to inhibit the autophagy process in uninfected cells [START_REF] Van Grol | HIV-1 inhibits autophagy in bystander macrophage/monocytic cells through Src-Akt and STAT3[END_REF][START_REF] Li | HIV-1 trans-activator protein dysregulates IFN-gamma signaling and contributes to the suppression of autophagy induction[END_REF], underlining the complex relationship between autophagy and this viral protein.

This also seems to be the case for another retroviral transactivator, the HTLV-1 Tax protein.

Indeed, the group of KT Jeang demonstrated that Tax is a substrate for autophagy degradation, but the underlying mechanism is still unknown. They also demonstrated that Tax is able to block the degradative phases of autophagy suggesting that this viral protein could control its own stability and turnover [START_REF] Tang | The cellular autophagy pathway modulates human T-cell leukemia virus type 1 replication[END_REF].

Highly active antiretroviral therapy (HAART) can efficiently block viral replication and reduce the levels of circulating virus below the detection limit. Despite its efficacy, the virus is not eradicated due to the establishment of latent but replication-competent proviruses in reservoir cells probably located in peripheral blood and tissues. Viral latency is characterized by HIV-1 LTR-transcriptional repression and, interestingly, a hallmark of this process is the low level of Tat. Thus, specific degradation of Tat by autophagy could adversely contribute to the establishment of HIV-1 latency which should be taken into account in the prospect of future antiviral strategies.

Since Tat is absolutely required for viral transcription, many therapeutic strategies targeting this viral protein have been considered, such as Tat antagonists [START_REF] Hsu | Inhibition of HIV replication in acute and chronic infections in vitro by a Tat antagonist[END_REF], dominant-negative Tat [START_REF] Green | Mutational analysis of HIV-1 Tat minimal domain peptides: identification of trans-dominant mutants that suppress HIV-LTR-driven gene expression[END_REF][START_REF] Pearson | A transdominant tat mutant that inhibits tat-induced gene expression from the human immunodeficiency virus long terminal repeat[END_REF], Tat inhibitory peptides [START_REF] Agbottah | Inhibition of HIV-1 virus replication using small soluble Tat peptides[END_REF][START_REF] D'orso | Targeting tat inhibitors in the assembly of human immunodeficiency virus type 1 transcription complexes[END_REF][START_REF] Hamy | An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication[END_REF][START_REF] Okamoto | HIV-1 replication is inhibited by a pseudo-substrate peptide that blocks Tat transactivation[END_REF], or siRNAs against Tat [START_REF] Coburn | Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference[END_REF][START_REF] Lee | Inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using Tat-or CCR5specific small interfering RNAs expressed from a lentivirus vector[END_REF][START_REF] Surabhi | RNA interference directed against viral and cellular targets inhibits human immunodeficiency Virus Type 1 replication[END_REF]. However, none of The results obtained in our study provide new evidence of the antiviral action of autophagy and underlie why the viruses have evolved strategies to block this process.

  statement. Primary CD4 + T cells were purified from blood obtained from the EFS (Etablissement Français du Sang). Procedures using human cells were approved by the Human Experimentation and Ethics Committee of the CNRS Institute. The HIV-1 infection experiments were performed in a Biological Safety Level 3 laboratory. Cell culture was performed in a Biological Safety Level 2 laboratory. Cell culture. The original HEK.293T cell line, HEK expressing wild type CD4 and wild type CXCR4 and HEK stably expressing Env at their surface were cultured in DMEM supplemented with antibiotics and 10% fetal calf serum (FCS). MAGIC5B cells (cell line expressing CXCR4 and CCR5 and the β-galactosidase under control of the HIV long terminal repeat (LTR) were obtained from T. Masashi (Tokyo, Japan) and cultured in DMEM supplemented with antibiotics and 10% FCS. MOLT cells are non-adherent human acute lymphoblastic leukemia cells. The chronically HIV-1-infected MOLT cells (MOLT-X4) and the uninfected parental cell line (MOLT) were provided by J. Blanco (Barcelona, Spain). The 8.E5 cell line is a CEM-derived T-cell line containing a single integrated copy of HIV-1 and unable to produce infectious virions. T cell lines were cultured in RPMI 1640 medium supplemented with antibiotics and 10% FCS. Primary CD4 + T cells were isolated from Peripheral blood mononuclear cells and purified by negative selection using the CD4 + Rosette separation technique (Stem Cell Technologies). Cells were phenotypically analyzed, stimulated with 2 μg of phytohemagglutinin (PHA) for 24h and maintained in culture by adding 100 U/ml of interleukin 2 (IL-2) (Boehringer Mannheim) every 2-3 days. Reagents and antibodies. Torin 1 (To) was purchased from Tocris, 3-methyladenine (3-MA), PHA, E64d and Pepstatin A (AP) were purchased from Sigma-Aldrich. T20 was obtained from the NIH AIDS Reagent Program. Anti-LC3 (ref. L7543), anti-GAPDH (reference G9545), anti-ATG7 (ref. A2856) and anti-FLAG-HRP (ref. A8592) were obtained from Sigma-Aldrich, anti-Tat (ref. sc65912) and anti-Beclin 1 (ref. sc11427) were purchased from Santa-Cruz Biotechnologies. The monoclonal anti-p24 antibody (Ab) was from the NIH AIDS Reagent program.

  these strategies has been successful to date. The autophagy-mediated degradation of Tat presented here could represent a new way to target this viral protein in infected cells, allowing to block viral transcription and thus viral replication. Indeed, while HAART acts directly on viral enzymatic proteins, the induced degradation of Tat by autophagy could represent a new strategy based on a cellular response. Interestingly, some sirolimus-derived compounds (rapamycin, rapamune), which are strong inducers of autophagy and already used in therapeutics were shown to possess anti-HIV-1 properties (62). Recently, Campbell and Spector demonstrated that vitamin D3 inhibits HIV replication through induction of autophagy (63). It would be, therefore, interesting to examine the autophagic-induced degradation of Tat in these contexts and its possible outcome on inhibition of HIV replication.

  JA, Bendtzen K, Nicoletti F. 2010. Potential use of rapamycin in HIV infection. British journal of clinical pharmacology 70:784-793.63.Campbell GR, Spector SA. 2012. Toll-like receptor 8 ligands activate a vitamin D mediated autophagic response that inhibits human immunodeficiency virus type 1. PLoS Pathog 8:e1003017.

Figure 1 :

 1 Figure 1: Autophagy has an anti-HIV-1 effect in CD4 + T lymphocytes Primary CD4 + T cells (A) or Jurkat cells (B) were infected with HIV-1 at a MOI of 1 for 3 days. Cells were then extensively washed to eliminate excess of free viruses and cultured for 24h in the presence of 2 µM Torin 1 to induce autophagy. HIV Gag-p24 levels were quantified by ELISA in the culture supernatants. (C) Chronically infected MOLT-X4 cells were treated for 24h with 2 µM Torin 1 and HIV Gag-p24 concentration was quantified in the culture supernatants by ELISA. Data are representative of triplicates from at least 3 independent experiments.

Figure 2 :

 2 Figure 2: Tat interaction with p62 leads to its selective degradation by autophagy (A) HEK cells were co-transfected with the FLAG-Tat vector and the GST or GST-p62 plasmid. 24h post-transfection, cells were lysed and lysates were subjected to GST pull-down before immunoblotting with anti-FLAG or anti-GST Ab. A fraction of lysates was preserved and used as an input control and immunoblotted with the anti-FLAG Ab. The result is representative of at least 5 different experiments. (B) HEK cells were transfected with plasmids expressing FLAG-Tat. After 24h, the transfected cells were treated or not with 2 µM Torin 1 in the presence or absence of antiproteases (AP: E64d+pepstatin A, 10µg/ml each)

Figure 3 :

 3 Figure 3: p62-dependent interaction of Tat with LC3(A) HEK cells were co-transfected with the FLAG-Tat vector and plasmids expressing either GST (lanes 1 and 4), GST-LC3 (lane 2), GST-LC3F52A (lane 3), GST-p62 (lane 5) or GST-

Figure 4 :

 4 Figure 4: Autophagy is induced at early steps of HIV-1 infection (A) HEK/CD4/CXCR4 cells, previously transfected with a plasmid expressing GFP-LC3, were co-cultured with either MOLT cells (CT, negative control, hatched line) or chronically infected MOLT-X4 cells (HIV, plain line) for indicated times. The number of autophagic HEK/CD4/CXCR4 cells was counted by 3 different experimenters and the result is the mean of 3 independent experiments. The percentage of autophagic cells obtained after co-culture

Figure 5 :

 5 Figure 5: Autophagy-mediated Tat degradation inhibits HIV-1 LTR activation and viral replication (A) HEK/CD4/CXCR4 cells transfected with the FLAG-Tat vector were co-cultured with uninfected (MOLT) or chronically HIV-1-infected cells (MOLT-X4) cells during 4h, in the presence or absence of 10 mM 3-MA. Tat expression was monitored by Western blot using the anti-FLAG Ab and quantified upon normalization with GAPDH levels. (B) HEK/CD4/CXCR4 cells, transfected as above, were co-cultured for 4h with effector control cells (Env negative) or cells expressing HIV-1 envelope (8.E5 cells). Tat detection and quantification was done as previously described. (C) MAGIC5B cells were co-cultured with MOLT-X4 cells during 4h or 8h in the presence or absence of 3-MA. After cell lysis, β-galactosidase activity was quantified by luminescence and normalized to the condition in absence of 3-MA for each time of coculture. Data represent the mean +/-SE of at least 3 independent experiments. (D) MAGIC5B cells were transfected with control (CT) or Beclin 1 (Bec1) or Atg7-specific siRNA during 16h. Transfected cells were then co-cultured with MOLT-X4 cells during 4h. The cells were washed, harvested, and then lysed. A fraction of lysates was kept to control Beclin 1, ATG7 and GAPDH levels by immunoblotting (left panels) while remaining lysates were used to measure β-galactosidase activity, normalized to the activity obtained from cells transfected with CT siRNAs for each time point. Data are mean +/-SE of 4 independent experiments. (E) MAGIC5B cells co-cultured with MOLT-X4 cells were incubated or not with 2 µM of Torin 1 for 16h in the presence or absence of AP. Fold induction of β-galactosidase activity was calculated as the enzymatic activity obtained in treated cells compared to the one in

ACKNOWLEDGMENTS

The work was supported by institutional funds from the Centre National de la Recherche Scientifique (CNRS) and the Montpellier University I and II (UMI and UMII), and grants from Sidaction and the Agence Nationale de Recherche sur le SIDA (ANRS). Sophie Sagnier was the recipient of a fellowship from ANRS and Sidaction. We thank Dr. M. Benkirane for providing the Tat-K71R construct, Dr. T. Johansen for the GST-p62 plasmids, Dr. T. Yoshimori for the GFP-LC3 construct and Dr. N. Chazal for the Gag expression vector. The anti-p24 antibody was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH.


Pauline Butaud

Patrice Le Moal

Gilles Bourbon

Vincent Placet

Emmanuel Ramasso

Benoît Verdin

Eric Joseph

Benoit Verdin

Towards a better understanding of the CMUTs potential for SHMapplications

Keywords: MEMS, CMUT, SHM, experimental, modeling, circular membranes array

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

Towards a better understanding of the CMUTs potential for SHM applications

Introduction

The Acoustic Emission technique which detects stress waves released as structural damage occurs is one of the Structural Health Monitoring (SHM) promising possibilities [START_REF] Saeedifar | Damage characterization of laminated composites using acoustic emission: A review[END_REF]. The development of a SHM system involves the enhancement of the data processing and consequently a good knowledge of the behaviour of the sensors used to record events. Different technologies are proposed for this purpose: optical fiber [START_REF] García | Optical fiber sensors for aircraft structural health monitoring[END_REF][START_REF] Bremer | Structural health monitoring using textile reinforcement structures with integrated optical fiber sensors[END_REF], piezoelectric sensor [START_REF] Chandarana | Early damage detection in composites during fabrication and mechanical testing[END_REF], MEMS transducers as PMUTs (Piezoelectric Micromachined Ultrasonic Transducers) [START_REF] Jung | Review of piezoelectric micromachined ultrasonic transducers and their applications[END_REF] and CMUTs (Capacitive Micromachined Ultrasonic Transducers) [START_REF] Oppenheim | Mems ultrasonic transducers for the testing of solids[END_REF][START_REF] Oppenheim | Electrical characterization of coupled and uncoupled mems ultrasonic transducers[END_REF][START_REF] Galisultanov | Squeeze film damping and stiffening in circular cmut with air-filled cavity: Influence of the lateral venting boundary conditions and the bias voltage[END_REF]. Our paper will be focused on the latter. More than ten years ago, Ozevin et al. [START_REF] Ozevin | Resonant capacitive mems acoustic emission transducers[END_REF] highlighted the potential of the CMUTs transducers for Acoustic Emission (AE) measurements for SHM. The authors studied a MEMS chip containing multiple resonance capacitive-type vibration transducers and demonstrated the possibility to detect acoustic emission events associated to crack initiation and growth. The only drawback emphasized for this technology was the poor signal-to-noise ratio, which could be improved, according to the authors, by a better packaging and shielding, or by increasing the number of cells. The transducers proposed at this time were designed using the poly-MUMPS surface micromachining process [START_REF] Koester | Mumps design handbook, Cronos Integrated Microsystems[END_REF]. Since then, other designs of CMUT have been proposed: a 3-D CMUT collocating two in-plane sensors and one out-of-plane sensor at the millimeter scale [START_REF] Greve | Development of a mems acoustic emission sensor system[END_REF], a polymer-based CMUT [START_REF] Hutchins | Structural health monitoring using polymer-based capacitive micromachined ultrasonic transducers (cmuts)[END_REF] produced using micro-stereolithography, capacitive MEMS sensors manufactured with the electroplating method using the MetalMUMPs [START_REF] Saboonchi | Mems acoustic emission transducers designed with high aspect ratio geometry[END_REF], all with promising properties, in particular a really interesting electromechanical coupling, and finally a signal-to-noise ratio suitable for the targeted applications. Nevertheless, as CMUTs supposedly required high bias voltage, this technology is less and less studied and PMUTs take the center stage [START_REF] Kabir | Piezoelectric mems acoustic emission sensors[END_REF].

CMUTs, especially in the context of SHM applications, still suffer from a lack of data and knowledge on their electromechanical dynamic behaviour from a theoretical and experimental point of view. The often recent and not yet fully mature micro-fabrication technologies used for their manufacture, and the multi-physical couplings at the various interfaces, are probably the main causes of this finding.

This article therefore aims to provide some answers to the ability of CMUTs to design broadband sensors for SHM applications. This mainly concerns two aspects:

• the multi-frequency aspect involving different individual membranes (theoretically addressed and experimentally validated) ;

• the bandwidth aspect including the intrinsic capabilities of an array of identical membranes (approached from a purely experimental angle).

In the future, the two aspects could be combined to benefit from each other but, as a first step towards this goal, the present work examines them separately.

For this purpose, a chip composed of CMUT unit membranes with different radii from 50 µm to 250 µm and CMUT arrays of 100 µm radius cells is designed. The target frequency range is between 80 kHz and 2 MHz. The manufacturing process, the constitutive materials and the key points of the geometries are outlined in this paper. Besides, in a numerical study, the impact of the geometry resulting from micro-fabrication technologies, and more particularly the influence of the fabrication process "Multi-Users MEMS Processes" (MUMPS) on the resonance frequency of CMUTs are studied. Indeed some geometrical elements are often ignored in the literature with regard to the effects of residual stresses and squeeze film damping. Residual stresses ranges are identified and briefly discussed on the basis of theoretical and experimental matching of resonance frequencies. However, the squeeze film damping effects will be neglected in this study due to the particular design of our CMUT membranes. Finally the pull-in voltage is determined for each different radius membrane.

Furthermore an experimental study on the CMUT membranes is conducted. The chip is integrated in a brass package to minimize parasitic effects. First of all, the resonance frequencies of the different radius membranes are measured to validate the design and the multi-frequency abilities of CMUTs. Then, the array of 100 µm radius membranes is experimentally characterized. The influence of the DC bias voltage on the sensitivity of the CMUTs array is highlighted through a single frequency excitation. The CMUT ability to respond to a frequency range below and beyond its resonance frequency is illustrated. Finally, the capacity of the array to detect AE events is demonstrated by a broadband excitation obtained by a pencil lead break; this despite a low sensitivity and a signal-to-noise ratio to be improved.

The paper is organized as follows. Section 2 outlines the design of the CMUT chip. Section 3 presents the numerical study of unit cells. Section 4 illustrates the CMUT membranes properties within the application SHM framework through experimental validation. In the last section, conclusions are drawn on the potential of CMUTs for SHM applications with regard to the two aspects considered, i.e. multi-frequency and bandwidth.

Sensor design

A CMUT sensor usually comprises several elementary capacitive micromachined ultrasonic cells acting as microphone membranes. The cell membranes are vibrating subjected to ultrasonic waves from the surrounding medium, either fluid or solid. In the present case, mechanical solid or surface waves propagating in monitored structures are transferred to the cells through the membrane clamping supports. Sensing the ultrasonic waves is achieved by converting vibration-induced membrane displacements into electrical energy via capacitive effects. The generic architecture, the mechanical properties and the geometrical key elements of the CMUT cells are described in this section.

The CMUT unit cells, based on perforated circular membranes clamped at periphery, are fabricated by surface micromachining using the standard fabrication process "Multi-User MEMS Processes" (MUMPS, run #120). The top and sectional views of an elementary CMUT cell layout, as proposed in Figure 1, show the successive constituent layers:

• a silicon nitride layer on the silicon handle substrate isolating both bottom and top electrodes,

• a first polysilicon layer which is patterned according to the ground, feed electrodes and electric tracks,

• two sacrificial silicon oxide layers which are afterwards removed to create the electrostatic gap,

• a second polysilicon layer constituting the vibrating membrane which clamping periphery contacts the feed electrode.

Once the second polysilicon layer is deposited, membranes, clamping parts and several holes are patterned by Reactive Ion Etching [START_REF] Emadi | A novel 6× 6 element mems capacitive ultrasonic transducer with multiple moving membranes for high performance imaging applications[END_REF]. The holes are etched to release the vibrating membrane, which becomes suspended by dissolving the sacrificial oxide layers underneath with hydrofluoric acid. MUMPS design rules [START_REF] Koester | Multi-user mems processes (mumps) design handbook, Cronos Integrated Microsystems[END_REF][START_REF] Miller | Characteristics of a commercially available silicon-on-insulator mems material[END_REF] recommend to design holes with a minimum diameter of 4 µm with 30 µm pitch. In our work, holes enlarged to 10 µm diameter with reduced 20 µm pitch are preferred in order to control in the design phase the minimization of the squeeze film stiffening of the air layer in the cavity. Mohite et al [START_REF] Mohite | Analytical solutions for the stiffness and damping coefficients of squeeze films in mems devices with perforated back plates[END_REF] introduces the notion of an equivalent pressure cell depending on the hole configuration (staggered with a hexagonal pattern or non-staggered with a square pattern) which radius is roughly 0.5 times the pitch. In this case, the radius of the pressure cell around 10 µm leads to a squeeze number lower than 0.5 for all the 50 µm to 250 µm radius membranes. Referring to works of Galisultanov et al [START_REF] Galisultanov | Squeeze film damping and stiffening in circular cmut with air-filled cavity: Influence of the lateral venting boundary conditions and the bias voltage[END_REF] based on solutions developed by Crandall et al [START_REF] Crandall | The air-damped vibrating system: Theoretical calibration of the condenser transmitter[END_REF], open cavity CMUTs for such very low squeeze number are strained by negligible elastic force from the compressed air layer. Meanwhile, open cavity CMUTs reveal higher damping forces that could contribute to wider bandwidth. On the other hand, holes ratio cannot be too much increased, because the CMUT capacitance would decrease and consequently the sensing sensitivity as well.

Our design based on 5×5 mm 2 chips includes single elementary CMUT cells for the frequency response and pull-in voltage determination, and cell arrays for the sensor tests under harmonic and broadband excitation. The thickness of the polysilicon membrane (so-called "Poly 2" in [START_REF] Koester | Multi-user mems processes (mumps) design handbook, Cronos Integrated Microsystems[END_REF]) and the electrostatic gap formed by the silicon oxides etching (so-called "Oxide 1 and Oxide 2" in [START_REF] Koester | Multi-user mems processes (mumps) design handbook, Cronos Integrated Microsystems[END_REF]) are experimentally measured by profilometry respectively around 1.30 µm and 2.40 µm.

The radius of the fabricated CMUT cells are: 50 µm, 75 µm, 100 µm, 150 µm, 200 µm and 250 µm and the measured thickness about 1.30 µm allowing a frequency range between 80 kHz and 2 MHz to be swept. This frequency domain is consistent with the AE scope usually involving transient signals in the 100 kHz -1 MHz range. show respectively the layout and the fabricated chip with elementary CMUT cells at the top left and the bottom right side and an array of 9 membranes of 100 µm radius at the middle (studied in section 4). On the photography of Figure 2(b), color gradients indicates that some membranes have a deformed shape after fabrication resulting from unexpected residual stresses. As expected, the deformed shapes are greater as the membrane radius is larger. Figure 2(c) focuses on the electrical connections and a top view of a 100 µm radius perforated circular membrane array.

The manufacturing process and the operating specifications induce four geometric structural features which could potentially impact the frequency response of the CMUT cell. Firstly, a step (marked "step" in Figure 1(b)) in the radial cross-section of the membrane due to the transfer of the ground electrode pattern modifies the membrane stiffness. Secondly, the clamp-ing conditions (marked "clamp" in Figure 1(b)) are not perfect due to the sidewall link between the membrane and the anchor posts. Thirdly, holes (marked "holes" in Figure 1(a)), patterned in the CMUT cell for the releasing of the suspended membrane, reduce the mass and the stiffness of the polysilicon membrane. Lastly, an opening (marked "electrode opening" in Figure 1(a)) in the annular clamping walls, needed for the connection between the electric track and the ground electrode under the membrane, affects the clamping conditions. The next section is dedicated to the study of those features by a numerical study.

Numerical modelling

The frequency response (resonance frequency, bandwidth and maximal vibration amplitude) of the elementary cells determines the sensor characteristics. Controlling the bandwidth and the maximal vibration amplitude is very challenging depending on different loss mechanisms (air loss, support loss, thermomechanical loss) [START_REF] Ghaffari | Accurate modeling of quality factor behavior of complex silicon mems resonators[END_REF][START_REF] Hao | An analytical model for support loss in micromachined beam resonators with in-plane flexural vibrations[END_REF][START_REF] Park | Optimum design of circular cmut membranes for high quality factor in air[END_REF]. On the other hand, the resonance frequency can be accurately defined by the geometrical dimensions, the mechanical properties of the constitutive material, the clamping conditions and the bias DC voltage for electrostatic devices.

The "stepped geometry", the "real boundary conditions", the "releasing holes" and the "electrode opening" directly involved by the surface micromachining steps are successively and accurately analyzed by finite elements simulations with ANSYS ® in terms of resonance frequencies. Then the pullin voltage is determined for each different radius membrane because it is an essential electromechanical parameter conditioning the operating range in terms of the DC bias voltage itself determining the sensor sensitivity.

Geometric key elements modelling

Leissa [START_REF] Leissa | [END_REF] develops closed-form solutions for mode shapes and resonance frequencies of circular plates. The resonance frequency is expressed as a function of geometric dimensions R and h (respectively the radius and the thickness of the membrane), of mechanical properties E, ν and ρ (respectively the Young's modulus, the Poisson's ratio and the density of polysilicon material) and of a parameter λ depending on the mode shapes and the boundary conditions:

f = λ 2 2πR 2 h E 12ρ(1 -ν 2 ) . (1)
The CMUT circular cells involve the first mode shape and are clamped at the periphery. Thus, the parameter λ is around 10.2158. Moreover, MUMPS fabrication capabilities lead to consider h = 1.30 µm, E = 160 MPa, ν = 0.22 and ρ = 2330kg/m 3 . As reported in Table 1, membrane radii from 50 µm to 250 µm determine resonance frequency from 80 kHz to 2 MHz. The top and sectional views of the membrane (Figure 1) present the geometry of each fabricated CMUT cell showing some significant discrepancies with the analytical approach assumptions. As previously described in the manufacturing process section, four key discrepancies can be noticed: a stepped geometry, non-optimal clamping conditions at the periphery, perforated membranes and an opening in the clamping ring. FEM modal analyses with the ANSYS ® finite element software are performed considering accurate 3D models meshed by 20 node hexahedral SOLID186 elements. The successive models take into account an additional key discrepancy in order to point out the influence of each geometrical particularity.

First of all, an initial benchmark model based on a quarter of the structure allows to validate at the same time the analytic results and the meshing size of the FEM model. The results in terms of the first resonance frequency are identical and not reported in the following.

Stepped geometry. This modelling includes the stepped geometry of the membrane (Figure 3). It shows a stiffening of the structure involving an increasing of the resonance frequency around 7% which is, until now, rarely, if ever, commented about surface micro machined architectures according to the authors. The higher the radius, the stiffer the structure is, with a slight variation between 6.3% and 7.6% for the 50 µm -250 µm radius range (Table 1). Real boundary conditions. This modelling considers the specific clamping conditions (Figure 4). It introduces a softening of the structure leading to a slight decrease in resonance frequencies, decrease of maximum 2.2% for the radius of 50 µm. The higher the radius, the less the clamping conditions have to be taken into account. For example, the variation of the resonance frequency for the 250 µm radius is at 0.50% (Table 1). Releasing holes. This modelling involves a perforated membrane as it can be seen in the Figure 5. It highlights a significant softening of the structure: the relative decrease in resonance frequencies varies from 2.0% to 7.7% for a radius between 50 µm and 250 µm (Table 1). This relative decrease of resonance frequencies is all the more remarkable as it results from two opposite contributions: one tends to increase them by reducing the mass of the membrane varying from 12% to 19%, and the other tends to decrease them by reducing the stiffness of the membrane. Thus, the holes in the membranes seem to induce a quite significant reduction of the stiffness membranes. Electrode opening -complete model. Lastly, the modelling takes into account the opening in the annular clamping and therefore includes the complete structure due to the symmetry breakage (Figure 6). Due to time-consuming 3D numerical simulations, this model requires, compared to the previous models, a very slight worsening in mesh size for radius above 75 µm. Practically, the size element is increased by 1 µm (100 µm / 150 µm: 1 µm to 2 µm, 200 µm: 2 µm to 3 µm, 250 µm: 3 µm to 3.9 µm). Therefore, the resonance frequency variations for the highest radius are partly linked to the meshing differences. In order to accurately evaluate the influence of the opening in the annular clamping, the "releasing holes" model is also run with the same worsened meshing (cf. right result values in the line "releasing holes model" in Table 1). In this case, the intrinsic influence is clearly proved showing a slight decreasing of the resonance frequency between 0.10% and 1.7% in the radius range.

To conclude on this numerical study, two geometrical configurations, i.e. the boundary conditions and the opening in the annular clamping, can be reasonably neglected with very small influences on the first resonance frequency (below 2%). These influences are all the more negligible the larger the radius of the membrane is. On the other hand, the stepped geometry and the perforated membranes respectively lead to significant stiffening and softening of the structure with relative variations of up to 8% for the first resonance frequency. Overall, these effects cancel each other out in this case, but reliable design requires careful consideration. Indeed, it should be different if the parameters of the stepped geometry and perforated membranes are changed: for example, the radius and thickness of the bottom ground electrode, thickness of the membrane and holes density.

Pull-in voltage

Another key point for the membrane design is the identification of the pull-in voltage corresponding to the membrane collapse. The pull-in voltage determines the operating range in terms of the DC bias voltage of the sensor: indeed no insulating layer protects the bottom ground electrode from the top supplied electrode (i.e. the membrane itself). The sensing parameters here are the electrostatic charges or electrical current which is directly proportional to the DC bias voltage. As the previous dynamic study, the complete model is used to discriminate the static influence of realistic structure geometries on the pull-in voltage. Static analyses are performed using the electromechanical line transducer element TRANS126 in ANSYS ® . These elements are generated from the bottom face of the perforated membrane and more particularly under the Poly 0 step which is the image of the bottom ground electrode. However, such elements do not intrinsically include edge effects. Figure 7(a) shows a simplified model with only a few TRANS126 elements to maintain readability. Identification of the pull-in voltage is achieved incrementally with 0.1 V steps. Figure 7(b) illustrates the deformation of a 100 µm radius membrane just beyond the pull-in voltage. To do so, a "virtual" insulating layer of 0.05 µm is added for convergence convenience. Table 2 reports numerical pull-in voltages from finite element simulations to be compared with the analytical values calculated for a circular, perfectly clamped, uniform, and non-perforated membrane on the basis of the approach described by Nikoozadeh et al [START_REF] Nikoozadeh | Analytical calculation of collapse voltage of cmut membrane [capacitive micromachined ultrasonic transducers[END_REF]. As can be seen, numerical and analytical results are very similar, fully confirming the findings of the dynamic study. In dynamics, numerical and analytical resonance frequencies result from stiffening of "the stepped geometry", softening of "the perforated membrane", and from the reduction of the membrane mass. In the same way, numerical and analytical pull-in voltages result from the same two effects of stiffening and softening and from reduction of resulting electrostatic forces. Indeed, while holes in dynamics reduce the membrane mass, they also reduce in statics the opposing electrode surfaces and thus the resulting electrostatic forces by the same proportion. The following section, as this one from a numerical point of view, is focused on determining the electromechanical characteristics of CMUT based on an experimental tests campaign.

Experimental characterizations

Resonance frequency of the membranes

The first step of the experimental campaign is to check the validity of the manufacturing process by measuring the resonance frequency of the membranes and to compare it with the numerical model. At this stage, the electromechanical coupling is not taking into account, i.e the response of a membrane is captured through the displacement of the membrane and not with the electrical signal generated by the MEMS.

Materials and methods. The CMUT chip is wire-bonded in a ceramic package and a probe station is used to supply the cell (Figure 8). Elementary CMUT cells are tested one after the other (at the bottom right in Figure2(a)). A bias voltage V DC (small compared to the pull-in voltage in order to avoid the electrostatic softening effect) is applied to the membrane terminals and a frequency sweep is performed with an alternating current V AC (small compared to V DC in order to avoid electrostatic non-linearities). A laser vibrometer (Figure 8) is directed at the center of the membrane to measure the velocity amplitude of the membrane according to the excitation frequency. The resonance frequency of the membrane is determined when the maximum of amplitude is reached. The measurements have been repeated on three different chips. 3 synthesises the measurements. The experimental resonance frequencies are highly reproducible for radii up to 150 µm with a maximal relative standard deviation around 2%. The manufacturing process is then quite stable. In this radius range, the numerical resonance frequencies calculated from the complete model correctly approximate experimental ones by a relative excess of up to 12%. By introducing an initial stress state into the complete model, an average level of homogeneous residual stresses has been estimated for each radius by matching experimental and numerical resonance frequencies (Table 3). For radii less than 150µm, the stress level is perfectly consistent with the values given by MUMPS (between -20 MPa and 0 MPa with a nominal value at -10 MPa). Beyond a radius of 150 µm, on the one hand, the measurements are not reproducible and, on the other hand, the calculated stress levels do not match the MUMPS range. This could be explained by membranes slender enough to induce critical buckling stresses in the MUMPS stress range (i.e. about -14 MPa and -9 MPa respectively for 200 µm and 250 µm radius). Thus, membrane buckling can lead to a redistribution of internal stresses by gradients into the thickness and to a modification of the structure (arch shape) inducing a dynamic stiffening of the membrane. This stiffening then results in the identification of a homogeneous initial tensile state.

To conclude, the correlation of experimental and theoretical resonant frequencies of moderate slenderness membranes (radius to thickness ratio below 150) demonstrates the validity of assumptions of the complete numerical model taking into account realistic residual stresses. Moreover, the experimental and theoretical multi-frequency aspect of membranes of different radii is highlighted by a broad covered frequencies range from 100 kHz to 2 MHz.

Electro-mechanical characterization

In this section, the objective is to quantify the electrical response of a CMUT array of identical membranes to an external mechanical excitation at a specified frequency. The bias voltage influence is also studied.

Materials and methods

The CMUT chip is integrated in a brass package as it can be seen in the Figure 9. The 5 mm square chip is surrounded by a PCB. The brass package is linked to the electric mass of the CMUT chip. This setup makes it possible to drastically reduce the electro-magnetic noise picked up by the cells. It can be noticed that the assembly is a 24 mm square, but on the underside 9(c), in contact with the test piece, a 10 mm square is in over-thickness in order to improve the contact between the sensor and the specimen. Measurements are focused on the array of 9 membranes of 100 µm radius (at the middle in Figure 2(a), and in Figure 2(c)) for which it is recalled that the resonant frequency and the pull-in voltage are about 450 kHz and 116 V respectively. For the experiment, a Cooknell power supply SU3/C and an amplifier CA7/C (Figure 10) are used to power the CMUT chip and to measure the electrical charges generated by the membranes vibrations. The DC bias voltage applied on the CMUT is varying between 0 V and 60 V by 10 V step. The specimen used is an aluminum beam with 200 mm length, 30 mm width and 3 mm thick, hanging by wires (Figure 10). The acoustic waves propagation through the beam is studied with two kinds of sensor: one is the CMUT, the other is a classical µ80-type sensor (Mistras Group Ltd.). Spring clamps are used to attach the sensors on one end of the beam. A permanent contact is obtained thanks to an industrial coupling gel which ensures a good transmission of the signal. A Picoscope4824 ® (5 MS/s) is used to record the sensor signals. In order to control the frequency of the acoustic waves, a µ80 is clamped at the opposite end of the beam from the measurement (Figure 10). The µ80 transmitter (Mistras Group Ltd.) is first used at 300 kHz which corresponds to the main resonance frequency of the sensor as it can be seen on its data sheet (Figure 11). The objective is to limit the impact of the transmitter on the excitation signal and to avoid interference frequencies on the excitation as much as possible. In addition, the µ80 is also used at 530 kHz, a frequency close to the resonance frequency of the studied CMUT, and at 800 kHz, on the plateau of the µ80, in order to map the behaviour of the CMUT over a wide frequency range. A 6-cycle sine wave windowed excitation is generated (amplitude ±1 V) with a Picoscope4824 ® and amplified with a fixed gain of 50 (Tabor Electronics 9100 A).

Results -harmonic excitation

First of all, the raw signals for excitation at 530 kHz, which is close to the resonant frequency of our membranes, are studied. Figure 12(a) and(b) show the temporal signals measured respectively by the CMUT (with different bias voltage) and by the µ80. The reported amplitudes in mV are the image of the mechanical -electrical transduction carried out by the CMUTs and the µ80 sensor. Figure 12(c) and(d) show the FFT (Fast Fourier Transform) of the temporal signals.

Concerning the µ80, can be noticed that not only the 530 kHz is detected, the frequency response of the transmitter (Figure 11) maybe affecting the signal and the 300 kHz resonance frequency is then clearly detected by the sensor. The CMUT response is principally centered around the 530 kHz excitation frequency but the resonance frequency of the 100 µm membrane (at 450 kHz) is also detected. Besides, Figure 12(a) and (c) give an idea of the impact of the V DC on the response of the CMUT sensor. The higher is the DC bias voltage V DC , the higher is the electrical signal from the CMUT, as expected. However, the electrical performances with regard to this voltage are slightly lower than expected highlighting a non perfect sum of the elementary contributions of each cell. Indeed, to ensure the operation of CMUT arrays, the DC bias voltage is at most about 50% of the pull-in voltage. In this case, the static deflection of the membranes remains low in front of the electrostatic gaps. Thus, the amplitude of the detected electrical signal should be theoretically proportional to the DC bias voltage, which is not accurately shown by Figure 12(a) (i.e. an amplitude ratio about only 3.5 between 1.4 and 4.9 mV compare to a voltage ratio of 6 between 10 and 60 V DC ).

To go further, the other excitation frequencies are studied. Figure 13(a) presents the FFTs obtained by the µ80 sensor and Figure 13(b) those obtained by the CMUT sensor with 60 V DC . The three excitation frequencies are compared: it can be observed that the amplitude of the µ80 sensor responses is really sensitive to the excitation frequency. All frequencies can effectively be captured (the scale can be misleading here but Figure 12(d) shows that the 530 kHz-frequency is well measured), but the amplitude ratio is highly variable according to the excitation frequency and could lead to misinterpretation in a data analysis. On the contrary, the CMUT sensor is more stable over the frequency band tested in this study. As for the µ80 sensor, the CMUT reacts more at its resonance frequency, but for the CMUT sensor, all frequencies are detected in a ratio of 1 to 3, as opposed to a ratio of 1 to 45 for the µ80 sensor.

This analysis has also been performed for several bias voltages. The Signal-to-Noise Ratio (SNR), defined as

SN R = 10 × log 10 ( U S U N ) 2 , (2)
with U S the maximum amplitude measured on the temporal signal and U N the amplitude of the noise measured by the sensor, has been estimated for both sensors (Figure 14). Each sensor obviously has a better SNR at their resonance frequency. As expected, the commercial sensor µ80 has a much higher SNR than the CMUT, even with the highest bias voltage considered in this study. Nevertheless, as previously it can be observed that the SNR is varying according to the excitation frequency: the SNR of the CMUT is more stable (a variation amplitude of 10 dB compared to 20 dB for the µ80). Finally, the CMUT, even far away from the commercial sensor in terms of SNR, seems promising especially since the CMUT sensor sensitivity can still be improved: the numbers of membrane can easily be increased, the packaging should be enhanced with regard to acoustic waves energy transfer and parasitic noises, the elementary contribution of each cell and the efficient sum of all of them need to be further analyzed, etc.

To conclude on the results of harmonic excitation, arrays of identical CMUT membranes can be sensitive to a broad range of frequencies below and beyond the resonance frequency of the elementary cell. The next section consists of the presentation of a classical test case in the field of Acoustic Emission in order to highlight possible capabilities of CMUT sensors in SHM applications.

Acoustic emission test simulation

In this section we use an artificial source commonly used in acoustic emission: the Pencil Lead Break (PLB) [START_REF] Sause | Investigation of pencil-lead breaks as acoustic emission sources[END_REF] which allows to generate a broadband excitation.

Materials and method

In this configuration, to avoid rigid body motion, the aluminium beam is clamped at one end (Figure 15). The 2H lead has a 0.5 mm diameter, and it is broken with a length of about 2 mm. At the other end of the beam, spring clamps are used to attach the two sensors, the CMUT and the µ80. As previously, array of 9 membranes of 100 µm radius is involved.

Results -broadband excitation

The temporal signal and the FFT of the signals are studied for both sensors on the Figure 16. A higher magnitude on the FFT, centered around 300 kHz for the µ80, and on 450 kHz for the CMUT can be observed. These frequencies correspond to the resonance frequencies of the sensors. This kind of excitation then permits to highlight the footprint of the sensor.

The SNR is not post-processed here, but as it can be seen on the Figure 16(a) the temporal signal of the CMUT sensor is quite noisy. The noise, before and after the PLB test, is around 0.4 mV. With the highest bias voltage studied of 60 V the maximum amplitude observed is 1.3 mV . It is quite small but it still allows the CMUT to pick up the event. The more the bias voltage decreases the more difficult it is to get out of the noise. However, the FFT of the signals reported on Figure 16(c) shows several amplitude peaks from a DC bias voltage of 40 V and more pronounced peaks beyond that up to 60 V in our study. This richness of the frequency spectrum remains to be studied in more details in order to discriminate their real meaning. This could be advantageously used in data processing methods required for the SHM process.

Conclusion and perspectives

A CMUT sensor based on circular membranes is developed and characterized. A design procedure is proposed to obtain a sensor sensitive over a wide range of target frequencies. Two aspects are considered: the multi-frequency aspect which involves different individual circular membranes (from 50 µm to 250 µm radius) and the bandwidth aspect which concerns the intrinsic capabilities of an array of 9 identical membranes of 100 µm radius. An analytical approach is used to determine the frequency range of the CMUT: between 80 kHz and 2 MHz according to the radius. A numerical study is then developed which highlights the geometrical parameters influencing the electromechanical behaviour of a CMUT cell. In particular, the stepped geometry of the membrane leads to a consequent stiffening of the CMUT and a significant shift of the resonance frequency. Besides, this study involves a perforated membrane and it appears that the releasing holes induce a quite significant reduction of the stiffness of the membrane. These geometric features should then be taken into account when dimensioning this kind of cell. In a second step, an exhaustive experimental study is performed. Taking into account realistic residual stresses, a good correlation is obtained between the resonance frequencies of the numerical model and the experimental measurements for moderate slenderness membranes. However, for membranes with a slenderness ratio greater than 150, buckling phenomena induced by residual stresses seem to modify the dynamic properties of the structure leading to an increase of the resonant frequencies. Besides, an electromechanical characterization of a CMUT array of identical membranes highlights the sensitivity of the CMUT according to the frequency excitation and for different bias voltages. The signal to noise ratio of the CMUT is fully dependent on the excitation frequency (higher when closer to the resonance frequency) and can be improved by increasing the bias voltage. Finally the CMUT sensitivity of the array studied is still far away from commercial sensors but seems very promising in terms of frequency range. Lastly, a broadband characterization is done thanks to a pencil lead break which highlights the ability of the CMUT array to detect acoustic emission events. The influence of the bias voltage on the CMUT sensitivity is again clearly demonstrated. Thus, CMUT sensors reveals actual capabilities in the targeted SHM application from both multi-frequency and bandwidth aspects considered separately in the present study. A next step in this work is to combine these two aspects by testing membrane arrays with different radii in order to further enhance the frequency band covered. In addition to the problems associated with the various possible optimal bias voltages, a compromise will have to be found, in terms of the sensor footprint, between frequency bandwidth and sensitivity. Therefore, the implementation of CMUT sensors in real situations will also require further theoretical and experimental works on the electromechanical parameters influencing the ability of an elementary cell to convert dynamic mechanical energy into electrical energy, on the optimization of the transfer of acoustic energy from the monitored structure to the elementary cell and on noise reduction. All these possible ways of improving the signal-to-noise ratio are the next challenges for the success of CMUT sensors.

Figure 1 :

 1 Figure 1: (a) Top and (b) sectional views of a typical membrane layout

Figure 2 :

 2 Figure 2: Design of the CMUT sensor: (a) layout and (b) fabricated chip, (c) electrical connections and top view of a 100µm radius membranes array.

Figure 2 (

 2 Figure 2(a) and Figure 2(b) show respectively the layout and the fabricated chip with elementary CMUT cells at the top left and the bottom right side and an array of 9 membranes of 100 µm radius at the middle (studied in section 4). On the photography of Figure 2(b), color gradients indicates that some membranes have a deformed shape after fabrication resulting from unexpected residual stresses. As expected, the deformed shapes are greater as the membrane radius is larger. Figure 2(c) focuses on the electrical connections and a top view of a 100 µm radius perforated circular membrane array.

Figure 3 :

 3 Figure 3: Structural meshing for the "stepped geometry" model configuration: membrane thickness modified by the Poly 0 step, (a) complete and (b) zoom.

Figure 4 :

 4 Figure 4: Structural meshing for the "real boundary conditions" model configuration: clamping conditions via the peripheral sidewall, (a) complete and (b) zoom.

Figure 5 :

 5 Figure 5: Structural meshing for the "releasing holes" model configuration: perforated membrane, (a) complete and (b) zoom.

Figure 6 :

 6 Figure 6: (a) Complete model of the membrane and (b) a zoom on the electrode opening in the annular clamping.

Figure 7 :

 7 Figure 7: (a) Mesh of the model with TRANS126 elements and (b) the deformation of a 100 µm radius membrane at the pull-in voltage (around 116 V).

Figure 8 :

 8 Figure 8: Experimental setup to measure the resonance frequency of membranes

Table 3 :

 3 range [MPa] -12.5/-9.0 -9.5/-8.0 -8.0/-7.0 -1.0/0.5 4.5/10.0 4.5/17.0 Resonance frequency f 0 according to the radius of the membrane, measured experimentally, compared to the complete model (without any residual stress) and calculated residual stress range for experimental and theoretical frequencies matching.

Figure 9 :

 9 Figure 9: CMUT chip integrated in a brass package, (a) box open, (b) box closed -top view and (c) bottom view.

Figure 10 :

 10 Figure 10: Experimental setup for harmonic excitation.

Figure 11 :

 11 Figure 11: Frequency response of sensor µ80, data sheet from Mistras Group Ltd.

Figure 12 :

 12 Figure 12: Experimental results at 530 kHz. (a) and (b) are the temporal signals measured respectively by the CMUT and the µ80. (c) and (d) are the FFT of the temporal signals. For the CMUT, the influence of the V DC is represented.

Figure 13 :

 13 Figure 13: FFT of the temporal signals (a) for the µ80 sensor, used as a reference and (b) for the CMUT with 60 V DC -for the different excitation frequencies.

Figure 14 :

 14 Figure14: Signal-to-Noise Ratio of the µ80 (dashed lines) and of the CMUT (dot points) at 300 kHz, 530 kHz and 800 kHz, according to the bias voltage.

Figure 15 :

 15 Figure 15: Experimental setup for pencil lead break excitation.

Figure 16 :

 16 Figure 16: Response to a pencil lead break excitation. (a) and (b) are the temporal signals measured respectively by the CMUT and the µ80. (c) and (d) are the FFT of the temporal signals. For the CMUT, the influence of the V DC is represented.

Table 1 :

 1 Resonance frequency according to the radius of the membrane, estimated analytically and by numerical simulation.

	Radius [µm]	50	75	100	150	200	250
	Analytic results [kHz]	2073 921	518	230	130	83
	Stepped geometry model [kHz]	2202 984	555	247	139	89
	Boundary conditions model [kHz] 2154 969	548	245	138	89
	Releasing holes model [kHz]	2111 931 515 -517 229-230 128 -128 82 -84
	Electrode opening model [kHz]	2074 924	516	229	128	84

Table 2 :

 2 Pull-in voltage according to the radius of the membrane of interest.

Acknowledgment

This works was supported by Bourgogne Franche-Comté Region in collaboration with EUR EIPHI Graduate School (contract ANR-17-EURE-0002). This work was partly supported by the french RENATECH network and its FEMTO-ST technological facility.