S Poncet
email: sandy.poncet@ifpen.fr

C Mehl

K Truffin

O Colin

Modified diffusion model adapted to non-unity Lewis number mixtures for low flame stretch using the thickened flame model

Keywords: Stretch, Diffusion, Non-unity Lewis number, Laminar premixed flame, Thickened Flame Model

This study proposes a methodology based on species diffusion adaptation to recover flame response to stretch in the context of the Thickened Flame Model for non-unity Lewis mixtures. First, the flame speed variation induced by low stretch, that is overestimated by the standard form of the TFM, is illustrated with stoichiometric C 8 H 18 /air (Le > 1) and lean H 2 /air (Le < 1) stretched thickened flame fronts. Secondly, the model correction parameter is deduced from spherical flame simulations for both mixture conditions. A posteriori validations, performed on spherical flames and flame-vortex interaction configurations, finally illustrate the effectiveness of this method.

Introduction

In Large Eddy Simulations (LES) of reactive flows, the flame thickness is usually small compared to the mesh size (∆x ≫ δ 0 L ). The Thickened Flame Model (TFM), consisting in the artificial widening of the flame front, is a competitive approach which ensures the proper resolution of flame fronts [START_REF] Butler | A numerical method for two dimensional unsteady reacting flows[END_REF], while preserving the correct propagation speed.

In TFM with full species and transport resolution, chemistry is solved using Arrhenius laws allowing for various phenomena such as flame-wall interactions, ignition and flame stabilization to be predicted accurately [START_REF] Veynante | instabilities in turbulent premixed burners large eddy simulation of combustion instabilities in turbulent premixed burners[END_REF][START_REF] Colin | Development of highorder taylor-galerkin schemes for les[END_REF]. However, a flame front thickened by a factor F is F times more sensitive to stretch than a real flame [START_REF] Veynante | instabilities in turbulent premixed burners large eddy simulation of combustion instabilities in turbulent premixed burners[END_REF]. Various studies [START_REF] Proch | Modeling heat loss effects in the large eddy simulation of a model gas turbine combustor with premixed flamelet generated manifolds[END_REF][START_REF] Han | Large eddy simulation/dynamic thickened flame modeling of a high karlovitz number turbulent premixed jet flame[END_REF][START_REF] Popp | An extended artificial thickening approach for strained premixed flames[END_REF] proposed a tabulated adaptation of the species diffusion coefficients to account for strain effect of turbulent flows for CH 4 /air mixtures (Le ∼ 1). With a similar approach, Comer et al. [START_REF] Comer | A modified thickened flame model for simulating extinction[END_REF] adapted the TFM approach in the context of strain-induced extinction limits for lean C 3 H 8 /air mixtures (Le > 1). Those studies only consider counter-flow strained flame configurations to determine their model correction parameters. Quillatre [8] proposed a diffusion-correction to limit the error of flame response to low stretch with TFM for non-unity Lewis mixtures based on asymptotic theories. However, restrictive assumptions of asymptotic theories didn't allow for the accurate preservation of flame speed for stretched-flame configurations.

The aim of this study is to propose a model to recover the flame response to low stretch in the context of the TFM approach for non-unity Lewis number mixtures. Unlike previous studies [START_REF] Proch | Modeling heat loss effects in the large eddy simulation of a model gas turbine combustor with premixed flamelet generated manifolds[END_REF][START_REF] Han | Large eddy simulation/dynamic thickened flame modeling of a high karlovitz number turbulent premixed jet flame[END_REF][START_REF] Popp | An extended artificial thickening approach for strained premixed flames[END_REF][START_REF] Comer | A modified thickened flame model for simulating extinction[END_REF], we propose to address both strain and curvature effects by considering curved flames configurations. In Sec 1, we briefly recall the theory of the stretch influence on flame speed and the impact of the TFM on stretched flame configurations. In Sec 2, we develop a methodology, based on the modification of species diffusivities, to correct the over-sensitivity of thickened flame fronts to stretch. The newly introduced method is assessed for a lean H 2 /air (Le < 1) and a stoichiometric C 8 H 18 /air (Le > 1) mixtures. A posteriori validations are finally performed in Sec. 3 on spherical flames and flame-vortex interactions.

1 Theoretical background

Premixed flame stretch sensitivity

The balance between thermal energy released by oxidation reactions and the supply of chemical energy from reactive species, sustaining the combustion process in premixed flames, can be altered by flame stretching. Stretch is defined as the rate of change of the surface A normalised by its area and is evaluated as K = 1/A × dA/dt. Under the assumptions of the asymptotic theory, the flame speed exhibits a linear trend that scales with stretch [START_REF] Matalon | Flames as gasdynamic discontinuities[END_REF]. The flame speed is linearly dependant on stretch when either considering the flame consumption speed S c or the flame displacement speed S b d considered close to the burnt gas side [START_REF] Giannakopoulos | Consumption and displacement speeds of stretched premixed flames -theory and simulations[END_REF]. On the one hand, the flame consumption speed represents the speed at which the flame consumes the reactants such as:

S c = - 1 ρ u Y k u -Y k b x + x -ωk dn (1)
where ρ, Y k , ωk and n are respectively the density, the mass fraction of species k, the reaction rate of species k and the distance along the flame normal.

x -and x + represent the coordinates in the unburnt and burnt gas side of the reactive front. Indexes u and b refer to the unburnt and burnt gases, respectively. On the other hand, the flame displacement speed S b d corresponds to the velocity of an iso-surface of the flame considered in the burnt gas. The impact of stretch on flame speed is characterized by the Markstein length L. Giannakopoulos et al. [START_REF] Giannakopoulos | Consumption and displacement speeds of stretched premixed flames -theory and simulations[END_REF] distinguish the consumption Markstein length L c and the displacement Markstein length L b d .

S c = S 0 L -L c K (2) S b d = S 0 L -L b d K (3)
where S b d is the normalized displacement speed defined by S b d = σ -1 S b d with the expansion ratio σ = ρ u /ρ b . The difference L b d -L c is always positive (see Eq. (34) in [START_REF] Giannakopoulos | Consumption and displacement speeds of stretched premixed flames -theory and simulations[END_REF]), i.e. L b d > L c , implying that these Markstein lengths can have opposite signs.

Stretch sensitivity of thickened flames

Based on the asymptotic theory, the consumption Markstein length scales with the flame thickness as

L c ∝ δ 0 L (Le i -1)
, where Le i is the Lewis number of the limiting reactant of the mixture and is defined as the ratio between thermal diffusivity α and mass diffusivity of species D i . Hence, for mixtures with large disparities between species mass diffusion and thermal diffusion, i.e Le i far from unity, the flame speed is significantly modified by stretch. As a consequence, with TFM, L c is proportional to the thickened flame thickness, Fδ 0 L where F is the thickening factor. A modification of the original TFM model is then necessary to account for stretch effects in flames modeled with preferential diffusion. Quillatre [8] proposed a corrected Lewis number Le * k for all species k based on the conservation of L c , estimated from the asymptotic theory:

Le * k = 1 + Le k -1 F ⇔ Sc * k = Pr + Sc k -Pr F (4)
This model was shown to reduce the over-estimation of the stretch effect with TFM [8]. However, the asymptotic theory being only qualitative for real multi-species chemistries, this approach still leads to important errors. Therefore, we propose here a model based on flame simulations which allows to accurately recover the real Markstein length of the flame.

TFM with corrected stretch sensitivity 2.1 Model formulation

Following previous approaches [START_REF] Comer | A modified thickened flame model for simulating extinction[END_REF]8] we propose to modify species diffusivities to correct the flame response to stretch. We define effective diffusion coefficients D * k , and thus Lewis numbers Le * k , as follows:

D * k = D k γ or Le * k = α D * k = γLe k ( 5
)
where γ is a model parameter determined so that the flame sensitivity to stretch is correctly retrieved. γ is a priori dependent on the local thermo-chemical conditions (pressure, temperature, equivalence ratio,...) and the thickening factor F. The modification of the diffusive fluxes however leads to an alteration of the flame speed and thickness. To recover the unstretched laminar flame speed, the pre-exponential factors of all reaction rates are multiplied by a parameter A. It is evaluated as the ratio A = S 0 L /S γ L 2 , with S γ L the flame speed obtained by using the effective diffusivity D * k without correcting the pre-exponential factor.

Model parameter evaluation

The method to estimate the model parameter γ is now described. In practice, we want to find the value of γ for each thickening factor F, written γ F , which satisfies:

L(F, γ F ) = L(F = 1, γ = 1) (6)
where L denotes here either the consumption or displacement Markstein length. We consider the Markstein number Ma defined as:

Ma(γ) = L(F, γ) Fδ 0 L (7)
Dividing Eq. ( 6) by Fδ 0 L leads to the following identity:

Ma(γ = γ F ) = Ma(γ = 1) F ( 8
)
The methodology adopted to determine γ F is then as follows: (i) we compute a series of stretched flames with varying values of γ, using each time the adequate correction factor A(γ), and we record the Markstein number Ma(γ). These flames may be computed at any fixed thickening F by virtue of the proportionality relationship given in Eq. ( 7). (ii) We solve Eq. ( 8) for the unknown variable γ F using an adequate optimization algorithm. The procedure is illustrated in Fig. 1.

Step (i) might be performed by considering either curved or strained flames. In the present paper, the estimation of γ F is performed by considering spherical curved flames.

A more detailed discussion of this choice is left for future work. Additionally, γ F might be determined considering the consumption or the displacement Markstein, leading to different estimations. A discussion on this aspect will be given in Sec. 3.

Figure 1: Illustration of the method to determine γ F

Validation conditions

The proposed methodology is assessed on stoichiometric C 8 H 18 /air (Le > 1) and lean H 2 /air (Le < 1) mixtures. Both mixtures are considered at temperature T = 300K and pressure P = 1bar. A 2-step mechanism involving 6 species [START_REF] Suillaud | Direct numerical simulations of high karlovitz number premixed flames for the analysis and modeling of the displacement speed[END_REF] is used for the isooctane case while a detailed mechanism (21 reactions, 10 species) [START_REF] Conaire | A comprehensive modeling study of hydrogen oxidation: A Comprehensive Modeling Study of Hydrogen Oxidation[END_REF] is used for the lean hydrogen mixture (Φ = 0.47). The laminar flame characteristics, obtained from premixed adiabatic laminar flames computed with the CANTERA 1D solver [START_REF] Goodwin | Cantera: An objectoriented software toolkit for chemical kinetics, thermodynamics, and transport processes[END_REF], are (i) S 0 L = 0.35m/s and δ 0 L = 357µm for the C 8 H 18 case (ii) S 0 L = 0.41m/s and δ 0 L = 466µm for the H 2 case.

Results and discussion

The application of the corrected TFM model to H 2 and C 8 H 18 cases is explored here. It is first applied on spherical flames in Sec. 3.1, which serve both to fit the γ parameter and as a first validation set-up. The model is then validated on flame-vortex interactions in Sec. 3.2.

Spherical flames 3.1.1 Numerical set-up

Spherical-flame simulations are performed with the CONVERGE CFD software [START_REF] Richards | [END_REF], which features a finite volume solver on Cartesian meshes. The present work considers in particular the resolution of 1-D spherical flame equations. This formulation enables an efficient computation of these flames and avoids the presence of intrinsic 3-D flame instabilities. The simulation domain length a is scaled with the thickening factor F such that a = F × 0.73m for both mixture cases. Mesh size is set as ∆ x = F × (δ 0 L /10). Hence, for every thickening factor considered, the flame front is solved on approximately n ∼ 10 grid points.

Markstein lengths for spherical flames

We consider a spherical flame front propagating in the outwards direction into quiescent premixed fresh gases. The rate of change of the flame surface is directly related to the flame radius R BG , determined in the burnt gas side:

K = 2 R BG dR BG dt = κS b d (9)
S b d and κ represent respectively the flame front displacement speed and the flame front curvature considered in the burnt gas. The flame front radius is evaluated from the burnt gas mass in the computational domain. The flame displacement speed considered in the burnt gas side is S b d = dR BG /dt. The consumption and displacement Markstein lengths are determined by a linear regression of S c (K) and S b d (K) on the stretch rate interval K = [20/F; 100/F]s -1 . Figure 2 shows the displacement and consumption Markstein numbers as a function of γ for the H 2 case.

Table 1 provides values of the Markstein lengths, for thickening factors values equal to 1 and 8. It emphasizes The methodology presented in Sec. 2 is used to determine the appropriate γ F to retrieve the reference Markstein length for a thickened flame. In particular, the evolution of Ma(γ) computed from spherical flames, as represented in Fig. 2 for the H 2 case, is used to determine γ F . Table 2 gives the correction factor values γ F for various thickening factors F, based either on Ma b d (γ) or Ma c (γ). lean H 2 /air mixture (Le < 1), the consumption Markstein length is negative and S c is greatly overestimated for the F = 8 thickened flame. A simulation with the TFM correction proposed by Quillatre [8] is also added in Fig. 3. According to Eq. ( 4), the corrected Lewis numbers rapidly tends towards 1 as F increases. This is evidenced here in Fig. 3 The initial vortex position is adjusted so that the centre of the vortex is equidistant from the peak reaction location for the real and thickened flame cases. We consider here the C 8 H 18 case only, since planar lean H 2 /air flames are subject to thermo-diffusive flame instabilities due to the low Lewis number. Hence, the methodology can't be assessed on the lean H 2 case because small-scale instability structures are nearly suppressed at F = 4 while they will appear at F = 1. A more quantitative comparison is performed considering space averaged properties of the flame. We define the flame length as:

Results

L f lame = S ∥∇c∥dS ( 10
)
Where c is the progress variable defined as c = 1-Y C 8 H 18 /Y u C 8 H 18 , where Y u C 8 H 18 corresponds to the value in fresh gases. S refers to the 2-D computational domain. The evolution of L f lame in time is represented in Figure 6: Flame length with respect to flame times. Fig. 6. It shows a significant under-estimation of the flame length for the thickened case with standard TFM compared to the non-thickened case. This is due to the flame speed underestimation induced by TFM. For all thickened flame fronts, with or without the corrected diffusion coefficients, the maximal flame length is the same. The underestimation of the flame length maximum value for thickened flames compared to the reference flame is due to a loss of flame surface for highly curved zone encountered after the penetration of the vortex in the flame (see the snapshots in Fig. 5). For the thickened flame front without correction, the maximal flame length is reached with a delay equivalent to 1.5τ f lame compared to the reference case (Fig. 6). On the contrary, the corrected TFM case based on displacement Markstein numbers reaches the maximal peak of flame length with an advance of 0.5τ f lame . The maximal flame length is obtained simultaneously for the real flame and the thickened front with diffusion correction based on consumption Markstein numbers.

For a given flame geometry, here estimated by the flame length L f lame , an averaged flame consumption can be defined as: [START_REF] Suillaud | Direct numerical simulations of high karlovitz number premixed flames for the analysis and modeling of the displacement speed[END_REF] with index f referring to the fuel species. Fig. 7 displays the averaged consumption speed as a function of the flame length. The laminar flame speed deduced from the simulation of a 1D planar flame with the CONVERGE solver [START_REF] Richards | [END_REF], which equals S 0 l = 0.37m/s is added to the plot. The flame-vortex interaction induces a flame length increase with a predominantly concave curvature towards the burnt gases. As Le > 1, this configuration leads to a decrease of the flame speed as illustrated by the reference case in Fig. 7. The thickened flame front without correction features an overestimated decrease of the averaged speed, in line with the over-sensitivity of the TFM model to stretch. The averaged consumption speed obtained with the correction based on the displacement Markstein length is on the contrary over-estimated which is consistent with conclusions drawn from Fig. 3. For the thickened flame front with diffusion correction deduced from the consumption speed, S c coincides with the reference case. Hence, the change of consumption speed induced by preferential diffusion effects is accurately recovered for a thickened flame front when modifying species diffusion according to the proposed methodology performed with the consumption Markstein number Ma c .

S c = 1 L f lame × -1 ρ u Y f u -Y f b S ω f dS (

Conclusions

The present study proposes a framework to correctly take into account stretch effects on artificially thickened flames. The standard TFM approach induces an oversensitivity of thickened flame fronts to stretch, which leads to a multiplication of the Markstein length by the thickening factor factor F. To overcome this limitation, we proposed a TFM-adapted methodology to recover the flame speed of weakly stretched flames. The model, based on the adaptation of species Lewis numbers, was evaluated both for a Le < 1 mixture (lean H 2 /air case) and for a Le > 1 mixture (stoichiometric C 8 H 18 /air case). The present work illustrates the distinction between the influence of stretch (i) on flame consumption speed and (ii) on flame displacement speed, characterized respectively by a consumption Markstein length L c and a displacement Markstein length L b d [START_REF] Giannakopoulos | Consumption and displacement speeds of stretched premixed flames -theory and simulations[END_REF]. It was shown that our TFM-adapted diffusion model can be used either to recover the reference value of L c or L b d . However, it is not possible to preserve both Markstein lengths simultaneously. Both approaches were shown to provide improved results compared to the standard TFM on a flame-vortex interaction case. Choosing L c as a target allows in addition to recover nearly exactly the mean consumption speed. Both approaches will need to be evaluated on practical burner cases in a future study.

Figure 2 :

 2 Figure 2: Markstein numbers with respect to γ for H 2 mixture (Φ = 0.47, T = 300K, P = 1bar)

Table 1 : 3

 13 Markstein lengths evaluated from the linear regression of flame consumption speed S c (Eq2) and flame displace-Estimation of γ F

Fig. 3

 3 Fig.3illustrates the evolution of S c with the stretch K for the H 2 spherical flames. The reference case is represented by the continuous black line. For the present

Figure 3 :Figure 4 :

 34 Figure 3: Flame consumption speed with respect to stretch for the H 2 mixture (Φ = 0.47, T = 300K, P = 1bar)

  by the fact that S c computed with Quillatre's correction is insensitive to curvature. The flame consumption speed is overestimated when using γ F=8 estimated from Ma b d (γ), as seen in Figure 3. On the contrary, S c is perfectly recovered when using γ F=8 estimated from Ma c (γ). Similarly, stretched-flame displacement speeds S b d (K) obtained with those various approaches are provided with Fig. 4. The flame displacement speed is overestimated for the non-corrected thickened flame while it is underestimated with Quillatre correction and diffusion correction based on Ma c (γ). The evolution of S b d (K) is accurately recovered with the corrected TFM based on Ma b d (γ). Finally, the proposed corrected TFM enables to recover an accurate flame response to stretch, either preserving the flame consumption speed or the flame displacement speed of an iso-surface considered in the burnt gas. However, it appears that preserving both consumption Markstein lengths L c and L b d is impossible. It is then necessary to understand which formulation performs better in actual flame-turbulence interactions. A first answer to this question is attempted in the next section, where flame/vortex interactions are considered.

3. 2

 2 Validation on flame-vortex interactions 3.2.1 Numerical set-up The validation set-up consists of a planar flame propagating towards a vortex. The 2D computational domain is square with l = 200δ 0 L side length. The incoming vortex is defined with radius R = 10δ 0 L and its azimuthal velocity at R is u ′ = 5S 0 L . Hence, the characteristic vortex time is τ vortex = R/u ′ = 2τ f lame , where τ f lame = δ 0 L /S 0 L is the flame time. The base mesh size is such that the vortex radius is discretized on 12 grid points, i.e. ∆x = R/12 = 0.8δ 0 L , and the TFM-AMR model is used to ensure a flame front resolution of n = 10 points in the flame using Adaptive Mesh Refinement (AMR) [15]. Four simulations are carried out to evaluate the performance of the newly developed corrected TFM model: (i) a reference simulation with F = 1; (ii) a simulation with F = 4 and the standard TFM; (iii) a simulation with F = 4 and the corrected TFM based on L b d ; (iv) a simulation with F = 4 and the corrected TFM based on L c .

Figure 5 :

 5 Figure 5: C 8 H 18 mass fraction profiles at t = 17τ f lame with (F = 1; γ = 1) (top left),(F = 4, γ = 1) (top right), (F = 4; γ = 0.622) (bottom left) and (F = 4; γ = 0.545) (bottom right). The white line represents the T = 400K isotherm.

Fig. 5

 5 Fig. 5 shows the fuel mass fraction field for various configurations. The white line localizes the isotherm T = 400K. The reference case (F = 1) is a fully resolved non-thickened flame front. Results using the standard TFM model for F = 4 is represented on the top right plot. A clear difference of flame front surface is observed between the F = 1 and F = 4 cases. The concave region towards burnt gases enters deeper into the fresh gases for the reference flame than for the thickened flame. In fact, local flame speed is underestimated since Markstein length is overestimated by a factor F = 4 in the thickened case. Using diffusion corrections based on L c and L b d , respectively on the bottom left and right in Fig. 5, the surface of the thickened flame is well retrieved.A more quantitative comparison is performed considering space averaged properties of the flame. We define the flame length as:

Figure 7 :

 7 Figure 7: Averaged consumption speed with respect to the flame length.

Table 2 :

 2 Correction factor values γ F based on Ma c and Ma b d .

	Fuel	Method	γ F=4 γ F=8 γ F=16
	C8H18 displacement 0.545 0.510 0.495
		consumption 0.622 0.589 0.574
	H2	displacement 1.621 1.829 1.959
		consumption 1.860 2.211 2.472

3.1.4 A posteriori validation on spherical flames

Acknowledgements

The authors gratefully acknowledge Damien Aubagnac-Karkar (IFPEN) for his technical and scientific assistance on the implementation of the model in the Cantera software.


Dr Emmanuel Fenouillet
email: fenouillet.e@jean-roche.univ-mrs.fr

Rym Barbouche

Joe ¨l Courageot

Raymond Miquelis

The Catalytic Activity of Protein Disulfide Isomerase Is Involved in Human Immunodeficiency Virus Envelope-Mediated Membrane Fusion after CD4 Cell Binding

Protein disulfide isomerase (PDI) is a multifunctional protein with thiol-disulfide redoxisomerase activities. It catalyzes thiol-disulfide interchange reactions on the cell surface that may cause structural modifications of exofacial proteins. PDI inhibitors alter human immunodeficiency virus (HIV) spread, and it has been suggested that PDI may be necessary to trigger HIV entry. This study examined this hypothesis by using cell-to-cell fusion assays, in which the HIV envelope (Env) expressed on the cell surface interacts with CD4 + lymphocytes. PDI is clustered at the lymphocyte surface in the vicinity of CD4-enriched regions, but both antigens essentially do not colocalize. Anti-PDI antibodies and 2 inhibitors of its catalytic function altered Env-mediated membrane fusion at a post-CD4 cell binding step. The fact that the PDI catalytic activity present on lymphocytes is required for fusion supports the hypothesis that catalysts assist post-CD4 cell binding conformational changes within Env.

. For these reasons, PDI is considered to be a multifunctional protein with an important role in the processing of secretory proteins in the endoplasmic reticulum.

, PDI has been detected in association with the cell surface of various secretory cells (e.g., rat exocrine pancreatic cells [5], platelets [6], lymphoid cells [7], the erythroleukemia

cell line [START_REF] Zai | Cell-surface protein disulfide isomerase catalyzes transnitrosation and regulates intracellular transfer of nitric oxide[END_REF], and thyrocytes [START_REF] Mezghrani | Protein disulfide isomerase in FRTL-5 cells: pH dependent thyroglobulin/PDI interactions determine a novel PDI function in the post endoplasmic reticulum of thyrocytes[END_REF]). Some reports have indicated that cell surface PDI catalyzes thiol-disulfide interchange reactions. This activity may control the functions of extracellular proteins through the induction of structural modifications. For example, PDI influences the cleavage of disulfides required for the activation of the diphtheria toxin through a thiol-disulfide interchange reaction [START_REF] Ryser | Cell surface sulfhydryls are required for the cytotoxicity of diphtheria toxin but not of ricin in Chinese hamster ovary cells[END_REF], and it mediates functional conformation changes that lead to the shedding of the human thyrotropin receptor ectodomain through the cleavage of disulfide bonds [START_REF] Couet | Cell surface protein disulfide isomerase is involved in the shedding of human thyrotropin receptor ectodomain[END_REF].

The mature human immunodeficiency virus (HIV) envelope (Env) is composed of the outer membrane glycoprotein (gp) 120 and transmembrane gp41 subunits. HIV binding to CD4 ϩ lymphocytes is mediated by gp120 interaction with CD4 ϩ cells [START_REF] Ugolini | HIV-1 attachment: another look[END_REF]. After CD4 ϩ cell binding, various gp120 domains interact with various viral coreceptors present on the lymphocyte surface, including chemokine receptors and enzymes [START_REF] Ugolini | HIV-1 attachment: another look[END_REF][START_REF] Avril | Identification of the U937 membrane-associated proteinase interacting with the V3 loop of HIV-1 gp120 as cathepsin G[END_REF][START_REF] Broder | Chemokine receptors and HIV[END_REF].

Conformational changes within Env follow these interactions between the virus and the surface of the target cells [START_REF] Broder | Chemokine receptors and HIV[END_REF]. These post-CD4 binding events are necessary to activate the fusogenicity of gp41 and, consequently, to induce virus-cell membrane fusion and virus entry [START_REF] Chan | HIV entry and its inhibition[END_REF]. These structural modifications are usually thought to result primarily from the intrinsic properties of the Env interacting with HIV receptors. It may be assumed, however, that catalysts also assist in these conformation changes, which may include a partial reorganization of the network of the 10 Env disulfide bonds [START_REF] Leonard | Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells[END_REF]. Such a hypothetical modification requires a redox-isomerase activity, and PDI is a prime candidate to exert this function. The observation made in a pioneering work by Ryser et al. [START_REF] Ryser | Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction[END_REF] supports this hypothesis. These authors showed that the catalytic function of PDI is required for HIV spread in cell cultures, since various inhibitors blocking this activity interfere with HIV infection. On the basis of this result, they postulated, but did not show, that PDI is necessary for virus-cell fusion and HIV entry. Their work [START_REF] Ryser | Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction[END_REF] is the only one on the role of PDI in HIV infection, and it is not clear why so much time has elapsed without further studies on this topic. Several years after the discovery of the coreceptors, however, the molecular basis of the receptor-induced conformational changes that take place in Env remain to be elucidated. The role that PDI plays in Env conformation changes is intriguing and deserves further study.

Materials and Methods

Reagents. We used PDI (Pierce) and anti-PDI rabbit polyclonal antibodies (SPA-890; StressGen). LY181984 was a generous gift from Lilly Laboratories [START_REF] Morre | A protein disulfidethiol interchange activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea[END_REF]. Chemical products were of analytical grade (Sigma). VV9-1 is a recombinant vaccinia virus (rVV; gift of M. P. Kieny, Transge ´ne S. A.) that encodes the native fusogenic HIV Lai Env [START_REF] Kieny | Improved antigenicity of the HIV env protein by cleavage site removal[END_REF]. VV1163 is an rVV that encodes a soluble form of gp160 Lai resulting from the disruption of both cleavage sites present on the native gp160 precursor and from the elimination of the transmembrane domain of the gp41 subunit; consequently, this antigen does not associate on the cell surface [START_REF] Kieny | Improved antigenicity of the HIV env protein by cleavage site removal[END_REF].

Confocal laser scanning microscopy (CLSM). We cultured human lymphoid CEM, MolT4, and C8166 CD4 ϩ cells in RPMI 1640 medium, supplemented with 5% fetal calf serum (FCS) and 1% glutamine (Gln). In 2 of 4 experiments, cells were activated by the phaseolus vulgaris leucoagglutinin lectin (PHA; Sigma; 5 mg/mL) for 12 h [START_REF] Hasegawa-Sasaki | Phytohemagglutinin induces rapid degradation of phosphatidylinositol 4,5-bisphosphate and transient accumulation JID 2001;183 (1 March) of phosphatidic acid and diacylglycerol in a human T lymphoblastoid cell line, CCRF-CEM[END_REF][START_REF] Clevers | Wheat germ agglutinin activates human T lymphocytes by stimulation of phosphoinositide hydrolysis[END_REF]. After a wash step, cells were incubated with blocking buffer (PBS; 10% bovine serum albumin [BSA]) and then were incubated with the anti-PDI antibodies SPA890 (1:50 in PBS-0.1% BSA) for 1 h at 4ЊC [START_REF] Mezghrani | Protein disulfide isomerase in FRTL-5 cells: pH dependent thyroglobulin/PDI interactions determine a novel PDI function in the post endoplasmic reticulum of thyrocytes[END_REF][START_REF] Courageot | Intracellular degradation of the HIV-1 envelope glycoprotein[END_REF]. After a wash with PBS-10% BSA, anti-rabbit antibodies (1:100) coupled to Texas red dye (Jackson Immunoresearch) were added for 1 h. The mouse monoclonal antibody (MAb) CD4 OKT4 (1:30; Ortho Diagnostics Systems) and anti-mouse antibodies (1:100) coupled to fluorescein isothiocyanate dye were used to label CD4 cells. Cells then were washed, were fixed in 4% (wt/vol) paraformaldehyde for 15 min, and were mounted on slides in Mowiol (Calbiochem). Samples were submitted to CLSM (Leica TCS 4D; Leica Lasertechnik).

We monitored a focal series of 8-10 horizontal section planes (0.5 mm apart) for each staining. The individual 8-bit-encoded pixel images, 1 for each channel for each section plane, 512 ϫ 512 were visualized with a true color display screen and were processed for analysis of colocalization. A single horizontal section plane representative of the cell sample was used for illustration. To assess fluorescence colocalization, the pixel images correspond-512 ϫ 512 ing to the single horizontal section plane used for illustrations were processed with our own computer analysis software. This method selects the regions of true overlap of green and red staining from the non-colocalized staining and allocates white dots to pixels corresponding to the regions where a yellow (red-green colocalization) signal is detected.

Aggregation and cell-to-cell fusion assays. CEM cells cultured in 48-well plates ( cells/300 mL) were infected by VV9-1 (2 5 3 ϫ 10 pfu/cell), as described elsewhere [START_REF] Fenouillet | Functional role of the glycan cluster of HIV-1 transmembrane glycoprotein gp41[END_REF]. Various molecules (Bacitracin, 5,5 -dithiobis 2-nitrobenzoic acid [DTNB], PDI, anti-PDI antibodies, and LY181984 at concentrations indicated below) were added to wells 5 h after rVV infection and before Env-mediated cell aggregation and cell-to-cell fusion appeared in the control well, which was free of inhibitor throughout the experiment. We obtained similar data when the compounds were added together with VV9-1 at the onset of the experiment.

We assessed the ratio (percentage) of the total surface occupied either by cell aggregates (before syncytia appeared) or by syncytia (24 h after infection) versus the surface of the field of vision in 5 fields of vision as follows. First, we scanned a photograph of each field of vision. Then, with Adobe Photoshop software (Adobe Systems) and our own computer analysis software, the areas occupied by aggregates or syncytia were outlined on the photograph, and the sum of the corresponding surfaces was calculated and plotted versus the surface of the field of vision. The cytotoxicity of DTNB and bacitracin was assessed by a thiazolyl blue (MTT) assay (Sigma): cells were incubated with MTT (0.5 mg/mL) for 3 h at 37ЊC. The reaction was stopped with a lysis buffer (50% dimethylformamide and 20% SDS, pH 4.5). The amount of MTT formazan product was determined (570/655 nm).

In some experiments, CEM or baby hamster kidney (BHK-21) cells (10 5 cells/mL) were infected by VV9-1 (1 and 5 pfu/cell, respectively) in 24-well plates, as described elsewhere [START_REF] Fenouillet | Functional role of the glycan cluster of HIV-1 transmembrane glycoprotein gp41[END_REF][START_REF] Fenouillet | Recombinant HIV envelope expressed in an alpha glucosidase I deficient CHO cell line and its parental cell line in the presence of deoxynojirimycin is functional[END_REF][START_REF] Fenouillet | Biological properties of recombinant HIV envelope synthesized in glycosylation mutant CHO cell lines[END_REF], and were incubated with PDI inhibitors 8 h later. CD4-negative BHK-21 cells were cultured in Glasgow medium supplemented with 5% FCS, 5% tryptose phosphate, and 1% Gln. Such low cell densities do not enable cell aggregation or syncytium formation at postinfection day 2 in CEM cell culture [START_REF] Barbouche | An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the Env precursor interferes with membrane fusion at a post-CD4 binding step[END_REF]. Cell supernatants were removed 36 h after infection to prevent (see also [START_REF] Fenouillet | Recombinant HIV envelope expressed in an alpha glucosidase I deficient CHO cell line and its parental cell line in the presence of deoxynojirimycin is functional[END_REF][START_REF] Fenouillet | Biological properties of recombinant HIV envelope synthesized in glycosylation mutant CHO cell lines[END_REF]) the subsequent infection by VV9-1 of the MolT4 fusion partner cells ( cells/mL) that expressed the various HIV receptors that 5 5 ϫ 10 were added in the presence or absence of inhibitors for another 10-15 h. Cell aggregates and fusions were assessed as described above. Finally, C8166 cells chronically infected with HIV MN were washed and were incubated in 96-wells plates (10 4 cells/100 mL) [START_REF] Benjouad | Effect of sialic acid removal on the antibody response to the third variable domain of human immunodeficiency virus type-1 envelope glycoprotein[END_REF]. MolT4 cells ( cells/100 mL) were then added in the 4 5 ϫ 10 presence or absence of DTNB. Syncytia were counted 18 h later, as described elsewhere [START_REF] Benjouad | Effect of sialic acid removal on the antibody response to the third variable domain of human immunodeficiency virus type-1 envelope glycoprotein[END_REF].

Detection of production of Env species by Western blot. To express Env, CEM cells were infected with VV9-1 (2 pfu/cell) in the presence or absence of DTNB (10 Ϫ3 M). Cell pellets (10 6 cells) were washed once in PBS (pH 7.4) and were lysed in sample buffer, as described elsewhere [START_REF] Barbouche | An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the Env precursor interferes with membrane fusion at a post-CD4 binding step[END_REF]. Cleared lysates and cell-free supernatants were analyzed by 10% SDS-PAGE. Gels were blotted onto a nitrocellulose filter. After blocking with PBS-2% casein (PBSC) and 0.5% Tween 20 for 1 h, the nitrocellulose strips were incubated for 1 h at 25ЊC with a pool of human HIV-positive or HIV-negative serum samples (1:100) [START_REF] Fenouillet | Functional role of the glycan cluster of HIV-1 transmembrane glycoprotein gp41[END_REF][START_REF] Fenouillet | Immunological analysis of human immunodeficiency virus type 1 envelope glycoprotein proteolytic cleavage[END_REF]. Incubations and washes were done in PBSC and 0.5% Tween 20. After 2 washes, staining was performed by using peroxidase-coupled antibodies (1:100; Dakopatts) and diaminobenzidine.

Detection of expression of Env at the cell surface. CEM cells were infected as described above. After a wash with PBS, cells were incubated for 2 h at 25ЊC with various dilutions (1:30-1:10,000 in PBSC) of a pool of HIV-positive serum samples to detect membrane Env, as described elsewhere [START_REF] Fenouillet | Recombinant HIV envelope expressed in an alpha glucosidase I deficient CHO cell line and its parental cell line in the presence of deoxynojirimycin is functional[END_REF][START_REF] Fenouillet | Biological properties of recombinant HIV envelope synthesized in glycosylation mutant CHO cell lines[END_REF][START_REF] Barbouche | An anti-HIV peptide construct derived from the cleavage region of Env acts on Env fusogenicity through the presence of a functional cleavage sequence[END_REF]. The use of a pool of serum samples instead of MAbs to quantify surface Env expression avoids potential drawbacks due to immunoreactivity changes within Env in relation to the use of inhibitors [START_REF] Fenouillet | Recombinant HIV envelope expressed in an alpha glucosidase I deficient CHO cell line and its parental cell line in the presence of deoxynojirimycin is functional[END_REF][START_REF] Fenouillet | Biological properties of recombinant HIV envelope synthesized in glycosylation mutant CHO cell lines[END_REF]. Incubations and washes were performed in PBSC in the presence of 0.1% NaN 3 , which blocks endocytosis [START_REF] Barbouche | An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the Env precursor interferes with membrane fusion at a post-CD4 binding step[END_REF][START_REF] Barbouche | An anti-HIV peptide construct derived from the cleavage region of Env acts on Env fusogenicity through the presence of a functional cleavage sequence[END_REF] and enables antibody binding to the cell surface Env to be monitored. To assess whether the signal induced by the pool of HIV-positive serum samples depended on the presence of cell surface-associated Env, we used the following controls: VV9-1-infected cells were incubated with a pool of HIV-negative serum samples and VV1163-infected or VV1163-uninfected cells were incubated with the pool of HIVpositive serum samples. After a wash step, peroxidase-coupled antihuman antibodies were added (1:100 in PBSC) for 1 h. After 2 washes, we added ortho-phenylenediamine to the cells. We measured the absorbance (492 nm/620 nm) of the cell supernatant 30 min after cell pelleting, to prevent interference with absorbance measurement. These conditions enable specific detection of surfaceassociated Env in an antibody concentration-dependent manner [START_REF] Fenouillet | Functional role of the glycan cluster of HIV-1 transmembrane glycoprotein gp41[END_REF][START_REF] Fenouillet | Recombinant HIV envelope expressed in an alpha glucosidase I deficient CHO cell line and its parental cell line in the presence of deoxynojirimycin is functional[END_REF][START_REF] Barbouche | An anti-HIV peptide construct derived from the cleavage region of Env acts on Env fusogenicity through the presence of a functional cleavage sequence[END_REF].

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Env and CD4 binding assay. Soluble CD4 (gift of I. M. Jones via Medical Research Council [United Kingdom] Reagents Program) was labeled as described elsewhere [START_REF] Barbouche | An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the Env precursor interferes with membrane fusion at a post-CD4 binding step[END_REF]: 5 mg was incubated for 30 min at 25ЊC in a 4-nM iodogen-coated tube with 250 mCi of 125 I-labeled Na. The labeling reaction was stopped by the addition of an excess of tyrosine. The labeled antigen (30 mCi/mg) was purified by chromatography. CD4 Ϫ BHK-21 cells were infected with VV9-1 (10 pfu/cell) for 2 days, to enable a high level of Env expression. Cells (1.5 and

) that expressed equivalent 6 3 ϫ 10 amounts of cell surface Env (determined as described above), regardless of the presence of DTNB during Env synthesis, were washed in PBS and were incubated for 2 h at 37ЊC with 125 I-labeled CD4 cells ( cpm/100 mL) in PBSC-0.1% NaN 3 . After 3 5 3 ϫ 10 washes, cell-associated radioactivity was assessed. Uninfected and VV1163-infected cells were similarly processed, to determine the background binding of CD4 to cell surface exhibiting no Env species. The specificity of 125 I CD4 cell labeling was also assessed by incubating VV9-1-infected cells with soluble CD4 (1 mg/10 6 cells) for 30 min before the addition of 125 I-labeled CD4.

Results

PDI is present near CD4-enriched regions on lymphoid cell surfaces.

We verified whether the target of the PDI inhibitors was present on the surface of the CD4 ϩ lymphocytes used in our assays, and we investigated the surface distribution of CD4 and PDI labeling. About 80% and 50% of CEM cells exhibited a readily detectable CD4 (green) and PDI (red) labeling, respectively (figure 1). The labeling obtained by using the anti-CD4 antibody was rather homogeneous and was present on ∼50%-70% of labeled cell surfaces. In contrast, the labeling obtained with the anti-PDI antibodies was organized in small clusters on the cell surface.

To study whether the surface expression of PDI depended on the state of the lymphocyte activation, lymphocytes were treated with PHA in conditions that induce CEM cell activation [START_REF] Hasegawa-Sasaki | Phytohemagglutinin induces rapid degradation of phosphatidylinositol 4,5-bisphosphate and transient accumulation JID 2001;183 (1 March) of phosphatidic acid and diacylglycerol in a human T lymphoblastoid cell line, CCRF-CEM[END_REF][START_REF] Clevers | Wheat germ agglutinin activates human T lymphocytes by stimulation of phosphoinositide hydrolysis[END_REF]. This treatment did not substantially modify the intensity of the labeling of PDI, but it increased the CD4 cell labeling Downloaded from https://academic.oup.com/jid/article/183/5/744/891086 by guest on 31 May 2023 (figure 1). PDI and CD4 were also detected on the surface of C8166 and MolT4 cells (data not shown). Of note, in the cell lines, PDI labeling was usually near the cell surface regions where CD4 cell labeling was important. However, PDI and CD4 exhibited essentially no colocalization on the cell surface, as indicated by the low level of yellow (red-green colocalization) signal on the true color display screen of our equipment in the surface regions where both labelings coexisted. The data shown in figure 1 were processed to further demonstrate the regions of true overlap of anti-PDI and anti-CD4 cell labeling. With this approach, the low level of colocalization of CD4 and PDI is immediately obvious (figure 1).

PDI inhibitors, cell aggregation, and cell-to-cell fusion. The catalytic activity of PDI was specifically [START_REF] Ryser | Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction[END_REF] inhibited by anti-PDI antibodies and by 2 membrane-impermeant PDI inhibitors, DTNB and the antibiotic, bacitracin. Both have a narrow specificity: DTNB is a sulfhydryl blocker that covalently reacts with the catalytic sites of PDI and abolishes its capacity for cleaving disulfides, whereas bacitracin inhibits both its reductive and oxidative functions [START_REF] Couet | Cell surface protein disulfide isomerase is involved in the shedding of human thyrotropin receptor ectodomain[END_REF][START_REF] Ryser | Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction[END_REF][START_REF] Mandel | Inhibition of a reductive function of the plasma membrane by bacitracin and antibodies against protein disulfide isomerase[END_REF]. We first determined whether these inhibitors could inhibit the Env-mediated cellto-cell fusion process in human lymphocyte cultures infected by an rVV encoding mature native Env on the cell surface.

This assay is reliable for addressing both Env bioactivity and its ability to interact with a cell surface presenting the various membrane components required for membrane fusion [START_REF] Ashorn | Vaccinia virus vectors for study of membrane fusion mediated by human immunodeficiency virus envelope glycoprotein and CD4[END_REF]. It enables the efficient discrimination between Env binding to CD4 cells and the membrane fusion step [START_REF] Kieny | Improved antigenicity of the HIV env protein by cleavage site removal[END_REF][START_REF] Fenouillet | Functional role of the glycan cluster of HIV-1 transmembrane glycoprotein gp41[END_REF][START_REF] Barbouche | An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the Env precursor interferes with membrane fusion at a post-CD4 binding step[END_REF]. Indeed, the presence of cell aggregates depends on the interaction of cell surface-associated gp120 with CD4, as shown elsewhere [START_REF] Barbouche | An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the Env precursor interferes with membrane fusion at a post-CD4 binding step[END_REF]. In brief, the relevance of these aggregates in terms of gp120 and CD4 interaction is shown by the fact that these formations are blocked [START_REF] Barbouche | An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the Env precursor interferes with membrane fusion at a post-CD4 binding step[END_REF] when Env is expressed by using an rVV coding for a soluble form of Env that does not associate with the cell surface (soluble gp120 and soluble gp160 encoded by VV1132 and VV1163, respectively [START_REF] Kieny | Improved antigenicity of the HIV env protein by cleavage site removal[END_REF]), and not when soluble CD4 (1 mg/100 mL) is added into the cell culture medium or when the anti-CD4 neutralizing Leu3a antibody (500 ng/100 mL) is present in the cell medium.

When Env was expressed at the cell surface by use of an rVV coding for fusogenic Env (VV9-1) and when fusion partner cells expressed surface CD4 in a context of various HIV coreceptors [START_REF] Ashorn | Vaccinia virus vectors for study of membrane fusion mediated by human immunodeficiency virus envelope glycoprotein and CD4[END_REF], aggregates between Env-expressing cells and fusion partners developed in a few hours into large multinucleated cells (syncytia). These syncytia specifically reflect the coreceptordependent gp41-mediated membrane fusion process, as demonstrated by the following data. First, the presence of anti-V3 neutralizing antibodies interfered with cell-to-cell fusion but not with cell aggregation. Second, large aggregates, but no syncytium, appeared when Env was expressed on the cell surface by a gene coding for a nonfusogenic Env. The latter resulted from disruption of both gp160 cleavage sites (which impaired the activation of the gp41 fusogenicity [VV1139] [START_REF] Kieny | Improved antigenicity of the HIV env protein by cleavage site removal[END_REF]), removal of the cleavage site (REKRrNEHQ, which is essential for unmasking the gp41 fusion peptide [VV1134] [START_REF] Kieny | Improved antigenicity of the HIV env protein by cleavage site removal[END_REF]), and elimination of the gp41 fusion peptide (VV1136) [START_REF] Kieny | Improved antigenicity of the HIV env protein by cleavage site removal[END_REF]. These control procedures were carried out during development of the assays, to assess routine experimental procedures and to determine the reliability of the test format (see [START_REF] Barbouche | An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the Env precursor interferes with membrane fusion at a post-CD4 binding step[END_REF] for details).

In each experiment, we also determined the specificity of the assay. First, cell aggregation was inhibited by 70%-80% by the addition of soluble CD4 (1 mg/well). Second, infection by VV0 (a VV vector coding for a membrane Env that exhibits CD4 cell binding capacity but not fusogenicity [START_REF] Fenouillet | Functional role of the glycan cluster of HIV-1 transmembrane glycoprotein gp41[END_REF]) induced cell aggregation but not cell-to-cell fusion. Third, infection by VV1163, which codes for soluble gp160, did not induce cell aggregation or syncytium formation.

Compared with control conditions (i.e., CEM cell cultures infected by VV9-1 in the absence of inhibitor), the presence of either bacitracin or DTNB in the culture medium from the time that VV9-1 was added to the cell culture exerted a dosedependent inhibition of cell-to-cell fusion when cultures were observed 24 h after infection. Inhibition was important at M (figure 2A). Syncytium formation was abolished at Ϫ4 3 ϫ 10 10 Ϫ3 M, in contrast to cell aggregation, which remained unchanged. Similar data were obtained when cells were infected by VV9-1 in the absence of inhibitors to allow VV infection to proceed under routine conditions and when anti-PDI compounds were added to the culture 5 h later. Cell viability in uninfected cell cultures was assessed in parallel experiments after 2 days of treatment with either bacitracin or DTNB and by MTT assay and by monitoring cell counts (figure 2A). These parameters were compared with those obtained in untreated cell cultures. DTNB inhibited syncytium formation at concentrations that lacked cytotoxicity, in contrast to bacitracin. For these reasons, we used 10 Ϫ3 M DTNB in the following experiments to block cell-to-cell fusion.

The sulfonyl urea compound LY181984 inhibits thiol-disulfide interchange activities associated at the surface of human cell lines in the micromolar range but not that of PDI [START_REF] Morre | A protein disulfidethiol interchange activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea[END_REF]. Thus, its lack of effect on syncytium formation at concentrations of 10 Ϫ4 to 10 Ϫ10 M (figure 2B shows results at 10 Ϫ4 and 10 Ϫ6 M) argues against a specific role for PDI in fusion. Direct evidence of the involvement of PDI in the fusion process was shown with anti-PDI antibodies (1:50), which inhibited cell-tocell fusion, in contrast to irrelevant (preimmune) rabbit antibodies; cell aggregation remained unchanged. We also tested the effect of the presence of recombinant soluble PDI in cell medium on cell-to-cell fusion and observed that a high concentration of the molecule (16 mg/mL) did not modify syncytium formation.

Env production and membrane expression in the presence of DTNB.

We examined whether the inhibition by 10 Ϫ3 M DTNB of syncytium formation in the system described above was due to the inhibition of rVV infection or to an alteration Downloaded from https://academic.oup.com/jid/article/183/5/744/891086 by guest on 31 May 2023 of the synthesis, processing, and membrane expression of HIV Env.

CEM cells express Env with or without DTNB. Cell pellets were lysed by using sample buffer. Cleared lysates and cell supernatants were analyzed by Western blot. Under either condition, similar quantities of the various Env species were detected in the cell pellet by using a pool of HIV-positive serum samples; gp120 was efficiently secreted into the cell supernatant (figure 3A). The migration profiles of the Env species obtained here were similar to those reported with this expression system [START_REF] Fenouillet | Functional role of the glycan cluster of HIV-1 transmembrane glycoprotein gp41[END_REF][START_REF] Fenouillet | Recombinant HIV envelope expressed in an alpha glucosidase I deficient CHO cell line and its parental cell line in the presence of deoxynojirimycin is functional[END_REF][START_REF] Fenouillet | Biological properties of recombinant HIV envelope synthesized in glycosylation mutant CHO cell lines[END_REF][START_REF] Barbouche | An anti-HIV peptide construct derived from the cleavage region of Env acts on Env fusogenicity through the presence of a functional cleavage sequence[END_REF]. The 160-, 120-, and 41-kDa species detected with a pool of HIV-positive serum samples were HIV Env, since a pool of HIV-negative serum samples precluded their detection in samples derived from cells cultured under control conditions.

Alternatively, we incubated cells with a pool of HIV-positive serum samples to semiquantify the surface expression of Env [START_REF] Fenouillet | Recombinant HIV envelope expressed in an alpha glucosidase I deficient CHO cell line and its parental cell line in the presence of deoxynojirimycin is functional[END_REF][START_REF] Fenouillet | Biological properties of recombinant HIV envelope synthesized in glycosylation mutant CHO cell lines[END_REF]. When we used saturating concentrations of serum samples (1:30-1:100), the amount of Env expressed in the presence or absence of DTNB on the surface of the same number of cells led to similar signals (figure 3B). Nonsaturating concentrations (1:300-1:1000) also indicated that the alteration of Env expression by DTNB treatment could not obviously account for the potent anti-Env activity of the compound. Cell surface labeling depends on the presence of surface-associated Env, as demonstrated by the following results. First, the signal obtained by incubation with HIV-positive serum samples of cells infected with VV1163 that codes for soluble gp160 represented 3%-8% of the signal obtained when VV9-1 was used. Second, a similar background signal was obtained when we used either a pool of HIV-negative serum samples to detect Env at the surface of VV9-1-infected cells (data not shown) or when the HIV-positive serum pool was used for incubation with uninfected cells. These data indicate that 10 Ϫ3 M DTNB lacks important cytotoxicity: the amount of recombinant Env produced in its presence was not significantly affected, and it does not significantly inhibit cell surface expression of mature Env in our conditions.

DTNB acts on the fusion process at a post-CD4 cell binding step.

We did additional aggregation/syncytium assays, in which DTNB was present during the synthesis of Env and its routing to the cell surface and/or during the coincubation between Env-expressing cells and CD4 ϩ fusion partner cells. Our goals were to study whether DTNB exerts its inhibitory activity only during the coincubation between Env-expressing cells and fusion partners and to further determine whether DTNB inhibits gp120 binding to CD4 cells or the membrane fusion step.

Table 1 shows the data when Env was expressed at the CEM cell surface and when fusion partners were MolT4 cells. This format efficiently discriminates between the CD4-gp120 binding step and the membrane fusion step: the specificity of cell aggregation in terms of gp120-CD4 interaction and of syncytium formation in terms of coreceptor-and gp41-mediated fusion were demonstrated as described above and elsewhere [START_REF] Barbouche | An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the Env precursor interferes with membrane fusion at a post-CD4 binding step[END_REF]. In this assay, the presence of either DTNB (table 1) or anti-PDI antibodies during the coincubation of Env-expressing cells and CD4 ϩ fusion partners (contact ϩ condition) significantly altered cell-to-cell fusion, compared with control conditions (synthesis Ϫ /contact Ϫ ). This confirmed the results shown in figure 2A. Moreover, a substantial inhibition of fusion was obtained when MolT4 cells were pretreated for 1 h with either DTNB or anti-PDI antibodies, to block surface PDI before being 3 ϫ 10 3 ϫ 10 with soluble CD4 (CD4) was also determined ( experiments; duplicates done). n p 2 washed and their subsequent incubation with Env-expressing cells for 10 h (Molt4 ϩ DTNB/wash/fusion); no potent effect was detected when DTNB only was present during Env synthesis (synthesis ϩ /contact Ϫ ). These 2 observations are consistent with the idea that the PDI molecules, whose catalytic activity is required for membrane fusion, are present at the surface of the fusion partners and are not those associated with Env ϩ cells. The fact that the presence of DTNB only during Env synthesis (synthesis ϩ /contact Ϫ ) could not potently inhibit the subsequent cell-to-cell fusion with fusion partner cells further demonstrates that 10 Ϫ3 M DTNB did not substantially alter the surface expression of mature and bioactive Env (figure 3A,3B).

When DTNB was present during coincubation with fusion partner cells, cell aggregation occurred. Similar data were obtained with anti-PDI antibodies. The lack of a potent effect of both DTNB and anti-PDI antibodies on the cell aggregation process is consistent with the results obtained in the VV9-1-infected CEM cell cultures (figure 2). The observation that DTNB did not block the interaction between gp120 and the CD4 cell receptor present on adjacent cells was further illustrated by a molecular CD4-gp120 binding assay: the presence of DTNB during the incubation of Env-expressing BHK-21 cells with 125 Ilabeled CD4 cells did not alter CD4 cell binding to membrane Env, compared with control conditions in which DTNB was absent during both Env expression and coincubation with CD4 cells (figure 3C). Env expressed in cells treated by DTNB also bound 125 I-labeled CD4 cells.

We controlled the specificity of the CD4 binding reaction several ways. First, the binding of 125 I-labeled CD4 to Env-expressing cells was ∼50% of that obtained with 6 1.5 ϫ 10 cells. Second, the binding to uninfected BHK-21 cells 6 3 ϫ 10 or to cells infected with VV1163 represented ∼5% of the binding to VV9-1-infected cells. Third, the binding was inhibited by ∼70% by preincubation of Env-expressing cells with soluble CD4 (1 mg/assay) before the addition of 125 I-labeled CD4. The results obtained with both the aggregation/fusion assays and the molecular CD4 binding assay indicate that DTNB interferes with the cell-to-cell fusion process at a post-CD4 binding step. This conclusion was further supported by other fusion assays. When Env was expressed at the surface of CD4 Ϫ BHK-21 cells, the fusion between the Env-expressing cells and the fusion partners [START_REF] Barbouche | An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the Env precursor interferes with membrane fusion at a post-CD4 binding step[END_REF] was inhibited only when DTNB was present during their coincubation. A significant inhibition also was achieved when MolT4 cells were preincubated with DTNB for 1 h before being washed and after incubation with Env-expressing BHK-21 cells. When C8166 cells chronically infected by HIV MN were washed and further incubated with MolT4 cells overnight to allow cell-to-cell fusion, a strong reduction (180%) in syncytium was induced by the presence of DTNB, bacitracin, or anti-PDI antibodies, compared with results in the absence of the inhibitors. The specificity of the syncytium formation was verified by addition of the anti-CD4 neutralizing Leu3a antibody (500 ng/100 mL) into the incubation medium, which blocks this process. Inhibition of syncytium formation also was obtained with neutralizing anti-V3 antibodies, as described elsewhere [START_REF] Benjouad | Effect of sialic acid removal on the antibody response to the third variable domain of human immunodeficiency virus type-1 envelope glycoprotein[END_REF]. HIV MN infections were also performed as described elsewhere [START_REF] Fenouillet | Role of N-linked glycans of envelope glycoproteins in infectivity of human immunodeficiency virus type 1[END_REF]. C8166 cells were incubated for 3 h with serial dilutions (10 1 -10 Ϫ2 mL) of a viral supernatant with or without DTNB. Cells then were grown for 12 days. A cytopathic effect correlated with p24 antigen production in the culture medium, Downloaded from https://academic.oup.com/jid/article/183/5/744/891086 by guest on 31 May 2023 NOTE. The human immunodeficiency virus envelope (Env) was expressed on surface of CEM cells in the presence (synthesis ϩ ) or absence (synthesis Ϫ ) of DTNB under conditions that do not enable cell aggregation or syncytium formation. After washing, Env-expressing cells were incubated for 10 h with fusion partner cells in the presence (contact ϩ ) or absence (contact Ϫ ) of DTNB. Alternatively, fusion partners were incubated for 1 h with DTNB before washing and fusion assay (MolT4 ϩ DTNB/wash/fusion). Ratio of total surface occupied either by cell aggregates or by syncytia vs. the surface of the field of vision was assessed ( experiments; representative experiment is shown; means of results obtained n p 4 in 5 fields of vision are presented). and p24 antigen production correlated with the virus dose used for infection. HIV infectivity was reduced by ∼3 orders of magnitude in the presence of DTNB: p24 antigen production after infection with 10 mL of the virus supernatant in the presence of DTNB was lower than that in the absence of DTNB with 10 Ϫ1 mL.

The data demonstrate the inhibition by specific PDI inhibitors (anti-PDI antibodies and DTNB) of a post-CD4 binding step of the Env-mediated membrane fusion process in various cell systems in which either recombinant or viral Env interacts with cell surface presenting CD4 in a context of various membrane components required for fusion.

Discussion

The main objective of this study was to address the postulate that PDI is involved in HIV Env-mediated membrane fusion. Ryser et al. [START_REF] Ryser | Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction[END_REF] provided convincing evidence that "HIV infection of human lymphoid cells is markedly inhibited by DTNB, by bacitracin, and by anti-PDI antibodies" (p. 4559). The presence of inhibitors of the catalytic activity of PDI during virus-cell interaction in these conditions, however, may have altered HIV infection at several steps of the infectious pathway. Inhibition of HIV entry is likely and can occur at 3 different steps: CD4 binding, the gp41-mediated membrane fusion process, and during uncoating events. It is also possible that HIV expression or budding events may be modified by cell metabolism changes or by cell membrane alterations that result either from entry of the inhibitors into the cells by endocytosis or from cell surface contact with these chemical agents. The first step of HIV infection, CD4 binding, may be impaired by PDI inhibition, inasmuch as this interaction is strongly dependent on the correct folding and disulfide network of both gp120 and CD4. Subsequent to CD4 binding and before virion fusion with the membrane of the host cell, PDI inhibition may also preclude the unfolding of gp120 as part of its interaction with the various HIV receptors (e.g., CXCR4 on CD4 ϩ lymphocytes). Thus, the gp41-mediated membrane fusion is also a step that may be altered by DTNB, bacitracin, and anti-PDI antibodies, because it requires a stepwise reorganization of the conformation of Env to trigger the ability of gp41 to mediate fusion. Finally, PDI inhibitors may have interfered with virion disassembly, which releases the reverse-transcription complex. This uncoating step is poorly documented and difficult to study but is highlighted by the use of compounds (catalysts [START_REF] Fenard | Secreted phospholipases A(2), a new class of HIV inhibitors that block virus entry into host cells[END_REF], antibodies [START_REF] Corbeau | Ig CDR3-like region of the CD4 molecule is involved in HIV-induced syncytia formation but not in viral entry[END_REF][START_REF] Mcinerney | A human IgG1 (b12) specific for the CD4 binding site of HIV-1 neutralizes by inhibiting the virus fusion entry process, but b12 Fab neutralizes by inhibiting a postfusion event[END_REF], and bicyclams [START_REF] De Clercq | Potent and selective inhibition of human immunodeficiency virus (HIV)-1 and HIV-2 replication by a class of bicyclams interacting with a viral uncoating event[END_REF]) that inhibit virus entry without altering CD4 binding or membrane fusion.

We addressed these issues by using biologically sound systems that reasonably mimic the natural interaction of HIV with the native lymphocyte surface. Cell surface-associated native Env was expressed via a vaccinia virus vector [START_REF] Kieny | Improved antigenicity of the HIV env protein by cleavage site removal[END_REF][START_REF] Fenouillet | Functional role of the glycan cluster of HIV-1 transmembrane glycoprotein gp41[END_REF]. This approach is recommended [START_REF] Ashorn | Vaccinia virus vectors for study of membrane fusion mediated by human immunodeficiency virus envelope glycoprotein and CD4[END_REF] and is widely used to specifically investigate the fusion mediated by Env and CD4 in the absence of other viral proteins that may skew the results, including those discussed by Ryser et al. [START_REF] Ryser | Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction[END_REF]. Alternatively, expression of Env at the lymphocyte surface has been obtained by HIV infection.

In our format, membrane Env interacted with adjacent CD4 ϩ lymphocytes that presented PDI on their surfaces in the vicinity of CD4-enriched membrane regions, as shown here for the first time, and in the context of HIV receptors and fusion partner molecules. First, we investigated whether PDI inhibitors modify the CD4 cell binding capacity of Env. Ryser et al. [START_REF] Ryser | Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction[END_REF] did not deal with this point but observed that CD4 immunolabeling at the cell surface is unchanged by treating cells with DTNB and that virions pretreated with DTNB can subsequently interact with the CD4 ϩ cell surface in the absence of DTNB. In contrast, we used semiquantitative assays to show that the presence of PDI inhibitors during Env synthesis or during interaction with CD4 cells had no significant influence on Env binding to the primary viral receptor: aggregation between CD4 ϩ and Env ϩ cells occurred in the presence of either DTNB or anti-PDI antibodies, and DTNB had no effect on Env binding to CD4 at the molecular level. In contrast, when Env was expressed in the absence of DTNB on the cell surface, the presence of the PDI inhibitor or of anti-PDI antibodies during the incubation of Env-expressing cells with CD4 ϩ fusion partner cells blocked syncytium formation. The various steps of the biosynthetic pathway of Env, including Env synthesis, processing, and membrane expression, apparently were unchanged by the inhibitor. This shows that both DTNB and anti-PDI antibodies act on the HIV receptor-dependent gp41-mediated fusion process per se through inhibition of a post-CD4 binding step.

Our data are also consistent with the hypothesis that the PDI involved in membrane fusion was on the target CD4 ϩ lym-phocytes: syncytium formation was inhibited when fusion partner cells were pretreated with either DTNB or PDI antibodies before being washed and before subsequent incubation with Env ϩ cells, whereas no significant effect was observed when cells expressing Env were similarly pretreated with DTNB. Moreover, the lack of an effect of soluble PDI on syncytium formation supports the notion that only surface-associated PDI is involved in fusion.

The fact that DTNB exerts its anti-HIV effect by inhibiting the catalytic activity of PDI is supported by the following observations: 2 other inhibitors of the catalytic activity of PDI (PDI antibodies and bacitracin) interfered with syncytium formation in various systems, whereas LY181984, which inhibits some disulfide thiol exchange activities but not that of PDI [START_REF] Morre | A protein disulfidethiol interchange activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea[END_REF], had no activity in our assays. In addition, the concentration of DTNB that blocked fusion was /10 Ϫ3 M, which is Ϫ4 3 ϫ 10 comparable with that active in HIV infection experiments and with that required to inhibit the catalytic activity of PDI [START_REF] Ryser | Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction[END_REF].

Altogether, our data suggest that lymphocyte surface PDI assists changes in the redox status of HIV Env. This, in turn, may destabilize the gp120 subunit and allow remodeling of the Env disulfide network and induction of profound conformation changes within Env, to trigger membrane fusion, in accord with current models [START_REF] Ugolini | HIV-1 attachment: another look[END_REF][START_REF] Broder | Chemokine receptors and HIV[END_REF][START_REF] Chan | HIV entry and its inhibition[END_REF].

Finally, we show that PDI labeling is present in the vicinity of regions of the lymphocyte surface enriched in CD4, whereas CD4 labeling also appears in membrane regions where PDI is lacking. This result, together with the finding that PDI catalytic activity is required for the fusion process at a post-CD4 binding step, suggests that only the subset of CD4 receptors present in PDI-enriched surface regions can be involved in HIV into cell entry. If one considers that PDI directly catalyzes disulfide changes within Env after CD4 binding, this suggests that Env binding to CD4 ϩ cells should subsequently induce the redistribution of PDI and the Env/CD4 complex at the cell surface, to induce their colocalization, and hence contact between the catalyst and Env. Alternatively, it also suggests that Env/CD4 binding and the subsequent membrane remodeling [START_REF] Ugolini | HIV-1 gp120 induces an association between CD4 and the chemokine receptor CXCR4[END_REF] induce the colocalization of PDI and the viral coreceptor CXCR4 at the target lymphocyte surface. Future work will focus on these points. In contrast, if one considers that PDI indirectly influences the redox status of Env through the thiol content of defined membrane regions, such membrane remodeling may not be necessary to trigger fusion.

In conclusion, our results support and expand earlier findings [START_REF] Ryser | Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction[END_REF] that the catalyst function of PDI at the lymphocyte surface plays a role in HIV entry at the membrane fusion step. We propose that it assists post-CD4 binding Env conformation changes through changes of some of its critical disulfides. Our data also indicate that the cell surface PDI is a novel target for anti-HIV therapies based on new sulfonyl urea compounds with activity on isomerase functions, such as those already used in anticancer trials [START_REF] Morre | A protein disulfidethiol interchange activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea[END_REF].

Figure 1 .

 1 Figure 1. Lymphocyte cell lines, CD4, and protein disulfide isomerase (PDI). Living CEM cells (a, control conditions; b, cells activated by phaseolus vulgaris leucoagglutinin) were incubated with mouse anti-CD4 monoclonal antibody (Ab) or rabbit anti-PDI polyclonal Abs. Staining was done with anti-mouse Abs coupled to fluorescein isothiocyanate (green; column 1) or anti-rabbit Abs coupled to Texas red (red; column 2), respectively. Both labelings are shown in column 3. Processing of images in column 3 enabled selection of regions of true overlap of green and red staining from non-colocalized staining; regions of overlap are shown as white dots (column 4).

Figure 2 .

 2 Figure 2. A, Effect on syncytium formation and cytotoxicity of 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) and bacitracin (BCT). Ratio (percentage) of total surface occupied by syncytia vs. surface of field of vision was assessed (Sync.) in VV9-1-infected CEM cell cultures done in presence of DTNB or bacitracin ( ; representative experiment shown with means of values obtained in 5 fields of vision; control, absence n p 5 of inhibitor: syncytia). Cell viability (Viab.) was assessed in parallel. B, Effect of protein disulfide isomerase (PDI), LY181984, and 40% ע 5% anti-PDI antibodies on syncytium formation. Syncytium formation was assessed as described above in presence of 16 mg/mL PDI, sulfonyl urea compound LY181984 (LY1, 10 Ϫ4 M; LY2, 10 Ϫ6 M), anti-PDI rabbit polyclonal Abs (PAbs), or irrelevant rabbit polyclonal Abs (CAbs; n p 2 experiments shown with mean of data from 5 fields of vision; C, control).

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Figure 3 . 6 3 ϫ 10 1 . 5

 3615 Figure 3. A, Human immunodeficiency virus (HIV) envelope (Env) production in presence of 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB). Env was expressed in presence (ϩDT) or absence (ϪDT) of DTNB. Env in cell lysates (Cell) or secreted into cell supernatant (Supnt) was characterized by electrophoresis and Western blot with HIV-positive serum sample pool for staining. In parallel, lysate and supernatant of Env-expressing cells were analyzed with pool of HIV-negative serum samples (C). B, Membrane expression. Env was expressed at surface of VV9-1-infected CEM cells with or without DTNB. Semiquantification of membrane Env was done with pool of HIV-positive serum samples (1:100 or 1:300) and then with anti-human antibodies coupled to peroxidase. In parallel, cells infected with recombinant vaccinia virus (VV) encoding soluble gp160 and uninfected cells (C) were similarly processed ( experiments; duplicates were made). C, Labeled CD4 binding (bind.) to Env synthesized in n p 2 presence (Env synt. ϩDT) or absence (Env synt. ϪDT) of DTNB at surface of VV9-1Ϫinfected baby hamster kidney (BHK-21) cells (gray column, cells; white column, cells) in presence (CD4 bind.: ϩDT) or absence (CD4 bind.: ϪDT) of DTNB. Cell-associated radioactivity 6 6 3 ϫ 10 1.5 ϫ 10 was counted. Binding to cells ( ) infected by recombinant VV encoding soluble gp160 and to cells ( ) expressing native Env preincubated 6 6

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Table 1 .

 1 Effect of 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) on cell aggregation and syncytium formation.

	DTNB	Synthesis ϩ /contact ϩ	Synthesis Ϫ /contact ϩ	Synthesis ϩ /contact Ϫ	Synthesis Ϫ /contact Ϫ	MolT4 ϩ DTNB/wash/fusion
	Aggregation index	23% ע 4%	26% ע 4%	24% ע 2%	35% ע 7%	29% ע 7%
	Syncytium index	1% ע 1%	3% ע 1%	20% ע 5%	28% ע 5%	5% ע 4%

SIDA (2000-2001 to E.F.).


Alexia Gobet

Christine Jaxel

Sandrine Magnard

Manuel Garrigos

Stéphane Orlowski

Nadège Jamin
email: nadege.jamin@i2bc.paris-saclay.fr

Pierre Falson
email: pierre.falson@univ-lyon1.fr

Vincent Chaptal
email: vincent.chaptal@ibcp.fr

Chapter Title: Overexpression of the ABC transporter BmrA within intracellular caveolae in Escherichia coli

Keywords: ABC transporter, membrane protein overexpression, membrane caveolae, cytoplasmic vesicles

We described here the overproduction and oriented membrane insertion of membrane protein inside intracellular vesicles named heterologous caveolae within E. coli. The method is described with BmrA, a multidrug efflux pump from Bacillus subtilis. BmrA is produced in these vesicles, thanks to the co-expression with the canine caveolin-1b, one of the two isoforms of caveolin-1. Enriched by sucrose gradient, the caveolae-containing fraction allows to probe the ATPase and Hoechst 33342 transport activities, the latter displaying a higher specific activity than the same without caveolin-1b.

Introduction

Membrane proteins are key drug targets that drive many researches on these objects [1]. Overexpression is thus often used to obtain the large quantity of proteins required and achieve adequate purity and homogeneity levels. For membrane proteins, this overexpression has often been a major bottleneck. Indeed, overexpression can be toxic to cells, frequently requiring a mild overexpression rather than a massive production, and the quality of the produced protein requires meticulous and rigorous testing. Some documented examples show that a gentler expression of membrane proteins often results in a protein more active than the same protein produced in a larger quantity [2].

The orientation of the membrane protein can also be a critical point for their function, a problem especially concerning multidrug efflux pumps which translocate drugs from cytosol to the extracellular space. Membrane caveolae are submicroscopic pits of the membrane found in higher eukaryotes [START_REF] Parton | Caveolae as plasma membrane sensors, protectors and organizers[END_REF]. These 60-80 nm wide pits are distinct from typical membrane invagination, being formed by the recruitment of a specific caveolin-1 protein and showing features distinct from clathrin-coated pits. Caveolae are cell-type specific with cells being devoid of them (Kidney), or at the opposite can represent 50% of the plasma membrane surface for endothelial cells or adipocytes [START_REF] Parton | Caveolae as plasma membrane sensors, protectors and organizers[END_REF][START_REF] Razani | Caveolae: from cell biology to animal physiology[END_REF]. Recently, heterologous caveolae have been successfully described in E. coli by heterologous expression of human caveolin-1 [START_REF] Walser | Constitutive formation of caveolae in a bacterium[END_REF], opening the way to their use is membrane protein overexpression, being a reservoir for cell membrane [START_REF] Shin | Display of membrane proteins on the heterologous caveolae carved by caveolin-1 in the Escherichia coli cytoplasm[END_REF]. Membrane caveolae are made from the plasma membrane, which imposes the orientation of the expressed protein exposing the intracellular region of the protein to the cytosol once the caveolae is generated.

The Bacillus subtilis ABC transporter BmrA is a multidrug transporter recognizing and transporting many structurally unrelated drugs [START_REF] Orelle | Conformational change induced by ATP binding in the multidrug ATPbinding cassette transporter BmrA[END_REF][START_REF] Steinfels | Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis[END_REF][START_REF] Chaptal | Drug-bound and -free outward-facing structures of a multidrug ABC exporter point to a swing mechanism[END_REF]. Its overexpression confers resistance to B. subtilis against cervimycin C, an antibiotic secreted by a biotope competitor [START_REF] Krugel | Cervimycin C resistance in Bacillus subtilis is due to a promoter upmutation and increased mRNA stability of the constitutive ABC-transporter gene bmrA[END_REF]. Here, we report the functional overexpression of BmrA within heterologous caveolae in E. coli (Figure 1) allowing to probe the transport of drugs. -N, N, N', N'-tetramethylethylenediamine (TEMED, Sigma-Aldrich) -Running buffer stock solution: Tris-Glycine-SDS (TG-SDS) 10X (Euromedex) -Pre-stained molecular weight marker (10-170 kDa) (Euromedex) -Staining solution: Coomassie buffer: 0.025% (w/v) Coomassie blue R250, 1% (v/v) ethanol, 25% (v/v) isopropanol, 10% (v/v) acetic acid -Destaining solution: 10% (v/v) acetic acid 2.4. Cells and media for cell culture -Bacteria : E. coli C43(DE3) strain -SOC medium: 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose (NEB).

Materials

Lab equipments

-Antibiotics stock solution: 40 mg/mL kanamycin (Sigma-Aldrich) and 50 mg/mL ampicillin resuspended in dH20(Sigma-Aldrich). Kept at -20°C. -LB media: 10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl. The powder is dissolved in deionized water (making a total weight of 25 g/L) and sterilized by autoclaving 20 min at 121 °C. Right before use, the medium is supplemented with antibiotics: ampicillin 50 µg/mL for BmrA expression and kanamycin 40 µg/mL when caveolin is expressed. -LB-agar media: LB media supplemented with 15 g/L agar and antibiotic(s) -Isopropyl β-D-1-thiogalactopyranoside (IPTG) 0.5 M stock solution (Sigma-Aldrich)

Membrane preparation and sucrose gradient

-Bacterial lysis and membrane washing buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.

Methods

E. coli co-transformation with genes coding for BmrA and MBP-Cav1b

To generate heterologous caveolae and to have BmrA inserted into them, MPB-Cav1b and BmrA are overexpressed simultaneously. This is achieved by having the two plasmids at the same time within E. coli cells by co-transformation and selection using two different antibiotics.

1. Fifty nanograms of each supercoiled circular plasmid pET15b-bmra [START_REF] Chaptal | Drug-bound and -free outward-facing structures of a multidrug ABC exporter point to a swing mechanism[END_REF] and pETM40mbpcav [START_REF] Perrot | Production dans Escherichia coli de vésicules enrichies en cavéoline-1(32-178) canine ou son fragment[END_REF] are added to 100 µL C43(DE3) E. coli competent cells, for 30 minutes on ice. 2. Heat shock at 42 °C for 30 seconds, then place back on ice, 3. Add 300 µL SOC medium under biological safety cabinet and incubate 1 h at 37 °C under agitation. 4. Spread 50 µL of the cell mix onto LB-agar medium supplemented by ampicillin and kanamycin. 5. Incubate overnight at 37 °C, upside-down.

Controls for downstream experiments are achieved by co-transformation of mutant

BmrA E504A (inactive) with pETM40-mbpcav to compare to the WT protein. Controls of BmrA transformed alone, without the plasmid coding for MBP-Cav, are also performed to compare expression and activity levels.

Culture and overexpression

1. A freshly transformed colony is transferred into 20 mL LB-medium (in a 100 mL flask) supplemented with adequate antibiotics. 2. Incubate this pre-culture overnight at 37 °C under agitation (180 rpm) 3. Inoculate 4 x 500 mL of LB medium (with adequate antibiotics) in 5 L Erlenmeyer to a final OD600 of 0.1 with the overnight pre-culture. 4. Incubate at 37°C under agitation until OD600 reaches 0.6 and induce expression with 0.5 mM IPTG. Incubate overnight at 22°C under agitation. 5. Centrifuge cultures at 5,000 xg, 4 °C for 15 min with JLA 9.1000 rotor to collect bacteria 6. With spatula, transfer the pellet in 50 mL-tube, suspend them in 40 mL of 50 mM Tris-HCl pH 8.0, 150 mM NaCl to wash bacteria. Centrifuge again as previously and discard the supernatant. 7. If cell lysis is not performed immediately, store pellets at -80 °C.

Membrane preparation

1. Resuspend bacteria in 50 mL per 2-Liter of culture with lysis buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM EDTA and benzonase. From this point, keep everything at 4°C or on ice. 2. Lyse cells with a cell disrupter such as Cell-D Constant Systems Ltd with one pass at 1.4

kbar. If others cell disrupters are used, refer to the manufacturer's guidelines in order to keep vesicles intact (see Note 1). 3. Centrifuge cell lysate at 5,000 xg, 15 min, 4°C to pellet cell debris and intact cells. Collect supernatant and discard pellets. 4. Centrifuge supernatants at 100,000 xg, 1h, 4°C using 50.2 Ti rotor and suspend pellet with 1 mL of 50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5 mM EDTA, anti-protease. Use a glass homogenizer and pestle to homogenize.

Take a 10 µL aliquot to determine protein concentration in membranes using a BCA assay (following manufacturer's procedures, PIERCE) with an approximate 20-fold dilution of the membranes. 5. Homogenized membranes can be stored in cryo-tubes and frozen in liquid nitrogen until further use.

Probe

BmrA expression on SDS-PAGE, quantify expression using pre-purified protein 1. Prepare a 10% separating gel by mixing 9.6 mL of water, 5 mL of 40% acrylamide/bisacrylamide solution, 5 mL of 1.5 M Tris-HCl pH8.8, 200 µL of 10% SDS, 200 µL of 10% APS (see Note 5) and 20 µL of TEMED. Pour it between 2 glass plates and add water at the surface of the gel to ensure a flat surface. Let polymerize and prepare a 4% stacking gel: 3.1 mL of water, 550 µL of 40% acrylamide/bis-acrylamide solution, 1.3 mL of 0.5 M Tris-HCl pH6.8, 50 µL of 10% SDS, 50 µL of 10% APS and 5 µL of TEMED.

Remove water on separating gel and add stacking gel, place a 15-wells comb and let polymerize. 2. Pipet 10 µg of total protein, add water to a final volume of 10 µl and add 3 µl of 4x loading buffer. 3. Load 12 µL sample and run 10 min at 90 V then 1.5 h at 120 V 4. Add known amount of purified BmrA [START_REF] Chaptal | Drug-bound and -free outward-facing structures of a multidrug ABC exporter point to a swing mechanism[END_REF] on the gel for comparison. For pure protein, incubate only 5 minutes with loading buffer to minimize aggregating artifacts on the gel. 5. After electrophoresis, incubate gel in staining solution for 30 min and decolorating solution for 1 h. 6. Quantify the intensity of bands corresponding to BmrA by densitometry, using for example ImageJ.

ATPase activity

For each type of membranes, 2 tubes are prepared, with VO4 -and without VO4 -

Reaction buffer

Reaction mix -VO4 -mix + VO4 -mix 30 mM Tris-HCl pH8.0 100 mM NaCl 10 mM KCl 2 mM MgCl2 1. Calculate each necessary volume for "-VO4 -mix ", "+ VO4 -mix", "reaction mix" and each reagent 2. Prepare Na2S and NaN3 stock solution (they must be prepared just before the experiment and cannot be stored) (see Note 2). 3. Incubate VO4 -stock solution for 10 minutes at 95°C in dry bath before dilution (see Note 3). 4. Prepare reaction buffer and ATP-Mg solution. 5. Prepare "-VO4 -mix "and "+ VO4 -mix". 6. Place 24 µL of each type of membranes in 2 different tubes. 7. Add 46 µL of "-VO4 -mix" or "+ VO4 -mix" to the corresponding tube and incubate for 10 min at 30 °C under shaking. 8. Prepare reaction mix and incubate at 30 °C. 9. For each tube with or without VO4 -, dispatch in 3 x 20 µL in a 96-wells UV plate. 10. Add 180 µL of reaction mix in each well and read immediately NADH absorbance at 340 nm for 20 min at 30 °C.

Figure 2 shows a typical example of BmrA ATPase activity on membranes. BmrA specific activity is the difference between the total activity, and the vanadate inhibited activity. Overexpression of BmrA in presence of caveolin-1b increases BmrA specific activity, being the result of fewer amount of BmrA produced but showing better activity. Note that the use of a BmrA inactive mutant is also a good control to differentiate specific activities.

Sucrose gradient and SDS-PAGE analysis

1. Prepare 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% and 60% (w/v) sucrose in 50 mM Tris-HCl pH 8.0, 150 mM NaCl. 2. A sucrose gradient is prepared in SW41 rotor tube by pipetting first 1 mL of 60% sucrose solution and placing it at the bottom of the tube. Then, 1 mL of each sucrose concentration are added one after another in decreasing concentration order, by gentle pipetting to minimize disruption of the sucrose layers. At the top, add the volume of membranes corresponding to 10 mg of protein (see Note 4).

When adding the sucrose solution, pour the solution slowly against the tube walls to not disturb the sucrose layers. It is a good idea to store each fraction in aliquots of 0.5 mL in liquid nitrogen.

Resuspend the pellet in 1 mL of 50 mM Tris-HCl pH 8.0, 150 mM NaCl. 5. Analyze fractions of the gradient by SDS-PAGE by mixing 10 µL of sample with loading buffer and incubate 30 min at room temperature before loading on acrylamide/bisacrylamide gel, as described before. 6. Each fraction can be analyzed for activity as described above.

Figure 3 shows a representative result of sucrose gradient separation of a membrane fraction. Noticeably, BmrA, overexpression in the presence of caveolin-1b results in a lower amount of BmrA produced, but the ATPase activity is remarkably higher. This is not surprising as it has been observed after changing BmrA overexpression on other E. coli cell lines [2].

2. 1

 1 . Plasmids -The gene coding for BmrA WT and BmrA E504A are inserted in the pET-15b vector (Invitrogen) bearing ampicillin resistance. -The gene coding for the fusion protein Maltose-Binding Protein-caveolin-1β (MBP-Cav) is inserted into the pETM-40 vector (EMBL). The periplasm addressing sequence of MBP has been removed. The plasmid bears kanamycin resistance. Both proteins carry an N-terminal 6xHis tag.

3 .

 3 Centrifuge overnight (about 15 h) at 150,000 xg, 4 °C. 4. Collect 1 mL fraction one by one from top to bottom and keep them in Eppendorf tube.

3. 7

 7 Hoechst 33342 transport assays -The Hoechst 33342 transport is recorded at 25°C with λexcitation = 355 nm and λemission = 457 nm and bandwidth of 8 nm. -In the quartz cuvette, put membranes corresponding to 50 µg of proteins and complete to 1 mL with transport buffer. -One minute later add 10 µL of 200 µM Hoechst 33342, final [Hoechst 33342] = 2 µM. -One minute later add 10 µL of 200 mM ATP, final [ATP] = 2 mM. -Record fluorescence until equilibrium is reached.