Mathieu Caillau

Céline Chevalier

Pierre Cremillieu

Thierry Delair

Olivier Soppera

Benjamin Leuschel

Cédric Ray

Christophe Moulin

Christian Jonin

Emmanuel Benichou

Pierre Crémillieu

Pierre-François Brevet

Christelle Yeromonahos

Emmanuelle Laurenceau

Yann Chevolot
email: yann.chevolot@ec-lyon.fr

Jean-Louis Leclercq
email: jean-louis.leclercq@ec-lyon.fr

Sub-micron lines patterning into silica using water developable chitosan bioresist films for eco-friendly positive tone e-beam and UV lithography

Keywords: chitosan, riboflavin, water developable, biopolymer, environmentally-friendly, photolithography, electron beam lithography, etching, green nanotechnology, green-tech

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INTRODUCTION

Environmentally-friendly processes are more and more adopted in chemistry [START_REF] Clark | Green chemistry : challenges and opportunities[END_REF][START_REF] Anastas | Origins , Current Status , and Future Challenges of Green[END_REF] and in nanotechnology [START_REF] Karn | The road to green nanotechnology[END_REF][START_REF] Smith | Green nanotechnology[END_REF] . In microelectronics industry, efforts have already been undertaken to reduce the environmental impact of technology in particular the one of lithography. Indeed, lithography requires oil based chemicals like solvents, resists and developers. These chemicals and theirs syntheses may be hazardous for health and environment generating security and waste management issues and costs. First efforts focused on changing the solvents by harmless ones such as ethyl lactate [START_REF] Levinson | Lithography -Green and getting greener[END_REF] . Some researchers went further by replacing the current resists by biosourced polymers soluble in aqueous solutions. Takei et al. used derivatives of polysaccharides as negative tone resist for electron beam (e-beam) lithography [START_REF] Takei | Electron beam lithography using highly sensitive negative type of plant-based resist material derived from biomass on hardmask layer[END_REF][START_REF] Takei | Eco-friendly electron beam lithography using waterdevelopable resist material derived from biomass[END_REF][START_REF] Takei | Organic solvent-free water-developable sugar resist material derived from biomass in green lithography[END_REF][START_REF] Takei | Application of natural linear polysaccharide to green resist polymers for electron beam and extreme-ultraviolet lithography[END_REF] or for extreme ultra-violet (EUV) lithography [START_REF] Takei | Application of natural linear polysaccharide to green resist polymers for electron beam and extreme-ultraviolet lithography[END_REF] whereas Kim et al. tested silk as positive and negative tone resists for e-beam [START_REF] Kim | All-water-based electron-beam lithography using silk as a resist[END_REF] or EUV lithography [START_REF] Park | Eco-friendly photolithography using water-developable pure silk fibroin[END_REF] . The polysaccharides of Takei et al. were chemically modified with acryloyl groups. In addition, although they could achieve high resolution with 50 nm lines spaced by 450 nm, the selectivity of the chemically modified polysaccharides to fluorinated plasma etching was low. Therefore, a three layers process (resist, synthetic hard mask and synthetic etching layer) was required to enable transfer. With silk, although high resolution was also achieved (30 nm lines spaced by 100 nm for e-beam), selectivity to etching was not reported. Recently, a third group proposed egg white directly extracted from eggs as a resist [START_REF] Jiang | Water-Based Photo-and Electron-Beam Lithography Using Egg White as a Resist[END_REF] . But stripping of this resist was time consuming as the sample needed to be immersed in a solution of trypsin during 15 h which is not suited to a microelectronic process.

Herein, we proposed to replace current resists by another biopolymer, which is chitosan, for lithography and etching process as displayed in Figure 1. Chitosan is a polysaccharide constituted of D-glucosamine and N-acetyl-D-glucosamine units. The number of N-acetyl-D-glucosamine units along a chain corresponds to the degree of acetylation (DA, in percentage). Chitosan is prepared from chitin, the N-acetyl-D-glucosamine polysaccharide [START_REF] Rinaudo | Chitin and chitosan: Properties and applications[END_REF] (DA = 100 %). This polymer is the second most abundant on Earth after cellulose. It is present in the exoskeleton of arthropods and industrially extracted from the waste from sea food industry as crabs and shrimps shells. Chitosan is also a biodegradable, biocompatible and non-toxic polymer. Due to these properties, it was already used in biotechnology as an adhesion film for the cell culture [START_REF] Park | Low temperature, low pressure nanoimprinting of chitosan as a biomaterial for bionanotechnology applications[END_REF][START_REF] Fernandez | Microand nanostructuring of freestanding, biodegradable, thin sheets of chitosan via soft lithography[END_REF][START_REF] Cheng | Lithographic patterning of immobilized enzymes in chitosan thin films for multi-layer, chemical/biological sensors[END_REF] . Recent works used chitosan as positive tone resist for e-beam lithography [START_REF] Voznesenskiy | Study of Biopolymer Chitosan as Resist for Submicron Electronic Lithography[END_REF][START_REF] Voznesenskiy | Dose Characteristics of Multilayer Chitosan-Metal-Dielectric Nanostructures for Electronic Nanolithography[END_REF] . Patterns of holes of 500 nm spaced by 1.5 µm with development in water have been realized but no transfer of these patterns into the underlying substrate was reported. We demonstrated that unmodified chitosan could be used as a positive tone resist in a complete lithography/ etching process, using 30 kV e-beam lithography. 50 nm patterns were obtained and transferred by plasma etching into silica layer using a completely free organic solvent process [START_REF] Caillau | Fifty nanometer lines patterned into silica using water developable chitosan bioresist and electron beam lithography[END_REF] . The chitosan film resistance to etching by CHF 3 plasma was assessed through its selectivity corresponding to the ratio between etching rate of silica on etching rate of chitosan. The selectivity was more than 4.25 and appeared slightly higher than the resistance of PMMA (950-PMMA-A4 from Microchem) which is a common resist in lithography [START_REF] Caillau | Fifty nanometer lines patterned into silica using water developable chitosan bioresist and electron beam lithography[END_REF] . In this work, we aim to demonstrate that the unmodified chitosan can be a universal green resist for lithography (e-beam and UV) allowing sub-micron resolution with possibility of transfer into a silica layer.

EXPERIMENTAL

Materials

Highly deacetylated chitosan was provided by Mahtani Chitosan PVT, Ltd (India) from squid pens and further purified according to the procedure of Rinaudo [START_REF] Rinaudo | Chitin and chitosan: Properties and applications[END_REF] . Its molar mass of 570 kg.mol -1 was measured by size exclusion chromatography. Chitosans of DA of 2 % and 35 % were then prepared. DA was quantified by 1 H NMR using an AVANCE III 400 MHz spectrometer. Deionized water (ultrapure, 18 MΩ) and acetic acid (Fluka Analytical, glacial acetic acid) were used. Chitosan films were deposited on silica/ silicon substrates using classical spin-on technology. The silica layer (90 nm or 200 nm) was obtained by plasma enhanced chemical vapour deposition. In the case of photolithography at 248 nm, a natural dye, riboflavin (Sigma-Aldrich,  98 %, extracted from Ermothycium ashbyii), was added in the chitosan solution.

Methods for chitosan film preparation and characterizations

Aqueous diluted acetic acid solution was used to dissolve solid chitosan at concentration of 0.7 % (w/v) or 0.9 % (w/v), depending on the desired thickness. Acetic acid was stoichiometrically added with respect to the free amines of the glucosamine residues. Chitosan films were prepared on silica/silicon substrates by 30 s of spin-coating at 5000 rd.min -1 and 3000 rd.s -2 followed by a softbake at 100 °C for 1 min on a hotplate. It is well known that chitosan through hydrogen bonds have a strong adhesion on silica and silicon surfaces thanks to surface SiOH groups [START_REF] Laskowski | Current understanding of the mechanism of polysaccharide adsorption at the mineral/aqueous solution interface[END_REF] .

Film thickness was measured by ellipsometry with a spectroscopic ellipsometer UVISEL ™ from Horiba using the classical formula dispersion as model in the Horiba software. The model was adapted to chitosan by adjusting the refractive index between 1.5-1.6 and the extinction coefficient to 0 at 630 nm [START_REF] Nosal | UV-vis-infrared optical and AFM study of spin-cast chitosan films[END_REF] .

X-ray reflectivity was performed on an X-ray diffractometer from SmartLab using the Kα1 copper ray to determine the chitosan films density.

For photolithography at 248 nm, riboflavin was added in the chitosan (0.9 % (w/v)) solution at a concentration of 2.2 x 10 -4 mol.L -1 , maximum of riboflavin solubility in our conditions. Chitosan-riboflavin films were deposited on silicasilicon substrates following the same protocol as previously described.

In order to determine the distribution of riboflavin in the chitosan films, Two Photon Emission Fluorescence (TPEF) measurements (non-linear optic measurements) were carried out at Institut Lumière Matière. The non-linear optic experimental setup was based on a frequency doubled femtosecond Er-doped fiber laser source (Menlo Lasers, C-Fiber 780). The laser provided pulses with a duration of about 100 fs at a repetition rate of 100 MHz. The fundamental wavelength was set at 780 nm (spectral width of 13 nm) and an average power of about 65 mW was measured at the laser exit. The light beam was focused by a standard microscope objective (magnification X20, NA 0.75) mounted onto a microscope stand (Nikon Eclipse TE2000-U with 3D positioning in inverted geometry) holding the sample. Galvanometric mirrors allowed a beam swept to acquire non-linear images of various samples. The nonlinear optical signals, TPEF, were collected into an optical fiber feeding a spectrometer after going through mirrors and various color filters. These filters were placed on the emission light beam path to avoid any unwanted fundamental. A cooled photomultiplier tube was placed after the spectrometer.

Lithography and development

E-beam lithography was performed at 100 kV on an e-beam Writer JEOL JBX-9300FS with a ZrO/W thermal field emission source was used at the Plateforme Technologique Amont (PTA) in Grenoble. For 100 kV accelerating voltage, the electron beam has a diameter Gaussian spot of 4 nm, a speed of 50 MHz for a current range of 50 pA -100 nA.

Photolithography at 193 nm was performed at the Institut de Science des Matériaux de Mulhouse with a home-made DUV interferometric setup using a 193 nm ArF laser (Braggstar from Coherent) and phase masks generating regular sinusoidal patterns with periods ranging from 100 nm to 600 nm.

Photolithography at 248 nm was performed at PTA on a MBJB4 DUV SUSS Micro Tec device with a density power of 4 mW.cm -2 and a lamp power of 500 W.

Lithography is followed by development in deionized water (18 MΩ) during 60 s.

Etching process

Etching selectivity study of chitosan films was carried out in a Reactive Ion Etcher (RIE) Corial 200S using CHF 3 gas reactant (usually used for silica etching) during 2 min under 100 sccm of CHF 3 at a pressure of 50 mTorr at 20°C for a power of 140 W (corresponding to a bias voltage of 590 V). Chitosan was, then, selectively removed by O 2 plasma for 1 min under 100 sccm of O 2 at 100 mTorr, 20 °C, 100 W and bias voltage of 460 V.

Patterns characterizations

Patterns observations were realized by scanning electron microscopy (SEM) on a scanning electron microscope JEOL JSM 7401F at 2 kV, 5 µA and a working distance of 4 mm. The Image J software was used for the analysis of the SEM images to measure the size of the patterns.

The AFM characterization was carried out with a PicoPlus AFM from Molecular Imaging, in resonant mode.

UV-visible spectroscopy

The absorbance of the chitosan resist solutions for photolithography was measured by a SAFAS UVmc2 double beam spectrometer with quartz cuvettes of 1 cm. The blank was realized with an aqueous solution of acetic acid (0.05 mol.L -1 ) in a quartz cuvette.

The absorbance of the chitosan film was measured by a Cary 5G UV-vis-NI spectrophotometer with 2 reflexions at 45°. Data were treated with the Scan software to obtain the film absorbance.

RESULTS AND DISCUSSION

Film properties

In the introduction, the selectivity of chitosan resist to plasma etching was already presented. We previously demonstrated that chitosan films were stable at temperature up to 200 °C and were stable during development in water [START_REF] Caillau | Fifty nanometer lines patterned into silica using water developable chitosan bioresist and electron beam lithography[END_REF] . Herein, we will discuss particularly about the absorbance of the film for the photolithographic aspect and their thickness.

For photolithography, it is of prime importance that the resist can absorb the energy of the wavelength to generate chemical reactions. The absorbance of a chitosan (DA 2 %) film was measured as displayed in Figure 2.

Figure 2: Chitosan film absorbance (thickness of 180 nm). Substrate absorbance is given as reference.

On Figure 2, some interference fringes can be seen but the chitosan film absorbance is very low particularly at 248 nm corresponding to the wavelength of the photomasker we used. The low absorbance at this wavelength is confirmed for chitosan in solution for chitosan of low DA (2 %) but also at high DA (35 %) as displayed in Figure 3. This figure corresponds to the chitosan absorbance spectrum in solution for a concentration of 0.9 % (w/v). On the contrary, it seems that, for photolithography at 193 nm, chitosan absorbance in solution is higher than at 248 nm. Indeed, at 193 nm, absorbance is of 1.6.

Figure 3: UV-visible spectra of a solution of chitosan of DA = 2 and 35 % concentrated at 0.9 % (w/v) in deionized water and acetic acid (0.05 mol.L -1 ) solutions with and without riboflavin (concentrated at 2.2 x 10 -4 mol.L -1 ). Blank was made in a quartz cuvette with an aqueous acetic acid solution (0.05 mol.L -1 ).

In order to improve chitosan absorbance and so chitosan photosensitivity, a natural dye, riboflavin or B 2 vitamin, was added in the formulation of the resists. It is a natural and hydrosoluble dye present in fruits, vegetables and animals [START_REF] Sheraz | Photo, thermal and chemical degradation of riboflavin[END_REF] and extracted from flavinogenic microorganisms [START_REF] Demain | Riboflavin oversynthesis[END_REF] . It is known to generate the photodegradation of organic compounds (pesticides, food …) under UV-visible light [START_REF] Escalada | Photosensitized degradation in water of the phenolic pesticides bromoxynil and dichlorophen in the presence of riboflavin, as a model of their natural photodecomposition in the environment[END_REF] . Riboflavin was added at the concentration of 2.2 x 10 -4 mol.L -1 corresponding to its maximum solubility in deionized water in our conditions. At 248 nm, the optical absorption of the chitosan-riboflavin resist solution was significantly increased (Figure 3). Indeed the absorbance of the solution was of 2.5 with riboflavin whatever the chitosan DA and only of 0.25 without riboflavin.

The addition of riboflavin being required for photolithography at 248 nm, we studied the effect of its presence in the film particularly the possibility to make a film and its distribution into this film. Chitosan-riboflavin films were deposited by spin-coating at 3000 rd.s -2 for 30 s followed by a softbake at 100 °C for 1 min. Chitosan of DA 2 % and 35 % were tested for two concentrations. The film thickness as a function of the DA for two concentrations (0.7 % and 0.9 % (w/v)) is displayed in Figure 4. Film thicknesses are shown for chitosan with riboflavin (2.2 x 10 -4 mol.L -1 ) and without it.

Figure 4 : Spin-curves for chitosan bioresists for two DA (2 % and 35 %) and two concentrations (0.9 % and 0.7 % (w/v)) with or without riboflavin.

Chitosan-riboflavin films could be obtained by spin-coating. The spin-curves on Figure 4 for chitosanriboflavin films present the same profile and similar thickness than those obtained with chitosan alone. With or without riboflavin, the film thickness depends on the concentration of the polymer as expected but also on the DA. Indeed, spin-coated film thickness depends on inverse of the square of the dynamic viscosity of the polymer solution [START_REF] Emslie | Flow of a Viscous Liquid on a Rotating Disk[END_REF] . This viscosity relies on the quality of the solvent to dissolve the polymer and to the concentration of polymer. When a polymer is dissolved in a good solvent, its chains are completely spread leading to an increase in the viscosity [START_REF] Flory | Thermodynamics of High Polymer Solutions[END_REF] . In the case of low DA chitosan in an acidic solution, the chains were totally protonated. They could spread easily. At higher DA, less amino groups were protonated. Even though N-acetyl group is polar, the solvent quality, under similar conditions, decreased reducing the chitosan solubility as observed by Rinaudo et al. [START_REF] Rinaudo | Characterization of chitosan. Influence of ionic strength and degree of acetylation on chain expansion[END_REF] , Anthonsen et al. [START_REF] Anthonsen | Solution properties of chitosans: conformation and chain stiffness of chitosans with different degrees of N-acetylation[END_REF] and Wang et al. [START_REF] Wang | Viscosity and flow properties of concentrated solutions of chitosan with different degrees of deacetylation[END_REF] . Chains were less spread and the viscosity was consequently lowered. Therefore, viscosity and the film thickness decreased from low DA to high DA as we noticed. The increase in thickness with concentration could be explained, according to Rinaudo et al. [START_REF] Rinaudo | Characterization of chitosan. Influence of ionic strength and degree of acetylation on chain expansion[END_REF] , by the fact that, at these concentrations and in these conditions, chitosan could be assumed in a semi-diluted concentration. Chitosan chains would then be very close ones to others generating entanglements which would limit the flow. The higher the concentration, the higher the number of entanglements, hence the increase in viscosity and in film thickness.

In order to determine if riboflavin was homogeneously distributed in a chitosan film, chitosan -riboflavin films deposited by spin-coating on the silica-silicon substrate were analysed by a non-linear optic measurement Two Photon Excited Fluorescence (TPEF). For a maximal efficiency of riboflavin, it was of prime importance that riboflavin repartition was well distributed in the film. The TPEF measurements gave us access to a three-dimensional resolution. Indeed, the material was excited by two synchronous photons, thus the non-linear optical process only occurred at the focus of the beam. Firstly, TPEF spectra for a chitosan film and a riboflavin-chitosan film with a 780 nm wavelength excitation were recorded (Figure 5).

Figure 5 : TPEF spectra for a pure chitosan film and a mixed riboflavin-chitosan film with a 780 nm wavelength excitation.

In both cases, a peak of the half of the 780 nm excitation wavelength is observed on Figure 5 corresponding to a second harmonic response of these two films due to chitosan. We noticed that chitosan alone was not fluorescent. On the contrary chitosan-riboflavin film presented a high TPEF intensity centred at 512 nm. Thus this fluorescence was only due to riboflavin. TPEF signal integrated over the 410 -650 nm spectral region as a function of the film depth was then recorded (Figure 6). According to the previous TPEF spectra, the TPEF signal was only due to riboflavin. On the Figure 6, a TPEF peak appears in the region focused on the chitosan -riboflavin layer (thickness of 150 nm). The reference corresponding to the SiO 2 layer on silicon substrate was obtained in absence of chitosan and riboflavin and was shifted due to a different position of the sample. This depth profile measurement resulted from the convolution of the beam focus and the film Chitosan + riboflavin depth. Hence a TPEF maximum indicated that at the resolution imposed by the beam waist (few micrometers), riboflavin was distributed rather homogeneously within the film.

According to these studies, riboflavin enhanced the chitosan photosensitivity for photolithography at 248 nm and is homogeneously distributed in the chitosan film.

The following parts of this work will now focused on the results obtained in lithography (e-beam, 248 nm and 193 nm) and in transfer with this bioresist.

Electron beam lithography

Firstly, Monte-Carlo simulations were undertaken to simulate the behaviour of electrons through a chitosan film deposited on a silicon substrate with a silica layer of 90 nm. The results were then compared to the ones for a PMMA film (70 nm in the experimental conditions for 950-PMMA-A4 from Microchem). Results are displayed in the Figure 7.

Simulations were realized on the CASINO software for an electron beam with a diameter of 10 nm for 200 electrons accelerated at 100 kV. Density of the different material was 0.88 for chitosan for DA 2 % measured by X-ray reflectometry and already registered in the database 1.74 for silica, 2.33 for silicon and 1.18 for PMMA densities. Profile obtained for PMMA is similar to the one obtained for a 500 nm layer of PMMA by Urbánek et al. [START_REF] Urbánek | Monte Carlo Simulation of Proximity Effect in E-Beam Lithography[END_REF] with the CASINO software. In both case, the electron beam passes through the polymer layer without any scattering. Back scattering took place randomly from the silicon substrate for around 10 % of electrons. These simulations suggested that chitosan could behave as PMMA under an electron beam and validated the use of chitosan as resist for e-beam lithography. For electron beam lithography, we used chitosan of DA of 2 % concentrated at 0.7 % (w/v) on silicon substrate with a silica layer of 90 nm. The film thickness in these conditions was of 80 -90 nm. Development was performed in deionized water during 60 s.

At previous studies with our 30 kV e-beam writer, we demonstrated that high resolution patterns were obtained in a chitosan film with 50 nm lines spaced by 300 nm or 500 nm [START_REF] Caillau | Fifty nanometer lines patterned into silica using water developable chitosan bioresist and electron beam lithography[END_REF] . Line edge roughness was low in both cases, respectively of 19 nm and 14 nm. In comparison, Voznesenskiy et al. [START_REF] Voznesenskiy | Study of Biopolymer Chitosan as Resist for Submicron Electronic Lithography[END_REF][START_REF] Voznesenskiy | Dose Characteristics of Multilayer Chitosan-Metal-Dielectric Nanostructures for Electronic Nanolithography[END_REF] who also worked on chitosan films in e-beam lithography (25 kV, deionized water as developer) obtained 500 nm holes spaced by 1.5 µm. At this point, we went further by increasing the resolution and demonstrating the high quality of the final patterns. Such resolution is similar at the one obtained by Takei et al. [START_REF] Takei | Electron beam lithography using highly sensitive negative type of plant-based resist material derived from biomass on hardmask layer[END_REF] but for a lower electron energy than in their case (75 kV) and for an unmodified biopolymer.

In order to validate chitosan as e-beam resists, next studies were performed with a 100 kV standard e-beam writer.

Photolithography at 248 nm

UV light at 248 nm irradiation of chitosan was reported to generate the chitosan degradation by a free radical photooxidation mechanism [START_REF] Sionkowska | The influence of UV irradiation on the mechanical properties of chitosan/poly(vinyl pyrrolidone) blends[END_REF][START_REF] Wasikiewicz | Degradation of chitosan and sodium alginate by gamma radiation, sonochemical and ultraviolet methods[END_REF] . But degradation could occur only for high doses of exposure (2 h to 8 h at 248 nm with a dose density of 0.263 J.cm -2 .min -1 ) as presented by Sionkowska et al. [START_REF] Sionkowska | The influence of UV irradiation on the surface of chitosan films[END_REF] . Indeed, as demonstrated in section 3.1 film properties, the adsorption of chitosan (DA 35 %, film thickness 90 nm prepared from a solution concentrated at 0.9 % (w/v)) at 248 nm is low. We assessed that 50 minutes exposure (11988 mJ.cm -2 ) was not sufficient to develop the patterns throughout the chitosan layer: only 10 nm thickness of the exposed area could be dissolved. Given these results and the low absorbance of the film, the addition of a photoactive agent, riboflavin, in the chitosan resist appears of interest. For the two chitosans with riboflavin (films prepared from a solution concentrated at 0.9 % (w/v) in chitosan and 2.2 x 10 -4 mol.L -1 in riboflavin), patterns were obtained with high quality. For DA 2 %, a dose of 10800 mJ.cm -2 enabled to reveal patterns but only on the top surface (10 nm) of the chitosan film. On the contrary, using a DA 35 %, for the same dose of 10800 mJ.cm -2 , the patterns were reproduced into the whole thickness of chitosan film (Figure 9) with high fidelity to the mask. DA appeared to have a significant effect on the photosensitivity at 248 nm. At DA 2 % or 35 %, patterns were revealed but at 35 %, riboflavin efficiency was dramatically increased. In both cases, photodegradation took place partly due to the exposure at the 248 nm as demonstrated by Sionkowska et al. [START_REF] Sionkowska | The influence of UV irradiation on the surface of chitosan films[END_REF] and Wasikiewicz et al. [START_REF] Wasikiewicz | Degradation of chitosan and sodium alginate by gamma radiation, sonochemical and ultraviolet methods[END_REF] . At this effect, we can add the photodegradation caused by riboflavin. Under UV light, riboflavin is known to be excited which allowed the formation of reactive oxygen species (ROS) as O 2 in excited singlet or triplet state, O 2

•-or H 2 O 2 [START_REF] Escalada | Photosensitized degradation in water of the phenolic pesticides bromoxynil and dichlorophen in the presence of riboflavin, as a model of their natural photodecomposition in the environment[END_REF][START_REF] Pajares | Modelling the natural photodegradation of water contaminants A kinetic study on the light-induced aerobic interactions between riboflavin and 4-hydroxypyridine[END_REF] . These ROS can react with chitosan by radical reactions to break the glycosidic bond. Given that the apparent increase of photoefficiency for DA 35 %, an additional possible mechanism can also occur since for a polymer with C=O group in presence of another molecule or polymer with C=O group, a vibrational mixing between these C=O groups can be observed [START_REF] Amerik | The Photochemistry of Ketone Polymers. IV. Photolysis of Methyl Vinyl Ketone Copolymers[END_REF] . When the molecule was excited, an energy transfer could take place through this mixing leading to the excitation of the first polymer and, eventually, to its photodegradation. As chitosan at DA 35 % has more C=O groups than at DA 2 % and as riboflavin also has C=O groups, such mechanism could occur and explain this improvement of efficiency with DA 35 %.

On these bases, the study was pursued by increasing the resolution using mask with patterns of less than 0. As for e-beam lithography, it was possible to write sub-micronic patterns into a chitosan-riboflavin film. These preliminary results are very encouraging. However, the photosensitivity should be improved in order to reduce the exposure time. In photolithography at 193 nm, the two chitosans (low DA at 2 % and high DA at 35 %) were tested. The same chitosan films than for e-beam lithography (chitosan concentrated at 0.7 % (w/v)) were tested with DUV doses ranging from 100 mJ to 2000 mJ. Irradiation was followed by a development in deionized water during 60 s. For DA of 35 %, no patterns could be obtained in the bioresist whatever the applied dose. On the contrary, low DA chitosan films appeared of interest as we can see on the Figure 11. On Figure 11, patterns appeared for doses superior to 300 mJ (Figure 11 a andb). The dose was however too low to reveal patterns on the total thickness of the film. By increasing it steadily, reproduction of patterns was of better quality and their depth was steadily increased as we can observe on the Figure 11 (c to f). At 1000 mJ (Figure 11 e and f) SiO 2 layer was almost reached, the film thickness of chitosan was of 90 nm. Profile of the patterns was sinusoidal suggesting that the chitosan resist has a linear response under deep UV exposure instead of a binary one. Interestingly, the dose needed to pattern the chitosan bioresist is in the same order of magnitude to standard photoresists used in the same setup.

These first results need to be optimized and the photochemical pathway is still under investigation but they are promising for the use of chitosan bioresist for 193 nm.

Transfer by etching

In several applications and particularly in microelectronics, lithography is followed by a step of transfer. We studied the transfer by reactive ion etching by CHF 3 plasma. Transfer into silica by CHF 3 plasma etching during 4 min and stripping by O 2 plasma for 2 min were applied on chitosan and chitosan -riboflavin films after lithography. Previously, we assessed the chitosan selectivity at 4.25 [START_REF] Caillau | Fifty nanometer lines patterned into silica using water developable chitosan bioresist and electron beam lithography[END_REF] . In the Figure 12, examples of patterns after transfer and stripping after photolithography are also displayed. [START_REF] Caillau | Fifty nanometer lines patterned into silica using water developable chitosan bioresist and electron beam lithography[END_REF] and by photolithography at 248 nm (10800 mJ.cm -2 ) (b) 60 µm lines and (c) optical image of 0.3 µm patterns (6400 mJ.cm -2 ).

In Figure 12 a, were displayed the patterns obtained by 30 kV e-beam lithography after transfer and stripping from a chitosan DA 2 % film. We noticed that even for high resoluted patterns obtained by e-beam lithography was of high quality with very low line edge roughness of only 5 nm [START_REF] Caillau | Fifty nanometer lines patterned into silica using water developable chitosan bioresist and electron beam lithography[END_REF] . In photolithography, the transfer was also successfully done for chitosan DA 35 %-riboflavin films as shown in the Figure 12 b for photolithography at 248 nm. The size was still well respected with squares of (436 ± 5) µm and width of the lines of (60 ± 1) µm. Figure 12 c displays that such quality of transfer was also reached for sub-micronic resoluted mask patterns in photolithography.

It was demonstrated that addition of riboflavin to chitosan did not impair the selectivity of the chitosan allowing for the transfer of the patterns by CHF 3 plasma. Therefore, these experimental results open the possibility of using chitosan as a resist for e-beam lithography and photolithography followed by fluorinated gas plasma etching.

CONCLUSIONS

Aiming at developing green resist for lithography, a biopolymer, chitosan, issued from waste repurposing was proposed for a complete lithography and etching process. In addition, no chemical modification of chitosan nor any oil-based and toxic chemical were necessary. Only water was used as a solvent. We also demonstrated that chitosan is a ubiquitous positive tone resist allowing for ebeam lithography at 30 kV and 100 kV as well as photolithography at 193 and 248 nm. In the latter case, a natural, water soluble, non-toxic photoactivator was needed: riboflavin. We showed that the DA affect the effectiveness of the riboflavin promoted reaction.

Figure 1 :

 1 Figure 1 : Lithography and etching process applied on the chitosan bioresist film.

Figure 6 :

 6 Figure 6 : TPEF signal integrated over the 410 -650 nm spectral region as a function of the film depth for chitosan + riboflavin film and the reference (SiO 2 layer on Si substrate).

Figure 7 :

 7 Figure 7 : Monte-Carlo simulations of the trajectory of 200 electrons at 100 kV for a sample of resist (a) chitosan or (b) PMMA on silica and silicon. Primary electrons are in blue, backscattered electrons are in red.

  Submicronic 2 D holes (Figure 8 a) and 1 D lines (Figure 8 b) patterns were patterned at 200 µC.cm -2 at 100 kV. Figure 8 a and b show the preliminary results after development in deionized water. Holes and lines of 30 nm spaced by 200 nm were obtained. There are well consistent to the results obtained by Kim et al. 10 who have patterned 30 nm holes spaced by 100 nm with an e-beam writer at 125 kV. Next work will focus on the optimization of exposure dose.

Figure 8 :

 8 Figure 8 : SEM images of patterns obtained in a chitosan bioresist by e-beam lithography (100kV, 200 µC.cm -2 ) and development in deionized water (1 min) (a) 30 nm holes spaced by 200 nm and (b) 30 nm lines spaced by 200 nm.

Figure 9 :

 9 Figure 9 : (a) Optical and (b) SEM images of the patterns after lithography at 248 nm (10800 mJ.cm-2) and development in deionized water for 1 min. Patterns of the mask are non-exposed squares of 440 µm x 440 µm spaced of 60 µm exposed lines.

  3 µm. Results are displayed on the Figure 10 after development.

Figure 10 :

 10 Figure 10 : Optical image of 0.3 µm patterns obtained in a chitosan DA 35 % -riboflavin film by photolithography at 248 nm (6400 mJ.cm -2 ) and revealed by development in deionized water during 1 min.

Figure 11 :

 11 Figure 11 : AFM images and profiles of patterns obtained in a chitosan bioresist film after photolithography at 193 nm and development in deionized water for 1 min for (a, b) 300 mJ, (c,d) 500 mJ and (e, f) 1000 mJ.

Figure 12 :

 12 Figure 12 : SEM images of patterns after transfer by reactive ion etching of CHF 3 plasma and stripping by O 2 plasma obtained (a) by e-beam lithography (30 kV) 50 nm lines spaced by 300 nm from Caillau et al.[START_REF] Caillau | Fifty nanometer lines patterned into silica using water developable chitosan bioresist and electron beam lithography[END_REF] and by photolithography at 248 nm (10800 mJ.cm -2 ) (b) 60 µm lines and (c) optical image of 0.3 µm patterns (6400 mJ.cm -2 ).

beam, hν (248 nm, 193 nm)

	Si SiO 2 Si	Spin-coating	Si Chitosan resist Si SiO 2	Development Writing E-Si Si SiO 2 SiO 2
		Stripping		Etching

ACKNOWLEDGEMENTS

Nanolyon platform is acknowledged for equipment and support. Thierry Chevolleau and Christophe Lemonias from the Plateforme Technologique Amont of Grenoble are acknowledged for access, equipment and technical support. Green lithography project is supported by Pulsalys. EEA doctoral school is acknowledged for the doctoral scholarship for Mathieu Caillau.

Next works will focus on the increase of sensitivity for UV lithography particularly at 248 nm.


Philippe & Giuliani
email: p.giuliani@montpellier-bs.com

Marc Robert
email: m.robert@montpellier-bs.com

How to manage the obstacles related to management innovation implementation

Keywords: Industrial Company, Implementation, Management innovation

Recent literature on management innovation suggests that the implementation phase is a critical step in the entire process of management innovation. This phase is critical because of his many possible obstacles. The present study focuses on the obstacles and provides insight how to overcome them. We conducted an in-depth explorative case study of a major management innovation implemented (to a "top down model diffusion") in all business units of a multinational industrial company. First, we identified, different categories of obstacles. Then, we described how the company faces these obstacles using organizational and managerial adaptations. Finally, we suggest two principles of management innovation implementation and best managerial practices in order to successfully implement management innovation. Our findings suggest for a management innovation implementation to be successful using a "top down model" requires manager involvement through a systemic approach which linked all the hierarchical levels and specific objectives.

INTRODUCTION

A substantial body of literature shows that management innovation provides a competitive advantage to companies [START_REF] Mol | The sources of management innovation: when firms introduce new management practices[END_REF][START_REF] Battisti | How innovative are UK firms? Evidence from the fourth UK community innovation survey on synergies between technological and organizational innovations[END_REF][START_REF] Camison | Organizational performance as an enabler of technological innovation capabilities and firm performance[END_REF][START_REF] Volberda | Management innovation: management as a fertile ground for innovation[END_REF][START_REF] Lavastre | Pratiques interorganisationnelles innovantes (P2I) dans le Supply Chain Management: développement et validation d'un instrument de mesure[END_REF]. Some authors even argue that the competitive advantage resulting from management innovation is more enduring than a competitive advantage which can be obtained by product or process innovation [START_REF] Hamel | The why, what and how of management innovation[END_REF][START_REF] Hamel | The future of management[END_REF].

Nevertheless the implementation of management innovation is both under-researched and not without its problems [START_REF] Birkinshaw | Management innovation[END_REF][START_REF] Mamman | From management innovation to management practice[END_REF][START_REF] Ansari | Made to fit: how practices vary as they diffuse[END_REF][START_REF] Dubouloz | Innovation organisationnelle et pratiques de mobilisation des RH[END_REF].

Previous studies have identified various obstacles in implementing a management innovation.

Innovation scholars indicate that because of the unavoidable internal obstacles related to the implementation of new management practices, a management innovation may be rejected by the organization (Knights and McCabe, 1978;Lozeau et al., 2002). Given the gap between the practices and the rhetoric related to the management innovation, other authors argue that this is why only part of the innovation is adopted (Zbaracky, 1998, Hill and[START_REF] Hill | In search of TQM[END_REF].

Finally, management innovation scholars admitted that, when new management practices are introduced within a company, there is an interaction between the nature of the management innovation and the company environment, the result being that both elements interact [START_REF] Segrestin | Les chantiers du manager[END_REF][START_REF] O' Mahoney | The diffusion of management innovations: the possibilities and limitations of memetics[END_REF][START_REF] Damanpour | Managerial Innovation: conceptions, processes, and antecedents[END_REF][START_REF] Ansari | Made to fit: how practices vary as they diffuse[END_REF][START_REF] Ansari | How practices made to vary? Managing practice adaptation in a multinational corporation[END_REF]. To some up, the literature suggests that the management of the obstacles generated by the introduction of a management innovation is a critical factor in gaining competitive advantage [START_REF] Torbjorn | Organizational innovations in a long term perspective: legitimacy and souls-of-fires as critical factors of change and viability[END_REF][START_REF] Mamman | From management innovation to management practice[END_REF][START_REF] Ansari | Made to fit: how practices vary as they diffuse[END_REF][START_REF] Ansari | How practices made to vary? Managing practice adaptation in a multinational corporation[END_REF].

While scholars have examined the implementation of a management innovation which had been created "from the outside" (Lillrank, 1995;[START_REF] Dubouloz | Innovation organisationnelle et pratiques de mobilisation des RH[END_REF][START_REF] Peeters | Sources of variation in the efficiency of adopting management innovation: the role of absorptive capacity routines, managerial attention and organizational legitimacy[END_REF] there has been less research about the implementation of a management innovation created internally [START_REF] David | La place des chercheurs dans l'innovation managériale[END_REF][START_REF] Ansari | Made to fit: how practices vary as they diffuse[END_REF][START_REF] Ansari | How practices made to vary? Managing practice adaptation in a multinational corporation[END_REF]. In the present paper we address the following: how to manage the obstacles related to a management innovation created internally and how to implement it according to "top-down" model.

To address this question, we focus on the implementation of a management innovation which aims to deeply and completely transform the management style of an international company operating in an industrial sector.

We contribute in three ways. Firstly, we identify the main obstacles which appear during the implementation process of a management innovation. Secondly, we analyze how the organization faces them by initiating corrective actions which change its organization, and its management style. Finally, by studying these corrective actions, we will identify two principles for successfully management innovation implementation and we suggest best practices. We conclude with some theoretical and managerial implications of our work.

THEORETICAL BACKGROUND

When literature focuses on how companies adopt a management innovation, the specific phase of implementation is seen more as a blurred process than as a process in its own right.

Management innovation implementation is usually driven through a dialectical process [START_REF] Klein | The challenge of innovation implementation[END_REF][START_REF] Birkinshaw | Management innovation[END_REF].We can refer to different frameworks in literature related to the process of management innovation implementation. More precisely, it is possible to identify two main models: the "recursive interlinked model" and the "top down model" [START_REF] Daft | A dual core model of organizational innovation[END_REF][START_REF] Birkinshaw | Management innovation[END_REF].

Furthermore, literature allows us to identify traditional obstacles that disturb the implementation process of innovative management practices [START_REF] Brockman | The evolution of managerial innovations in distribution: what prospect for ECR?[END_REF][START_REF] Ayerbe | Les transitions entre innovations : études de cas et proposition d'une grille d'interprétation[END_REF]. Some authors demonstrate that absorptive capacity routines, and their underlying process of evolution, influence the efficiency of a management innovation implementation process [START_REF] Dubouloz | Innovation organisationnelle. S'ouvrir pour innover plus ?[END_REF][START_REF] Peeters | Sources of variation in the efficiency of adopting management innovation: the role of absorptive capacity routines, managerial attention and organizational legitimacy[END_REF]. Some recent studies highlight the point as to how management innovation practices vary as they diffuse and how to manage the tension between maintaining the innovative management practice's integrity while allowing for variation [START_REF] Ansari | Made to fit: how practices vary as they diffuse[END_REF][START_REF] Ansari | How practices made to vary? Managing practice adaptation in a multinational corporation[END_REF].

IMPLEMENTATION OF MANAGEMENT INNOVATION, DEFINITION AND MODELS

Usually scholars conceptualize the implementation of a management innovation as a multiphase and a multidimensional process [START_REF] Aiken | The organic organization and innovation[END_REF][START_REF] Klein | The challenge of innovation implementation[END_REF][START_REF] Canet | Phases of the adoption of innovation in organizations: effects of environment organization and top managers[END_REF][START_REF] Birkinshaw | Management innovation[END_REF]. Certain authors speak to four phases in the whole process of management innovation implementation from its emergence until it becomes completely absorbed and utilized by an organization. According to [START_REF] Aiken | The organic organization and innovation[END_REF] the four phases are: evaluation, initiation, implementation and routinization. [START_REF] Klein | The challenge of innovation implementation[END_REF] define it differently as: awareness, selection, adoption, implementation and routinization. [START_REF] Canet | Phases of the adoption of innovation in organizations: effects of environment organization and top managers[END_REF] distinguishes three different phases: initiation, adoption decision and implementation, where implementation is defined as: "Events and actions that pertain to modifying the innovation, preparing the organization for its use, trial use, acceptance of the innovation until it becomes a routine feature of the organization" (Damanpour and Schneider, 2006, p. 217). Focusing on management innovation, [START_REF] Birkinshaw | Management innovation[END_REF] distinguished four interlinked phases in the innovation process through which this kind of innovation comes about. These phases are: motivation, invention, implementation, theorization and labelling (Figure N°1). They define the specific phase of implementation as: "All the activity on the technical side of the innovation after the initial experiment, up to the point where the new management innovation is first fully operational" (Birkinshaw et al., 2008, p. 836). Later, other authors define implementation as: "The process by which an adopter strives to create a better fit between an external practice and the adopter's particular needs to increase its zone of acceptance during implementation" (Ansari et al., 2010, p. 71).

Implementation of management innovation according to the "recursive interlinked model"

According to the "recursive interlinked model" [START_REF] Birkinshaw | Management innovation[END_REF], the specific implementation phase of a management innovation corresponding with "idea testing" is divided into "in-vivo new practice" and "in vitro thought experiment". Here we consider mainly the "in-vivo new practice" and the role of internal change agents because as Birkinshaw et al state: "External change agent […] rarely play an active role in actually implementing new ideas in vivo" (Birkinshaw et al, 2008, p. 837). The "in vivo new practice" phase is related to two sub processes: "trial and error" and "reflecting experimenting". The first phase to engage by internal change agents is monitoring and making adjustments to the original concept of management innovation, with the aim of improving it after its first usage (trial and error). The second phase is evaluating the consequences of management innovation implementation in a way to know how to answer the tensions created by these changes (reflective experimenting) (Figure N°1). This means that the concrete management innovation implementation transpires through a series of interactions between the will of top management to promote new practices of management and the reality of the practices adopted by collaborators (Zbaracki, 1998, Hills and[START_REF] Hill | In search of TQM[END_REF].

Figure N° 1.

The "recursive interlinked model" (Birkinshaw et al., 2008 p. 832)

Implementation of management innovation according to "top-down model"

In 1978, Daft describes the process of innovation suggesting that the diffusion of an innovation could be divided into four essential steps: conception, proposition, adoption, and finally, implementation. He concentrates his study on the first two phases and does not cover the specific process of adoption and implementation, but he suggests a "dual core model" of diffusion which is always useful today. According to this "dual core model", technological innovations are implemented in a company according to a "bottom-up" process, although management innovation requires a "top-down model" to be implemented (Figure N° 2). [START_REF] Daft | A dual core model of organizational innovation[END_REF]

THE MAIN OBSTACLES OF MANAGEMENT INNOVATION IMPLEMENTATION

There is a consensus in literature on the fact that the specific phase of implementation of management innovation constantly requires the modification or adaptation of the core model of the management innovation [START_REF] Klein | The challenge of innovation implementation[END_REF][START_REF] Ansari | Made to fit: how practices vary as they diffuse[END_REF][START_REF] Ansari | How practices made to vary? Managing practice adaptation in a multinational corporation[END_REF]. As Akrich et al. state about implementing innovation: "To adopt is to adapt" (Akrich et al., 2002, p. 208). However, employee attitude toward change in the way of management is generally negative and sometimes leads to the rejection or the lessening of management innovation [START_REF] Knights | A road less travelled. Beyond managerialist, critical and processual approaches to total quality management[END_REF]. So, companies have to carefully manage the implementation phase with a determinate strategy [START_REF] Ansari | Made to fit: how practices vary as they diffuse[END_REF][START_REF] Ansari | How practices made to vary? Managing practice adaptation in a multinational corporation[END_REF]. Authors suggest that companies have to manage the implementation process and strive to strike a balance between extensive and high fidelity implementation and local adaptation to accommodate context idiosyncrasies [START_REF] Ansari | How practices made to vary? Managing practice adaptation in a multinational corporation[END_REF].

Resistance to change can take different forms depending on the nature of the innovation and the environment in which it is implemented. Literature allows us to identify traditional obstacles that disturb the implementation process of innovative management practices.

Some are attached to the learning process (Argyris, 2003;[START_REF] Ansari | Made to fit: how practices vary as they diffuse[END_REF][START_REF] Ansari | How practices made to vary? Managing practice adaptation in a multinational corporation[END_REF], others to the relationship between the culture of the company and the nature of the innovation [START_REF] Klein | The challenge of innovation implementation[END_REF][START_REF] Brockman | The evolution of managerial innovations in distribution: what prospect for ECR?[END_REF][START_REF] Ayerbe | Les transitions entre innovations : études de cas et proposition d'une grille d'interprétation[END_REF]. Other authors focus on the commitment of the managers (Torbjörn andAke, 1993, Birkinshaw et al., 2008).

Technological innovations

Organizational innovations

Another obstacle is related to the issue of the legitimation of the management innovation [START_REF] Peeters | Sources of variation in the efficiency of adopting management innovation: the role of absorptive capacity routines, managerial attention and organizational legitimacy[END_REF]. Finally, other studies focused on the difficulty a company faces in maintaining the changes introduced by the management innovation over time [START_REF] Dubouloz | Innovation organisationnelle et pratiques de mobilisation des RH[END_REF]. All these obstacles can endanger the entire implementation process.

Moreover, when "top-down" model is the diffusion mechanism, Lozeau et al (2002) demonstrates that in an international context, many business units may be reluctant to accept and adopt a management innovation.

So, what happens when a company decides to implement a management innovation throughout all its business units according to "top-down" process, without accepting local accommodations or reconfiguration of the core principle of the management innovation? This is precisely the point we will discuss.

More specifically, we focus on how the company succeeds in managing the obstacles allowing a successful adoption of a management innovation which dramatically changes the manager's tasks and the whole company way of management.

THEORETICAL GAP

We know that new management practices implementation, generate obstacle and resistance within an intra-organizational context, requiring redefinition and customization of the management innovation [START_REF] Canet | Phases of the adoption of innovation in organizations: effects of environment organization and top managers[END_REF][START_REF] Birkinshaw | Management innovation[END_REF][START_REF] Ansari | Made to fit: how practices vary as they diffuse[END_REF][START_REF] Ansari | How practices made to vary? Managing practice adaptation in a multinational corporation[END_REF]. The process of management innovation implementation necessitates careful consideration, to manage the tensions and the obstacles due to management innovation introduction. [START_REF] Canet | Phases of the adoption of innovation in organizations: effects of environment organization and top managers[END_REF][START_REF] Ansari | Made to fit: how practices vary as they diffuse[END_REF][START_REF] Ansari | How practices made to vary? Managing practice adaptation in a multinational corporation[END_REF].

Usually, literature states that malleability and customization enhance and favor the adoption of management innovation [START_REF] Ansari | Made to fit: how practices vary as they diffuse[END_REF][START_REF] Ansari | How practices made to vary? Managing practice adaptation in a multinational corporation[END_REF][START_REF] Peeters | Sources of variation in the efficiency of adopting management innovation: the role of absorptive capacity routines, managerial attention and organizational legitimacy[END_REF]. As far as we know, no prior studies suggest the main obstacles and how to remove these obstacles in order to implement a management innovation utilizing a "top down" model which doesn't allow local accommodations. That is precisely the gap we want to fill. How to manage the obstacles related to the management innovation implementation process in a case of enactment of "top-down model".

The present study provides "a success story" that aims to illustrate the obstacles and the solutions to remove it for implementing successfully a major management innovation. The statements below are from senior managers of the company and illustrate the fully management innovation implementation within the company.

"Management SIM method is spread throughout the group, you can move from one department to another, from one function to another and you will be not lost. It is important because in a large group you move a lot and therefore this common culture broadcasts a common operating mode" (Interview N° 5).

METHODOLOGY

The case research method is well-suited to reports an in-depth study focusing on the process of implementation of a major management innovation. Because the company has no authorized disclosure of its identity we call it firm SEC. The case study provides an opportunity to investigate in a real-life setting the organizational factors that can, not only inhibit the implementation of the managerial innovation, but also how the company succeeds to overcome these negative effects and finally succeeded to implement the management innovation throughout the organization. The main reason which motivated us to adopt a case research is because it's useful for understanding the specific mechanisms which modify an organization [START_REF] Pettigrew | Longitudinal field research on change: theory and practice[END_REF]Yin, 2009). Moreover, we refer specifically to the procession strategy research describe by Musca which is useful for a better understanding of complex longitudinal case including several sub-units involved in a large organizational change [START_REF] Musca | Une stratégie de recherche processuelle: l'étude longitudinale de cas enchâssés[END_REF].

SELECTION AND PRESENTATION OF THE CASE STUDY

Company SEC is a private French company, formed by the fusion of three main companies working for a long period in the same activity sector, each well known in their respective trades. At the turn of the 20 th century, the company decided to fuse all its trades into one trade mark name in order to enforce its reputation and visibility. When we conducted the interviews, the company employed more than 140,000 employees at approximately 150 locations in more than 100 countries. The company operates in an intensely competitive environment where it is crucial to improve its productivity to maintain its markets and the financial performance. Manufacturing business units are in competition with each other to reach the productivity objectives defined by the company and to increase efficiency. The implementation of an internal and unique method of management called Short Interval Management (SIM), is seen by the top management as the best means to reach the productivity objective defined by the company. That is why business units cannot change the core model of the management innovation but they have to apply this new way of management in their environments. This new way to organizing the hierarchical relationship and the management of the company was implemented throughout all business units for 5 years (figure N° 3).

Figure N° 3. SIM implementation in Schneider Electric in recent period

We also chose this particular company because we could be involved on a management level during the implementation process (2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012). The richness of data we could collect allowed us to analyze the direct and indirect effects generated by the introduction of management innovation and understand how the company succeeded in solving the inherent resistances to change. In this way, we contribute to increasing our knowledge about how management innovation could be successfully implemented in a company according to Mol and Birkinshaw's recommendations: "Future research should focus on poorly understood facets of management innovation, namely the processes of creation and implementation" (Mol andBirkinshaw, 2009, p. 1278).

DATA COLLECTION

We conducted 30 semi-directive interviews in five different business units at multiple hierarchical levels, getting abundance verbatim comments from the senior up to front line managers (Team-leaders). Each interview lasted at least one hour and sometimes all day. All the interviews were made in situ and during the period from 2010 to 2013. Certain key respondents were interviewed several times. In addition to registration and transcription of all the interviews, we physically assisted in the implementation of the SIM method in one business unit. This allows for cross-checking and verifying the accuracy of the data collected and also providing diversity of perspectives [START_REF] Eisenhardt | Theory building from cases: opportunities and challenges[END_REF]. At the beginning of each interview, informants were told that the purpose of the enquiry was to understand the main effects of SIM management innovation implementation on their way of managing and organizing their hierarchical relationship. We transcribed each interview and asked respondents to validate the verbatim. To do this we asked respondents to talk freely about the SIM management innovation and the direct or indirect effects in managing their daily tasks. The interviews were comprised of semi-structured questions addressing four main themes: the direct, indirect and meta effects of the introduction of SIM method on the way to manage, the main organizational obstacles related to the management innovation implementation, the response of the company to overcome these obstacles, and finally the key success factors according to their representation. Construct validity is supported by literature and also key company informants, in particular top managers in charge of the diffusion of the management innovation. This method is pertinent to investigate the direct and indirect interactions upon the organization [START_REF] Musca | Une stratégie de recherche processuelle: l'étude longitudinale de cas enchâssés[END_REF]Yin, 2009). For exploiting the verbatim we used two principle stages of data analysis. We imported all the verbatim into a computerized system (Nvivo), coding this data according to distinct units of meaning [START_REF] Dumez | Méthodologie de la recherche qualitative[END_REF].

However because a computerized system is too heavy to manage a large volume of data we identified pattern codes which were used to find causal relationships. To do this, we used matrices to identify the direct, indirect and "meta-effect" generating obstacles in the SIM implementation processes according to a specific segmentation of the respondents [START_REF] Miles | Analyse des données qualitatives[END_REF].Then we focused on these obstacles and we analyze how the company succeeded to solve these issues.

To improve the validity of our results, we adopt the triangulation method described by Miles and Huberman (1984). We used not only semi structured interviews with actors as part of our field study, but also immersion observations and analysis of internal documents and publication. All this data guarantees a multidimensional view and a wide range of research materials: « In fact, the various sources are highly complementary, and a good case study requires the use of the number of possible sources" (Yin, 2009, p. 101). Specific respondents are referred to with a number as to ensure their anonymity. The case study allowed us to observe, by immersion, the diffusion and the appropriation of the management innovation in five business units with agreement from the top management of the company.

TABLE OF THE MAIN OBSTACLES RELATING TO THE MANAGEMENT INNOVATION

IMPLEMENTATION

The obstacles related to the SIM management innovation implementation can be broken down into three broad categories. We have chosen to present our results by developing them in two samples of respondents. The first sample of respondents therefore, includes senior managers (executives and engineers). The second sample represents the middle and front line management, including collaborators such as Responsible of Team Manufacturing (RTM), technical officers and "team-leaders". To develop the table below, we counted each instance when someone declared obstacles in one of the categories mentioned. If someone made reference to the "commitment of the managers" several times during their interview we counted these as a single occurrence. On the other hand, if in a same interview a respondent indicated two different types of obstacles, we counted two different occurrences.

As expected, we have found different obstacle categories in our verbatim. R1: Actors consider that management innovation SIM allows productivity objectives to be reached as defines by the company legitimizing the rhetoric about management innovation (8 items)

A significant number of respondents argue that SIM management innovation allows to reach the productivity's objectives defines by the company. Successful implementation at the intra organizational level of SIM management innovation is supported by the feeling of the actors that this method allows the performance productivity level as defined by the company. SIM management innovation implementation has coincided with the implementation of measurement indicators to demonstrate the favorable evolution of specific processes. For example SIM indicators demonstrate a reduction in the processing time when manufacturing a specific product and consequently enhance of productivity. Another result credited to SIM indicators is the ability to put new equipment in use with less time required for experimentation. This first result is in accordance with [START_REF] Mol | The sources of management innovation: when firms introduce new management practices[END_REF] as well as Damanpour (2014) and [START_REF] Volberda | Management innovation: management as a fertile ground for innovation[END_REF] who argue that management innovation improve performance. However, our results are positioned at an intra-organizational level.

The testimony of a variety of managers allows us to exclude the bias of cross-effects.

"The bigger engine is the demonstration of the efficiency of the method; it's been 10 years since we changed gear in terms of performance and continuous improvement. Efficiency is our best ally" (Interview No. 4).

"It is not the technology that counts the most, because everybody has more or less the same.

Implementing technological innovation without the Short Management Interval Method, only allows the recovery of 10% of the potential productivity of the technological innovation.

While combining the two yields far better results, it is the synchronization of both which is important "(Interview N°7).

Second result:

R2: Management innovation fosters a more impartial individual and collective appreciation by using well-known indicators (18 items).

Transparent and relevant indicators continuously display the evolution of the production team's performance. Furthermore, the industrial performance measurement system is sufficiently sophisticated to take into account certain malfunctions which are not caused by individuals. Thus, the gap between the time standard to produce a deliverable is different from the allotted time, which takes into account the unpredictable external events, such as machinery failure.

Third result:

R3: Management innovation allows change of the decision-making circuit to generate greater speed and higher relevance (12 items).

We have seen that the SIM approach is based on different management sequences starting with the plan first level manager up to the plant manager. This systemic device guarantees not only taking into account the varying dysfunctions that may occur during each production shift, as well as the treatment of these dysfunctions by the action plans. This leads to two effects particularly important:

(i)

The voice of operators and the first levels of management are taken into account and lead to concrete improvement actions.

(ii) Adjustments regarding the requirement expressed in the action plans do not remain unanswered. They can be resolved at the level of loops 1 or 2, at the middle management level or the do through loops 4 or 5 until the plant manager is able to process them. Therefore, it is fundamental that the top-down decision circuit is as quick as possible because an inordinately slow process in decision-making leads inevitably to demobilization of lower level of management. In this case operators and team-leaders will stop suggesting new action plans and subsequently could endanger the entire device.

Result four:

R4: Management innovation increases the margin of autonomy of the first hierarchical levels of management and promotes the enrichment functions of this level of management (13 items).

We found that the first levels of management adopt well to SIM method because they find more autonomy and enrichment of their tasks, or diversity in their day to day work.

"The SIM approach as is often the case for managerial innovations induces reduction of hierarchical levels, and a reduction of staff in terms of organization by job enrichment, growth in versatility and skills, as well as productivity gains" (interview N° 2

Fifth result:

R5: Management innovation generates solidarity around common objectives between all hierarchical levels (14 items)

Through the system of successive loops, all the actors of the company are involved in solving action plans. No manager can operate outside this system of action plans related to the different loops. A lot of discrepancies between the expected results and the observed outcomes originate from unresolved or poorly managed action plans. Because upper hierarchical levels are also involved in the loops system it makes it visible. These higher hierarchical levels must also contribute to solve these action plans by attributing necessary resources in time or in asset. Thus, the SIM management innovation is successfully implemented in our opinion due to this solidarity around common objectives and through realization of action plans between all hierarchical levels linked in the loops system.

"SIM is a powerful method to ensure progress at the same pace and controlled tasks and projects" (Interview N ° 5).

THEORETICAL CONTRIBUTIONS

Figure N° 11. A proposal framework for successful MI implementation

MANAGERIAL CONTRIBUTIONS

The study offers important managerial implications for successfully management implementation innovation in companies. Firstly, we saw that the process of implementation and ownership of the managerial innovation is much better accepted that it demonstrated its effectiveness with regard to the objective announced. The first recommendation management that we can thus formulate is the need to build a theoretical corpus and internal practices relating to the nature of innovation management techniques and methods of implementation which is the most comprehensive possible. This document must contain a theoretical part which seeks to legitimize the implementation of innovation. This development and design phase may originate from the 'corporate' level of the company as part of a 'top-down' approach because the managerial innovation must be in line with the general strategy of the company and his general mode of organization. The corporate level is also the branch that has the

Principles of management for IM implementation:

-Principe of adhesion of all actors -Principe of solidarity between all actors

Best Managerial Practices

To use transparent indicators performance

To evaluate individual and collective performance

To increase margin and autonomy of first levels of management

To enrich tasks and missions of first level of management

To shorten decisionmaking circuits

To create solidarity between all hierarchical levels management

Obstacles of implementation:

-Obstacles related to directs effects -Obstacles related to meta-effects -Obstacles related to indirect effects competence to define the human and material resources implemented, as well as the modalities and timing of deployment.

Then the company has to dedicated specific teams in charge of the diffusion of the management innovation in the different business unit and to rely on the traditional line management to these teams.

Another recommendation is that management innovation must involve all the hierarchical levels to settle effectively and successfully in the business. The management innovation AIC by its systematic appearance, obliges the organized collaboration between all the actors of the hierarchical line, and does not allow managers to fall outside the system. They are necessarily involved in one of the loops of the SIM and any passivity on their part will appear immediately through a delay in the resolution of the action plans. The management innovation is based on the exemplary and organized involvement of each manager. This organization could be a good answer to the main problem of investment of the manager in the diffusion and appropriation of the management innovation. On the other hand, it's not possible to imagine a deep and radical change in management if the required examples do not come from the highest managers (Zbaracky, 1998).

This is why we give advice to a company wishing to implement a managerial innovation to be radical, and it involves all levels. the fact that real solidarity is organized between various managers through common practices and generalized methods we believe help the success of the process of implementation of innovation management. and finally the perseverance is a fundamental criterion.

LIMITATIONS AND FUTURES RESEARCH

Successful management innovation implementation is a complex construct, and capturing all its multidimensional aspects seems rather impossible. Our study therefore has several limitations. The boundary of our research in the first place is the specific approach of the case study. The use of unique survey ground, limits the generalization of the results obtained and the external validity of the research (Miles, 1979). However, we believe this argument have been weakened by the fact that our case study contains several sites which can be regarded to some extent as distinct entities although belonging to the same group. Another limitation is the fact that our analysis is part of a time "t" in the life of the company. As Wacheux wrote:

"A case analysis is an analysis of a spatial and temporal complex phenomenon by the conditions, events, actors and the implications" (Wacheux, 1996, p. 89). One could also object because our results are derived from a specific environment related to industry and manufacturing. What would be the result of SIM management innovation in a non-industrial sector? This is the next challenge that assigns herself the company with the aim of implement the SIM in its non-manufacturing units and departments. It would be relevant and interesting to analyze how this innovation could be declined in a service environment or in a commercial service. This is in our view a topic for future works of great interest.

CONCLUSION

This explorative research contributes to a better understanding of the obstacles and the key factors success of implementation of management innovation. In order to, we focus on the implementation of a management innovation in an international company operating in industrial sector. We propose a framework for a successful implementation in top-down model. This framework should be tested by researchers in future works.

Figure

  Figure N° 4. Characteristics of respondents and their function

Figure

  Figure N° 5. The main obstacles related to SIM implementation

search Trial and error Reflective experimenting Theory linking Idea testing Idea linking Agenda setting Idea contextualizing Idea refining Reflective theorizing Figure N° 2. The dual-core model of diffusion of organizational innovation

			Organizational context
		Problem-driven
	Actions of
	internal change
	agents
		1 Novel problem	2. Hypothetical new practices	3. In vivo new practice	4. Theorized new Practice (in context)
	Actions of	5. New threats		7. In vitro thought	8. Theorized practice (out of
	external change agents	or opportunities	6. New idea	experiment	Immediate context)
		Motivation	Invention	Implementation	Theorization and
			Environmental context	labelling

Table of

 of

		solutions for removing obstacles
		Middle and	Senior managers	TOTAL
	Best practices for success	Front line
		management
	SIM allows defined performance
	objectives to be reached	2	6	8
	SIM fosters a more impartial
	individual and collective	9	9	18
	appreciation by using well known
	indicators
	SIM allows change of the
	decision-making circuit to	4	8	12
	generate greater speed and higher
	relevance
	SIM increases the margin of
	autonomy of the first hierarchical	7	6	13
	levels and promotes the functions
	enrichment
	SIM generates solidarity around
	common objectives between all	7	7	14
	hierarchical levels
	5. DISCUSSION, THEORETICAL AND MANAGERIAL CONTRIBUTIONS
	5.1. DISCUSSION
	Our first result:


Ariane Bonnin

Adeline Danneels

Jean Dubuisson

Anne Goffard

Sandrine Belouzard
email: sandrine.belouzard@ibl.cnrs.fr

HCoV-229E spike protein fusion activation by trypsin-like serine proteases is mediated by proteolytic processing in the S2' region

Keywords: HCoV-229E, fusion, entry, spike protein Subject category Animal RNA Viruses

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

enveloped viruses, they need to fuse their viral envelope with a host cell membrane to deliver their genome to the target cell. Coronavirus spike proteins are major mediators of virus entry and fulfil a dual function by mediating receptor binding and fusion. Spike proteins consist of two distinct domains carrying these functions. The amino-terminal subunit, called S1 domain, is responsible for receptor binding, whereas the C-terminal part, called S2 domain, contains the fusion machinery. Proteolytic priming of coronavirus spike protein is a key feature of the fusion process, and coronaviruses exploit a wide variety of host proteases including cathepsins, furin and members of the transmembrane serine protease (TTSP) family [START_REF] Simmons | Proteolytic activation of the SARS-coronavirus spike protein: Cutting enzymes at the cutting edge of antiviral research[END_REF] to achieve this requirement. It is likely that the proteolytic processing differs according to the protease used. Depending on the cellular localization of the protease used to activate the fusion, entry can occur either at the cell surface or after internalization of the virus by endocytosis. For SARS-CoV spike protein, two cleavage events mediated by trypsin have been identified. The SARS-CoV virion harbors an uncleaved spike protein at its surface. First, the spike protein is primed by cleavage of the S1 and S2 domain, then to fully activate fusion, a second cleavage occurs at a position called S2' (R797) exposing the fusion peptide [START_REF] Belouzard | Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites[END_REF][START_REF] Madu | Characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with characteristics of a viral fusion peptide[END_REF].

Activation of the SARS-CoV spike protein fusion by elastase also involves recognition of a cleavage site in the S2' region [START_REF] Belouzard | Elastase-mediated activation of the severe acute respiratory syndrome coronavirus spike protein at discrete sites within the S2 domain[END_REF]. The importance of the S2' cleavage has also been demonstrated for MERS-CoV and MHV [START_REF] Burkard | Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner[END_REF][START_REF] Millet | Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein[END_REF].

HCoV-229E was identified in 1966 in students suffering from common cold [START_REF] Hamre | A new virus isolated from the human respiratory tract[END_REF], but children, elderly and immune-compromised persons can develop a more severe disease [START_REF] Van Der Hoek | Human coronaviruses: what do they cause?[END_REF]. It has been shown that HCoV-229E requires endosomal cathepsin L activity to infect Hela cells [START_REF] Kawase | Protease-Mediated Entry via the Endosome of Human Coronavirus 229E[END_REF].

However, Shirato et al. have recently shown that clinical isolates of HCoV-229E

preferentially uses the transmembrane protease serine 2 (TMPRSS2), a trypsin-like protease, rather than endosomal cathepsin for entry [START_REF] Shirato | Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry[END_REF]. TMPRSS2 is known to be present at the surface of the human airway epithelia and is likely the main activator of HCoV-229E fusion in vivo. In addition, TMPRSS2-mediated entry at the cell surface protects the virus from the inhibitory effects of interferon-induced transmembrane proteins (IFITMs) known to inhibit entry of several enveloped virus [START_REF] Bertram | TMPRSS2 Activates the Human Coronavirus 229E for Cathepsin-Independent Host Cell Entry and Is Expressed in Viral Target Cells in the Respiratory Epithelium[END_REF]. However, the detailed proteolytic processing of HCoV-229E spike protein to activate its fusion remains to elucidate.

To investigate this proteolytic maturation, we first cloned the spike protein sequence from a patient infected by HCoV-229E hospitalized in Lille, France in 2014 (Spike France/Lille/2014, accession number MH048989). The codon-optimized sequence of the France/Lille/2014 spike protein was inserted into the pCDNA3.1(+) vector and fused with a sequence encoding a C-terminal VSVG tag to facilitate the detection of the protein in immunoblot. Then, we analysed the proteolytic processing of the spike protein required for entry mediated by trypsin/trypsin like protease, focusing on the role of the S1/S2 junction and the S2' region. The sequence of the French isolate spike protein shows 99% identity with the spike of the Niigata, 0349 and USA/932-72/1993 isolates and only 94% of identity with the VR-740 laboratory strain. HCoV-229E spike protein contains 3 arginine residues in the S2' region (R679, R683 and R687) which are conserved in different strains of HCoV-229E, except for R679 that is absent in the VR-740 laboratory strain (Fig. 1(a)). First, to confirm the role of the HCoV-229E spike protein S2' region in entry, we inserted a furin cleavage site (Fur687) in the spike protein (Fig. 1 shows that the spike protein with the furin cleavage site does not rely on endosomal cathepsin for entry but relies on furin and this confirms the involvement of the S2' region for fusion activation of HCoV-229E, as observed for other coronaviruses [START_REF] Belouzard | Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites[END_REF][START_REF] Burkard | Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner[END_REF][START_REF] Millet | Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein[END_REF][START_REF] Yamada | Proteolytic activation of the spike protein at a novel RRRR/S motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells[END_REF]. It is interesting to note that the spike proteins incorporated on the pseudotyped virions were not cleaved by furin in producing cells, indicating that the protein is not processed during exit of the producer cell but during entry into the target cell (Fig 1(d)). This suggests that during exit and secretion, the cleavage site is inaccessible to furin and that during entry, conformational change induced by receptor binding may expose the cleavage site. To investigate the role of the S1/S2 junction and the specific role of the 3 arginine residues located in the S2' region (R679, R683 and R687) in the proteolytic activation of HCoV-229E spike protein by trypsin-like serineproteases, the arginine residues present at S1/S2 and at S2' were mutated individually (R565N, R679N, R683N or R687N). We chose to mutate the arginine residues into asparagine to minimize the effect of the mutations on the conformation/folding of the protein. Field strains were reported to use preferentially the TPPS pathway [START_REF] Shirato | Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry[END_REF], this result suggest that acquisition of the arginine residue at the position 679 is not involved in this adaptation. It is surprising that mutation of R687 had no effect on entry mediated by trypsin. Indeed, R687 is believed to be located at the N-terminus of the fusion peptide and the arginine residue located at the same position in the SARS-CoV spike protein is the major recognition site of trypsin. Unlike SARS-CoV, mutation of the arginine residue located at the S1/S2 junction (R565N) has little effect on entry mediated at the cell surface by trypsin. In vivo, the major route of entry of respiratory coronavirus is believed to be mediated by the TTPS pathway. To confirm our results observed with trypsin, we expressed the human TMPRSS2 in Huh-7 cells with a lentiviral vector. Transduction of cells with a lentivirus expressing the human TMPRSS2 resulted in an increase in transduction by particles pseudotyped with the wild type Taken together, our results show that activation of the fusion process by trypsin and trypsinlike serine protease, particularly TMPRSS2 expressed in human airway epithelia, strongly rely on the R683 residue as a cleavage site. It has been shown that the position of the cleavage site in the S2' region of the SARS-CoV spike protein can be shifted from the position relative to the fusion peptide but with some effects on fusion activation and infection. Indeed, for the SARS-CoV spike protein, cleavage at the N-terminus of the fusion peptide results in improved fusion and infection capacities [START_REF] Belouzard | Elastase-mediated activation of the severe acute respiratory syndrome coronavirus spike protein at discrete sites within the S2 domain[END_REF]. For HCoV-229E spike protein, the cleavage site by trypsin is not located at the N-terminus of the fusion peptide but is present 3 residues upstream. For the SARS-CoV spike protein, even if the location of the cleavage site presents some flexibility, certain position in the vicinity of the fusion peptide were also not accessible to protease. Indeed, no cleavage product was observed when R683 was mutated (Fig 3 ), suggesting that both arginine residues 679 and 687 were not accessible to the protease, otherwise, if these residues could be cleaved but without being functional to activate the fusion, the same band would have been observed with the R683N mutant.

As seen in

Author statements

The authors declare no conflict of interest. Funding A part of this study was supported by a grant " Soutien aux thématiques émergentes 2014" from the University Hospital of Lille, France.

FIGURE LEGEND

  (a)), at the N-terminal position of the potential fusion peptide. To analyse the effect of the mutation in the context of viral particles, we used a murine leukemia virus (MLV)-based pseudotyping system that produces particles inducing luciferase expression after transduction of permissive cells. Pseudoparticles fully mimic the functionality of the wild-type virus in terms of cell entry and were used because of their technical advantage. Huh-7 cells were inoculated with pseudoparticles harboring the wild type protein or the Fur687 mutant (Fig 1(b)). Neutralization of endosomal pH by ammonium chloride treatment inhibited transduction by HCoV-229E pseudoparticles showing that HCoV-229E infects Huh-7 cells via the endosomal route and uses low-pH active endosomal protease to mediate its fusion. As shown in Figure 1(b), pseudotyped virions with HCoV-229E spike protein containing a furin cleavage site at the S2' position were insensitive to endosomal pH neutralization, but were affected by furin convertase inhibitor. This result

  Fig 1(c), these arginine residues were not implicated in entry when the virions use the endocytic pathway to invade the cells. The endosomal route of entry of the different mutants was confirmed by inhibition of their entry by ammonium chloride treatment (Fig 1(c)). Indeed, when HCoV-229E is internalized, the virus is dependent on the endosomal cathepsin L to mediate its fusion. However, unlike other proteases, cathepsin L has a limited prime-site specificity in terms of recognition sites and the precise sites of proteolysis by cathepsin L remains unclear. In 2005, Simmons et al. showed that SARS-CoV fusion can be induced at the cell surface when virions are bound to the plasma membrane at 4°C and briefly treated by exogenous protease such as trypsin [13]. Particles pseudotyped with the different S1/S2 and S2' arginine spike mutants were bound at the surface of Huh-7 cells treated with ammonium chloride to neutralize the endosomal pH and block the endosomal entry pathway. Then, fusion was induced by a brief trypsin treatment (Fig 2(a)). For the mutant R683N, trypsin treatment was not able to bypass the requirement for cathepsin L activity during the entry process, suggesting that proteolysis by trypsin occurs at R683 residue. Mutations of R679 or R687 had no effect on entry mediated by trypsin. Spike proteins of field strains of HCoV-229E contain an arginine at the position 679 whereas the laboratory strain VR-740 contains a threonine.

S

  protein which was insensitive to ammonium chloride treatment (Fig 2(b)). As previously observed for the SARS-CoV [13, 14], HCoV-229E pseudotype entry mediated at the cell surface was more efficient than entry by the endocytic pathway. As seen with infection mediated by trypsin, mutation of the R683 abolished the capacity of the virus to use TMPRSS2 to infect the cells since its infectivity was sensitive to ammonium chloride treatment of the cells (Fig 2(c)). To further confirm that cleavage mediated by trypsin-like serine protease occurs at R683N, HEK293T cells transiently expressing the wild type or the different mutant proteins were treated by trypsin for 1h and then proteins at the cell surface were biotinylated at 4°C. After precipitation of biotinylated protein with streptavidinconjugated beads, cleavage products were analysed in western blotting with an antibody directed against a C-terminal tag of the protein. Spike proteins with a furin cleavage site introduced at the S1/S2 boundary (Fur565) or at the S2' position (Fur687) were used as controls (Fig. 3). Cleavage by furin between the S1 and S2 domain gave rise to a band with a size below 135 kDa corresponding to the S2 domain. Cleavage at the S2' position (Fur687) induces a cleavage product with a smaller size below 100 KDa. Contrary to other coronaviruses for which trypsin induces mainly a processing at the S1/S2 junction, cleavage of the wild-type HCoV-229E spike protein by trypsin resulted in the appearance of a cleavage product in western blot with a size similar to the one observed for the mutant Fur687, corresponding to the cleavage of the protein at the S2' position. Mutation of R683 abolished the cleavage induced by trypsin confirming the proteolytic processing of the spike protein at this position by trypsin. The absence of any S2 fragment detection suggests that either the S2 fragment is very short-lived, being rapidly processed at the S2' position or that the S1/S2 cleavage is not required for the S2' cleavage. If the S2 fragment was short-lived, then mutation of R683 should lead to the accumulation of the intermediate cleavage product. However, we did not observe any accumulation of cleavage product of bigger size when R683 was mutated, arguing again against the requirement of the S1/S2 cleavage to prime the cleavage at the S2' position as shown for the SARS-CoV and MERS-CoV spike proteins [2, 6].

Figure 1 (

 1 Figure 1 (A) Alignment of the S1/S2 and S2' region of different coronavirus spike protein (HCoV-229E isolated in France, 2014, HCoV-229E Niigata/01/08, HCoV-229E 0349, HCoV-229E USA/932-72/1993, HCoV-229E laboratory strain VR-740, MERS-CoV and SARS-CoV).R565 at the S1/S2 boundary is in red and R679, R683 and R687 are presented in green. For

Figure 2 (

 2 Figure 2 (A). Trypsin-mediated entry at the cell surface. Particles pseudotyped with the different mutated spike proteins were bound at the cell surface at 4°C in presence of 25 mM NH4Cl and were treated with a 5 min incubation with 2 μg/ml of trypsin to induce fusion. 48h later, the cells were lysed and luciferase activity was measured. Results are presented as the percentage of transduction observed with particles pseudotyped with the wild type protein and are expressed as the mean of three independent experiments. Error bars represent the standard error of the means (SEM). Data were analysed by using an ANOVA test (** P<0.01) (B) Huh-7 cells were transduced or not with a lentivirus expressing the human TMPRSS2. Then cells were infected with particles pseudotyped with the wild type spike protein in presence or absence of 25 mM NH4Cl. Results are presented as relative light units (RLU) and are expressed as the mean of three independent experiments. Data were analysed by using an ANOVA test (*** P<0.001). (C) Huh-7 cells transduced or not with a lentivirus expressing TMPRSS2 were inoculated with particles pseudotyped with the different mutated spike proteins. Results are presented as the percentage of infection observed with particles

Figure 3

 3 Figure 3Spike protein cleavage with trypsin. HEK293T cells were transfected with plasmid encoding the different mutated spike protein. 48h later, cells were treated for 1h with 2μg/ml of trypsin at 37°C. Then cell surface protein were biotinylated at 4°C and the cells were lysed. Biotinylated protein were precipitated with streptavidin-conjugated beads and analysed by western blotting with the C-terminal part of the protein detected with an anti-VSVG antibody.

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Acknowledgements

We thank all the members of the molecular and cellular virology laboratory for helpful discussions and input during the course of this work.