Mohamed Afqir
email: mohamed.afqir@yahoo.fr

Didier Fasquelle

Amina Tachafine

Yingzhi Meng

Mohamed Elaatmani

Abdelouahad Zegzouti

Abdelhamid Oufakir

Mohamed Daoud

Structure and dielectric properties of Lu-doped SrBi 2 Ta 2 O 9 synthesized by the molten salt method

Keywords: molten salt, diffuseness, dielectrics, microstructure, AC and DC conductivity

Lu-doped SrBi 2 Ta 2 O 9 (SrBi 2-x Lu x Ta 2 O 9 where x = 0, 0.025, 0.05, 0.75 and 0.1) powders were synthesized by combination of molten salt method and solid-state route. FTIR, Raman and XRD techniques were performed to follow the transformation of reactants into the desired products. Characterization of all samples shows pure and single-phase orthorhombic structured materials obtained with plate-like morphology that is composed of fine and coarse-grained particles. The prepared powders were pressed and sintered at different temperatures up to 1200 °C. Microstructure of the sintered samples is also likely to be affected by doping. The first study of dielectric measurements describes the effect of the application of DC bias, at room temperature, on the undoped and Lu-doped ceramics and shows that there is little or no effect of DC bias. The sample SrBi 1.95 Lu 0.05 Ta 2 O 9 had maximal dielectric constant (ε ′ ) and minimal dielectric loss (tanδ). In the second part of this work, the temperature dependence of ε ′ and tanδ was considered. It was concluded that Lu-doping not only reduces the Curie temperature, but also brings a diffused phase transition, showing a crossover between displacive and diffusive behaviour.

I. Introduction

Early research on compounds with Aurivillius phase started in the 1940s. One of the most attractive materials from this group is strontium bismuth tantalate (SrBi 2 Ta 2 O 9 ), which shows no polarization fatigue with electric field cycling and has been very successfully applied in FeRAM [START_REF] Senthil | Relaxation and conduction mechanism of Dy 3+ substi-tuted SrBi 2 Ta 2 O 9 ceramics[END_REF][START_REF] De Araujo | Fatigue-free ferroelectric capacitors with platinum electrodes[END_REF]. However, the biggest challenge for commercially viable SrBi 2 Ta 2 O 9 -based electronic devices is how to assess and enhance the electrical and dielectric properties [START_REF] Li | Modulation of photocatalytic activity of SrBi 2 Ta 2 O 9 nanosheets in NO removal by tuning facets exposure[END_REF][START_REF] Zhu | The superior piezocatalytic performance of SrBi 2 Ta 2 O 9 nanoflower : Mechanism of screening effect and energy band theory[END_REF].

The enhanced physical properties cannot be achieved only by doping, but also by choosing an adequate synthesis process. Compared with the huge energy consumption of the solid state process, chemical synthe-sis provides an appealing route under mild conditions. However, it still faces challenges in producing highpurity materials based on the SrBi 2 Ta 2 O 9 compound. Very few materials have been synthesized through the molten-salt route, largely because the current method involves additional chemicals arising from the molten mixture that may contaminate the materials, but also involves many steps in the synthesis process, which is time-consuming. Recently, we have proven that the molten salt technique is a promising method to prepare different Aurivillius compounds [START_REF] Afqir | Molten salt synthesis , structural and dielectric properties of SrBi 2-y Y y Nb 2-x V x O 9 (0 ≤ x ≤ 0.2 and 0 ≤ y ≤ 0.2) ceramics[END_REF][START_REF] Afqir | Molten salt synthesis of Gd-doped SrBi 2 Ta 2 O 9 ceramics with enhanced dielectric properties at room temperature[END_REF]. Substitution of ions in these complex structures is a popular technique to achieve desired material properties [START_REF] Wendari | Enhanced dielectric and ferroelectric responses in La 3+ /Ti 4+ co-substituted SrBi 2 Ta 2 O 9 Aurivillius phase[END_REF]. If the substitution is performed on Sr site, it favours the structural distractions in the TaO 6 octahedron that improves the ferroelectric behaviour, as observed in the enhancement of ferroelectric properties [START_REF] Senthil | Relaxation and conduction mechanism of Dy 3+ substi-tuted SrBi 2 Ta 2 O 9 ceramics[END_REF][START_REF] Senthil | Dielectric, ferroelectric, impedance and photocatalytic water splitting study of Y 3+ modified SrBi 2 Ta 2 O 9 ferroelectrics[END_REF]. However, if the site of substitution is Ta, this leads to the enhancement of dielectric properties. Enhanced electrical and optical properties have been achieved by partially replacing Bi 3+ ions in this oxide with mainly rare earth elements, such as Sm 3+ , Eu 3+ or Gd 3+ [START_REF] Zhong | High thermally Sm 3+ -activated SrBi 2 Ta 2 O 9 orangered phosphor: Preparation, characterization, and optical properties[END_REF][START_REF] Zhong | Synthesis and optical properties of new red-emitting SrBi 2 Ta 2 O 9 :Eu 3+ phosphor application for w-LEDs commercially based on InGaN[END_REF][START_REF] Afqir | Dielectric properties of SrBi 1.8 RE 0.2 Nb 2 O 9 (RE = Yb, Tm, Tb, Gd, Er, Sm and Ce) ceramics[END_REF][START_REF] Afqir | Dielectric properties of gadolinium-doped SrBi 2 Nb 2 O 9 ceramics[END_REF].

The purpose of this study was to optimize the structure of Lu-doped SrBi 2 Ta 2 O 9 by changing the dopant content and sintering temperature in order to improve physical properties. To this end, the work consists of two parts: collecting the evidence of structural and microstructural characteristics and conducting quantitative measurements of dielectric properties.

II. Experimental

Lu-doped SrBi 2 Ta 2 O 9 (SrBi 2-x Lu x Ta 2 O 9 where x = 0, 0.025, 0.05, 0.75 and 0.1) powders were synthesized using the stoichiometric amounts of SrCO 3 (99.9%, Aldrich), Ta 2 O 5 (99.9%, Aldrich), Bi 2 O 2 CO 3 (99%, Aldrich) and Lu 2 O 3 (99.5%, Acros Organics). The precursors were heated at 600 °C for 6 h and then ground manually for 30 min. The ground mixture was stirred for 3 h in the molten salt mixture containing 46 wt.% NaNO 3 and 54 wt.% KNO 3 to promote the crude powders. FTIR (VERTEX 70v FT-IR), Raman (Confotec, 532 nm), and XRD (SmartLab SE, Rigaku) techniques were performed to track the transformation of reactants into desired products. The obtained crude powders were heated at 1000 °C for 12 h in an electric furnace and then uniaxially pressed at 1 MPa to form pellets having thickness and diameter of 0.9 and 10 mm, respectively. The pressed pellets were sintered in air at different temperatures up to 1200 °C. Silver paste electrodes were fixed on both surfaces of the sintered pellets and fired at 400 °C for 20 min in air. XRD patterns were recorded in the 2θ range of 10-70°with a step of 0.05°. Scanning electron microscope SEM (TESCAN VEGA) was used to analyse microstructure on the cross section areas of the prepared ceramics. Dielectric measurements were carried out using Solartron 1260 impedance/gain-phase and IVYTECH LACR-106x analysers.

III. Results and discussion

Microstructure

XRD patterns of the synthesized SrBi 2 Ta 2 O 9 and SrBi 2-x Lu x Ta 2 O 9 powders (in Fig. 1) are almost identical. They are isostructural with the SrBi 2 Ta 2 O 9 phase as compared with the XRD patterns in the ICDD database. All the diffraction peaks can be indexed to the SrBi 2 Ta 2 O 9 structure based on the orthorhombic crystal system with the A2 1 am space group. It can be observed that specific fingerprint peaks, corresponding to (113), (115), (020) and (135) planes, are easily identifiable in all XRD patterns. Thus, the used synthesis strategy can produce homogeneous materials with the resulting final products free from impurities.

If considering substitution of Bi 3+ (1.02 Å) by Lu 3+ (0.85 Å) ions with this large mismatch in ionic radii, one would have expected lower lattice parameters. However, the calculations reported in Table 1 revealed the smooth change of unit cell parameters upon adding Lu ions. It is important to underline that these physical values have an accuracy of measurement associated with them. Here, XRD data acquisition options only allowed us to determine the compounds' fineness as a primary purpose. In this regard, Lu-doping has not shown a significant change in structure.

Theoretical (ρ th ) and experimental (ρ ex ) densities were estimated through the XRD method and the Archimedes method, respectively (Table 1). Densities of the ceramics sintered at 1120 °C for 6 h are lower than that for the samples sintered at higher temperatures. The sintering at 1120 °C is not effective in getting a high density of ceramics, leading to lower fracture toughness and brittleness. It is demonstrated that sintering at 1200 °C maximizes experimental density, but at this

Composition

Cell parameters temperature chemical stability in compounds is lower, e.g. bismuth might be readily volatilized. Relative density (i.e. the ratio of experimental density to theoretical density) increases with Lu-doping amount and reaches 96 %TD with the SrBi 1.9 Lu 0.1 Ta 2 O 9 ceramics. The increase seems small in terms of the amount of dopant used, but they have consequences for the density of ceramics and their physical properties. Lu-doping had the task of improving ceramic density and reducing the loss of bismuth. Thus, higher relative density and lower material loss were achieved with sintering at 1200 °C for 1 h.

ρ ex [cm 3 /g] ρ th D [nm] a [Å] b [Å] c [Å] V [Å
FTIR and Raman spectroscopies were used to probe different chemical bonds in a compound. As a function of the amount of dopant, both spectra are not only particularly smooth and sensitive to the amount of dopant introduced into a lattice structure, but also show the same chemical bond nature. Regarding FTIR spectra (Fig. 2), bands observed at around 556, 646 and 787 cm -1 are characteristic for SrBi 2 Ta 2 O 9 structure [START_REF] Senthil | Relaxation and conduction mechanism of Dy 3+ substi-tuted SrBi 2 Ta 2 O 9 ceramics[END_REF][START_REF] Ke | Crystallization and nanograin growth in SrBi 2 Ta 2 O 9 synthesized by a novel sol-gel process[END_REF][START_REF] Moret | Infrared activity in the Aurivillius layered ferroelectric SrBi 2 Ta 2 O 9[END_REF][START_REF] Lu | Synthesis of ultrafine strontium bismuth tantalate powder by colloid-emulsion technique[END_REF]. The absorption peaks at 3425 and 1632 cm -1 correspond to O-H stretching and bending modes of chemisorbed water. The peaks at 2924 and 2849 cm -1 could be assigned to the C-H stretching vibrations arising from traces of organic matter. The peak observed at 1384 cm -1 , which is more intense with some compounds, may be due to the carbonate group's improper decomposition in KBr [START_REF] Cao | Adsorption of NO on ordered mesoporous carbon and its improvement by cerium[END_REF].

Raman spectra of the Lu-doped SrBi 2 Ta 2 O 9 powders are illustrated in Fig. 3 and exhibit characteristic bands at approximately 168, 206, 595 and 801 cm -1 as well as at 321, 369 and 456 cm -1 [START_REF] Kojima | Soft phonon and bismuth content in ferroelectric SrBi 2 Ta 2 O 9[END_REF][START_REF] Wu | Effect of V 2 O 5 B-site substitution on the microstructure, Raman spectrum, and dielectric properties of SrBi 2 Ta 2 O 9 ceramics[END_REF]. The bands at ∼805 and ∼600 cm -1 are attributed to the oxygen stretching in O-Ta-O bonds in the TaO 6 octahedron. The band located at ∼212 cm -1 is associated with the rock salt structure of Sr-O. The band at ∼163 cm -1 is associated with the vibration of the Ta 5+ ion along the z-direction within the TaO 6 octahedron [START_REF] Perez | Effect of cationic substitution on Raman spectra of SrBi 2 Ta 2 O 9 ceramics and thin films[END_REF][START_REF] Liu | Lattice vibrational properties of SrBi 2 Ta 2 O 9[END_REF].

FTIR and Raman results show that there are no significant changes in intensities of characteristic bands as a function of the dopant content. Atoms in crystals behave like springs and in a certain respect, the amount of dopant can sometimes only slightly affect interatomic vibrations. Consequently, no significant interatomic shifts were observed from the vibrational spectra.

Figure 4 shows fractured surfaces of the Lu-doped SrBi 2 Ta 2 O 9 ceramics sintered at 1200 °C for 1 h. The SEM reveals a general distinction between the undoped and doped samples. The undoped sample exhibits plate- like morphology. Conversely, the doped ceramics had highly irregularly-shaped morphology and grain size did not vary significantly. Meanwhile, their microstructure is composed of both fine and coarse-grained particles. The relationship between Lu amount and grain size is less consistent. It would, on the whole, be affected by sintering conditions. Thus, during compaction and sintering, grain growth was obviously creating a coarser grain structure with several smaller particles filling the space between coarse grains and giving rise to porosity.

Dielectric properties

Comparisons of the dielectric constant (ε ′ ) and dielectric loss (tan δ) values of the Lu-doped SrBi 2 Ta 2 O 9 ceramics measured at V AC = 0.1 V and V DC = 0 V are given in Fig. 5. The dielectric constant was found to be as high as ε ∼ 267 above 1 kHz for the SrBi 1.9 Lu 0.1 Ta 2 O 9 (i.e. for x = 0.1), a value comparable to that of the undoped sample. The ceramics having x = 0.025 displayed the lowest ε ′ of ∼244. However, the dielectric constant above 1 kHz was the highest (∼277) for the sample SrBi 1.95 Lu 0.05 Ta 2 O 9 and at the same time this ceramics (together with the undoped sample) had a minimal tan δ. Adding an ion of the same valence state usually does not increase the carrier concentration. However, in the case of the samples having x = 0.05 and x = 0.1, it seems that substituting Lu 3+ ions at the Bi 3+ site in SrBi 2 Ta 2 O 9 decreased the carrier concentration, leading to a high ε ′ . In addition, the Lu ion substitution did not drastically reduce tan δ to less than 10 -3 . Dielectric constant (ε ′ ) and dielectric loss (tan δ) of the prepared ceramics are independent on DC bias, as shown in Fig. 6. None of the compounds exhibited ε ′ and tan δ degradation over the full DC range and did not result in any immediate change regarding ε ′ . Without a DC voltage, spontaneous polarization can happen freely. However, when a DC voltage was applied, spontaneous polarization was tied to the direction of the electric field in the dielectrics and independent reversal of spontaneous polarization was inhibited. As a result, tan δ becomes lower than before applying the bias. In an ideal capacitor, as the frequency increases, ε ′ will tend to be constant, but here, there exists a frequency boundary beyond which the dielectric constant (ε ′ ) and dielectric loss (tan δ) smoothly rise again at higher frequency. Meanwhile, a rapid decrease in the dielectric constant with increasing frequency may be attributed to the reduction of the space charge polarization effect [START_REF] Hong | Manganese effect on the relaxation behaviors of the space charge polarization in Pb(Fe 2/3 W 1/3 ) 0.9 Ti 0.1 O 3 ceramics[END_REF]. According to experimental results, neither ε ′ nor tan δ of the undoped and doped SrBi 2 Ta 2 O 9 compounds have a linear dependence on the dopant concentration. Three reasons might be responsible for this behaviour. The first is directly related to polarizability as a dominant factor. Bi 3+ ions possess the highest polarizability which is strongly responsible for the increase of ε ′ . The second reason is the possibility of substituting Sr 2+ (1.12 Å) with Lu 3+ (0.85 Å) where Lu 3+ ions might preferentially occupy Sr 2+ sites at low doping concentrations and start to occupy Bi 3+ sites when the Lu 3+ concentration increases. The third reason could be the influence of grain size heterogeneity.

Both the dielectric constant (ε ′ ) and dielectric loss (tan δ) as a function of temperature for different frequencies are shown in Fig. 7. The dielectric constant of the undoped and doped samples is independent of frequency in the temperature range up to 280 °C, and then shows pronounced frequency dependence. All doped samples exhibit the ferroelectric-paraelectric transition of normal ferroelectric materials, like the pure SrBi 2 Ta 2 O 9 , coupled with a broad peak indicating on a diffuse phase transition. The pure SrBi 2 Ta 2 O 9 sample has transition temperature around 300 °C. On the other hand, somewhat lower transition temperature of about 280 °C is characteristic of the doped samples. The decrease of the Curie temperature may be due to the absence of the lone pair of electrons on Lu 3+ ion when compared to Bi 3+ ion. The relatively high transition temperature of SrBi 2 Ta 2 O 9 is caused by the presence of bismuth atoms, which resonate with relatively high frequency within the crystal lattice. Because Lu is heavier than Bi, it resonates more slowly, so lower transition temperatures are expected for the doped samples.

Overall, an increase in temperature led to an increase in dielectric loss (tan δ) due to the increase in carrier concentration. It is evident that tan δ increases smoothly in the temperature range 40-200 °C, and has values up to 10 -2 . At higher temperatures, dielectric loss increases and this trend sped up from 280 °C.

In ferroelectric materials, the Curie Weiss's law illustrates the temperature dependence of the dielectric constant above the Curie temperature:

1 ε ′ = T -T CW C (1)
where C and T CW are the Curie constant and Curie-Weiss temperature, respectively. The Curie-Weiss plot is presented in Fig. 8. The transition between a low and high Curie temperature form of the pure and doped SrBi 2 Ta 2 O 9 ceramics is a second-order transition. This second-order transition begins at the point of intersection of the dashed lines, which represent the fitted linear regression curve. Meanwhile, the Curie constant of all compounds, whose order of magnitude is estimated to be around 10 5 °C-1 , is characteristic of displacive phase transitions. The doped samples show much larger deviations from ideal behaviour than the undoped one. This can be seen from the variation in temperature between T C and T CW .

The symmetry-breaking at the Curie temperature results in a phase transition to a higher symmetry phase, which is attributed to the well-known orthorhombic-totetragonal phase transition and consistent with a secondorder transition.

As the Lu content increases in the specimens, disorderliness increases, leading to a diffuse phase tran- sition [START_REF] Dhak | Dielectric diffuseness and conductivity study of CuNb 2 O 6 incorporated BaTiO 3 synthesized by chemical route[END_REF]. Based on the structural characterization of the samples sintered at 1200 °C for 1 h and possible volatilization of bismuth, it was determined that the increase in dielectric diffuseness around the Curie temperature, may not be attributed only to the rise in charge defects within the lattice structure, but also to a reduction in grain size while the Lu amount rises.

The AC conductivity as a function of temperature at different frequencies of the Lu-doped SrBi 2 Ta 2 O 9 ceramics is shown in Fig. 9. Up to 180 °C, the conductivity increases smoothly and after 200 °C it increases steadily. As the temperature increases, the charge carriers can move among the lattice structure, and as a result, these carriers may be thermally activated when the temperature reaches 200 °C. The increase in conductivity with an increase in temperature confirms the NTCR (negative temperature coefficient of resistance [START_REF] Li | Investigation of pyrochlore-type A 2 Sn 2 O 7 (A = La, Nd, Sm, or Gd) ceramics as negative temperature coefficient thermistors for high-temperature application[END_REF]) behaviour of the materials laying down the conduction process arising from space charge conduction.

AC conductivity as a function of temperature was analysed using the Arrhenius equation:

σ AC = σ 0 • exp - E a k • T (2)
where σ 0 , E a and k are temperature-independent constants, activation energy and the Boltzmann constant, respectively. The activation energy value was deduced from the slope of ln σ AC versus 1000/T . The activation energies at 200 kHz, over a temperature range of 240-290 °C, were estimated to be 1.33, 1.09, 1.03, 1.59 and 1.97 eV for x = 0, 0.025, 0.05, 0.075 and 0.1, respectively. For x = 0.025 and x = 0.05 the AC conductivities were 3.45 × 10 -4 S/m and 4.14 × 10 -4 S/m, respectively, when measured at 280 °C and 200 kHz. When comparing the activation energies of the Lu-doped SrBi 2 Ta 2 O 9 compounds, the x = 0.1 sample exhibited the highest activation energy, 1.97 eV, which is 1.48 and 1.23 times higher than that of the x = 0.05 and the undoped samples, respectively. As a result, the larger the Lu amount within the lattice is, the more energy is required to counter the thermal fluctuations of excited charge carriers at a selected temperature range. However, when a small amount of Lu is introduced into the structure, lower activation energy is obtained. This is due to a variety of factors, including a change in grain size and an increase in Sr-site disorder (Sr 2+ , Bi 3+ , and Lu 3+ ions) in the Bi 2 O 2 and perovskite layers with variable Lu content. The smaller ion in the case of Lu 3+ (0.848 Å [START_REF] Wu | Effect of V 2 O 5 B-site substitution on the microstructure, Raman spectrum, and dielectric properties of SrBi 2 Ta 2 O 9 ceramics[END_REF]) provides more space, which promotes the ionic displacement. In other words, the higher activation energy is attributable to the lower bottleneck size for oxideion migration, but also possible existence of chargeclusters, that may arise from the introduction of Lu ions. A group of charges clustered together would lead to an increase in the activation energy in the substituted compounds [START_REF] Senthil | Dielectric, ferroelectric, impedance and photocatalytic water splitting study of Y 3+ modified SrBi 2 Ta 2 O 9 ferroelectrics[END_REF].

Jumping from one side to the other, the hopping charge carrier has to overcome the activation energy. Meanwhile, a composition gradient in materials induces a chemical potential gradient, giving rise to activation energy as temperature increases.

The average AC conductivity, nearly 1.04 ×10 -4 S/m, in the doped samples rose gradually, equal to the undoped one when measured at 200 °C at 200 kHz. By contrast, AC conductivity for all samples accounted for most of that increase with the contribution of dopants, there is around 2.8 × 10 -4 S/m for x = 0 and 5.2 × 10 -4 S/m for x = 0.1 when measured at 280 °C and the same frequency. This inverted AC conductivity at high temperatures might either be attributed to the fact that the more diffuseness is pronounced, higher the conductivity will be, or the closer the Curie temperature is, higher the charge carrier density will be leading to increased AC conductivity.

IV. Conclusions

Lu-doped SrBi 2 Ta 2 O 9 (SrBi 2-x Lu x Ta 2 O 9 where x = 0, 0.025, 0.05, 0.75 and 0.1) compounds were synthesized by combination of molten salt method and solidstate route and sintering at different temperatures up to 1200 °C. The prepared samples had pure and singlephase orthorhombic structure with plate-like morphology that is composed of fine and coarse-grained particles.

It was shown that the sample SrBi 1.95 Lu 0.05 Ta 2 O 9 had maximal dielectric constant (ε ′ ) and minimal dielectric loss (tan δ). The prepared samples demonstrated their normal ferroelectric behaviour. The ferroelectricparaelectric transition is coupled with a broad peak indicating on a diffuse phase transition. The pure SrBi 2 Ta 2 O 9 sample had transition temperatures around 300 °C, however, somewhat lower transition temperature of about 280 °C was characteristic of the doped samples.

There was no linear dependence of the dielectric properties on the Lu content. Thus, it was impossible to determine whether ceramics heterogeneity or Lu substitution was the exact reason for different values of dielectric constant and dielectric loss.

Figure 1 .

 1 Figure 1. XRD patterns of Lu-doped SrBi 2 Ta 2 O 9 powders Table 1. Cell parameters, theoretical density (ρ th ), experimental density (ρ ex ) at different sintering times and temperatures, and crystallite size (D) of Lu-doped SrBi 2 Ta 2 O 9

Figure 4 .Figure 5 .

 45 Figure 4. FTIR spectra of Lu-doped SrBi 2 Ta 2 O 9 powders

Figure 4 .

 4 Figure 4. SEM images of Lu-doped SrBi 2-x Lu x Ta 2 O 9 ceramics with different x

Figure 5 .

 5 Figure 5. Comparison of ε ′ and tanδ values of different Lu-doped SrBi 2 Ta 2 O 9 ceramics measured at V AC = 0.1 V and V DC = 0 V

Figure 6 .

 6 Figure 6. Dielectric constant (ε ′ ) and dielectric loss (tanδ) as a function of frequency and bias voltage of Lu-doped SrBi 2-x Lu x Ta 2 O 9 ceramics with different x: a) 0, b) 0.025, c) 0.05, d) 0.075 and e) 0.1

Figure 7 . 1 Figure 8 .

 718 Figure 7. Dielectric constant (ε ′ ) and the dielectric loss (tanδ) of temperature-frequency-dependent Lu-doped SrBi 2-x Lu x Ta 2 O 9 ceramics with different x: a) 0, b) 0.025, c) 0.05, d) 0.075 and e) 0.1

Figure 9 .

 9 Figure 9. Frequency and temperature dependence of AC conductivity of Lu-doped SrBi 2-x Lu x Ta 2 O 9 ceramics with different x: a) 0, b) 0.025, c) 0.05, d) 0.075 and e) 0.1


Lucia Grenga

Fabrice Gallais

Olivier Pible

Jean-Charles Gaillard

Duarte Gouveia

Hélène Batina

Niza Bazaline

Sylvie Ruat

Karen Culotta

Guylaine Miotello

Stéphanie Debroas

Marie-Anne Roncato

Gérard Steinmetz

Charlotte Foissard

Anne Desplan

Béatrice Alpha- Bazin

Christine Almunia

Fabienne Gas

Laurent Bellanger

Jean Armengaud
email: jean.armengaud@cea.fr

Shotgun proteomics of SARS-CoV-2 infected cells and its application to the optimisation of whole viral particle antigen production for vaccines

Introduction

SARS-CoV-2 belongs to the B lineage of the beta-coronaviruses and is closely related to the SARS-CoV virus [START_REF] Jiang | An emerging coronavirus causing pneumonia outbreak in Wuhan, China: calling for developing therapeutic and prophylactic strategies[END_REF][START_REF] Velavan | The COVID-19 epidemic[END_REF]. It is the causative agent of COVID-19, a severe acute respiratory syndrome that spread world-wide within a few weeks starting on December 2019 in Wuhan [START_REF] Zhou | A pneumonia outbreak associated with a new coronavirus of probable bat origin[END_REF]. The major four structural genes encode the nucleocapsid protein (N), the spike protein (S), a small membrane protein (SM) and the membrane glycoprotein (M) with an additional membrane glycoprotein (HE) occurring in the HCoV-OC43 and HKU1 beta-coronaviruses [START_REF] Zhou | A pneumonia outbreak associated with a new coronavirus of probable bat origin[END_REF].

Based on the speed at which the outbreak of COVID-19 has developed, SARS-CoV-2 appears to spread easily in the human population. The reproductive number (R0) of the virus is currently thought to be around 3, suggesting the potential for sustained human-to-human transmission that appears to be through respiratory droplets and potentially a fecal-oral route [START_REF] Weston | COVID-19: Knowns, Unknowns, and Questions[END_REF]. In this pandemic situation, one of the outstanding questions concerns the possibility to contain the spread of SARS-CoV-2 and its persistence in the human population. Social distancing policy, lock-down and other containment measures have been worldwide implemented to slow down the spread. Current roadmaps to lifting the restrictions rely on the deployment of effective diagnostics, therapies, and eventually on the development of an effective vaccine.

Several platforms are being used to develop vaccines against SARS-CoV-2, including spike subunit, DNA, RNA, whole-virion, and nanoparticle vaccines. Most successful antiviral vaccines employ inactivated or attenuated whole viral particles as vaccine antigen and depend on the induction of neutralizing antibodies [START_REF] Plotkin | New vaccination strategies[END_REF][START_REF] Plotkin | The development of vaccines: how the past led to the future[END_REF] against structural proteins of the virus. However, virus yields from the dedicated cell culture systems could be relatively low compared to quantities envisioned to be required for massive vaccine production. In addition, the production campaigns are time-consuming and highly demanding due to the danger of working with these pathogens, and thus optimization of the production of whole viral particle antigen is of utmost interest for vaccines. Concomitantly with vaccine development, a better understanding of how the host responds to SARS-CoV-2 infection may help direct further therapeutic avenues.

Multiple proteomics strategies have been shown insightful for better understanding of coronavirus structure and its molecular mechanisms of infection. Tracheal tissues of chicken infected with infectious bronchitis coronavirus were analyzed by 2D-DIGE and MALDI-TOF tandem mass spectrometry to establish the host response [START_REF] Sun | Comparative proteome analysis of tracheal tissues in response to infectious bronchitis coronavirus, Newcastle disease virus, and avian influenza virus H9 subtype virus infection[END_REF]. Vero cells infected with porcine epidemic diarrhea virus (PEDV) were analyzed by shotgun proteomics [START_REF] Zeng | Proteome analysis of porcine epidemic diarrhea virus (PEDV)-infected Vero cells[END_REF]. Different PEDV coronavirus strains were compared with an iTRAQ-labeling quantitative approach showing differences of inflammatory cascade eliciting [START_REF] Lin | Differential Protein Analysis of IPEC-J2 Cells Infected with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strains Elucidates the Pathogenesis of Infection[END_REF]. The dynamics of the host proteins triggered by specific overexpressed coronavirus genes was also established [START_REF] Choi | Functional Characterization and Proteomic Analysis of Porcine Deltacoronavirus Accessory Protein NS7[END_REF]. While no literature is yet available on the proteomics characterization of SARS-CoV-2 virus, several studies of interest have been recently submitted and should be soon available [START_REF] Bojkova | SARS-CoV-2 infected host cell proteomics reveal potential therapy targets[END_REF][START_REF] Ortea | Re-analysis of SARS-CoV-2 infected host cell proteomics time-course data by impact pathway analysis and network analysis. A potential link with inflammatory response[END_REF][START_REF] Davidson | Characterisation of the transcriptome and proteome of SARS-CoV-2 using direct RNA sequencing and tandem mass spectrometry reveals evidence for a cell passage induced in-frame deletion in the spike glycoprotein that removes the furin-like cleavage site[END_REF].

Here, we describe the establishment of a workflow based on shotgun tandem mass spectrometry data that in addition to gaining more basic information about SARS-CoV-2 infection aims at guiding the optimisation of the conditions for whole viral particle antigen production and aiding SARS-CoV-2 vaccine development.

Materials and Methods

Cell culture and Virus preparation

Vero E6 (ATCC, CLR-1586) cells were cultured at 37°C in 9% CO2 in Dulbecco's modified Eagle's medium (DMEM, Gibco™, ThemoFisher) supplemented with 5% fetal calf serum (FCS) and 0.5% penicillinstreptomycin. Cells were passaged by trypsinization every 2 days. The SARS-CoV-2 strains 2019-nCoV/Italy-INMI1 (Genbank MT066156) was provided by the Lazzaro Spallanzani National Institute of Infectious Diseases (Rome, Italy) via the EVAg network (European Virus Archive goes global). SARS-CoV-2 stocks used in the experiments had undergone two passages on Vero E6 cells and were stored at -80°C. Virus titer was determined by standard plaque assay (1x10 7 pfu/ml).

Infection

For the kinetic, 1x10 6 Vero cells seeded into 25 cm 2 flasks were grown to cell confluence in 5 mL DMEM supplemented with 5% FCS and 0.5% penicillin-streptomycin for one night at 37°C under 9% CO2. They were infected at two multiplicities of infection (MOI): 0.01 and 0.001. Cells were harvested at 1, 2, 3, 4, and 7 days post infection (dpi). Supernatants of SARS-CoV-2 infected cells were saved for plaque assay titration to confirm production of infectious viral particles. Infected Vero cells were microscopically observed for cytopathic effect (CPE) at the same time points.

Quantification of viral RNA by qRT-PCR

SARS-CoV-2 RNA from cell culture supernatant samples and supernatant plus cells was isolated using the NucleoSpin RNA Virus, Mini kit for viral RNA from cell-free fluids (Macherey Nagel) according to the manufacturer's instructions. RNA was subjected to qRT-PCR analysis using the SuperScript III Platinum One-Step qRT-PCR Kit (ThermoFisher) and a CFX96 Touch Real-Time PCR Detection System Thermal Cycler (BioRad). Primers targeting IP2 and IP4 (RdRp) were as recommended [START_REF] Corman | Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR[END_REF] Standard curves were created using in vitro transcribed RNA derived from strain BetaCoV_Wuhan_WIV04_2019 (EPI_ISL_402124). The transcript contains the amplification regions of the RdRp and E gene as positive strand. Each microtube contains 1011 copies of target sequences diluted in presence of yeast tRNAs, and lyophilised. Mean and standard deviation were calculated for each group (n=3).

Sample preparation for mass spectrometry

At each of various time points, SARS-CoV-2 -infected Vero cells were washed twice with 5 ml of PBS to remove media and FCS used to culture the cells. A volume of 1.5 ml of PBS was added to the washed cells before harvesting. The virus was inactivated and the cells lysed by autoclaving the samples at 125°C for 40 min. Proteins were precipitated by adding cold trichloroacetic acid to a final concentration of 10% (w/v). After an incubation of 5 min at 4 °C, the precipitated material was recovered by centrifugation at 16000 xg for 10 min. The proteins in the resulting pellets were then dissolved in 100 μL LDS 1X (Lithium dodecyl sulfate) sample buffer (Invitrogen) and supplemented with 5% betamercaptoethanol (vol/vol) before sonication with a Hielscher UP50H disruptor operated for 20 sec at 60% amplitude with 0.25 sec impulsions and then incubation for 5 min at 99°C. A 20 µl aliquot of a 1/8 dilution in LDS 1X (invitrogen) of each sample was loaded on NuPAGE 4-12% Bis-Tris gel and subjected to short SDS-PAGE migration. The proteins were stained for 5 min with Coomassie SimplyBlue SafeStain (Thermo Fisher Scientific) prior in-gel trypsin proteolysis performed as described in Hartmann et al. [START_REF] Hartmann | Taking the shortcut for highthroughput shotgun proteomic analysis of bacteria[END_REF].

Liquid chromatography-mass spectrometry

Peptides were identified using an ultimate 3000 nano-LC system (Thermo Fisher Scientific) coupled with a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). Peptides were desalted on a reversed-phase PepMap 100 C18 μ-precolumn (5 μm, 100 Å, 300 μm i.d. × 5 mm, Thermo Fisher Scientific) before peptide separation on a nanoscale PepMap 100 C18 nanoLC column (3 μm, 100 Å, 75 μm i.d. × 50 cm, Thermo Fisher Scientific) at a flow rate of 0.2 μL per min using a 120 min gradient comprising 100 min from 4% to 25% of solvent B and 20 min from 25% to 40% of solvent B (solvent A consisted in 0.1% formic acid in water, solvent B was 80% acetonitrile, 0.1% formic acid in water). The mass spectrometer was operated in Top20 mode. Full MS were acquired from 350 to 1,500 m/z and the 20 most abundant precursor ions were selected for fragmentation with 10 s dynamic exclusion time. Ions with 2+ and 3+ charge were selected for MS/MS analysis. Secondary ions were isolated with a window of 2.0 m/z.

MS/MS Data Interpretation and label free protein quantification

The MS/MS spectra recorded on each sample were assigned to peptide sequences using the Mascot Server 2.5.1 (Matrix Science). A database [START_REF] Zhou | Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[END_REF]585

MS/MS data repository.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [START_REF] Perez-Riverol | The PRIDE database and related tools and resources in 2019: improving support for quantification data[END_REF] partner repository with the dataset identifier PXD018594 and 10.6019/PXD018594.

Data analysis

Principal component analysis was done as previously described [START_REF] Gouveia | Quick microbial molecular phenotyping by differential shotgun proteomics[END_REF]. Co-expression cluster analysis was obtained using the Bioconductor R package coseq v1.5.2 [START_REF] Rau | Transformation and model choice for RNA-seq co-expression analysis[END_REF]. The protein abundance matrix was used as an input in coseqR. Log CLR-transformation was applied to the matrix to normalize the abundance of proteins and the K-means algorithm was chosen to detect the co-expressed clusters across the different time points. The K-mean algorithm was repeated 20 times in order to determine the optimal number of clusters. The resulting number of clusters in each run was recorded, and the most parsimonious cluster partition was selected using the slope heuristics approach. Both the PCA and the coseq analysis were performed after removal of proteins with spectral counts lower than three (1402 host protein groups retained). Finally, proteins assigned to the different clusters were retained for cluster visualization and gene ontology (GO)-enrichment analysis per each cluster. Statistically enriched (FDR ≤ 0.05) GO terms on proteins that are differentially expressed between pairwise samples or on proteins assigned to each co-expression cluster were identified using Metascape [START_REF] Zhou | Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[END_REF]. The most statistically enriched GO terms were visualized in ggplot2 [START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF].

Results

Profiling of virus production by tandem mass spectrometry

To understand the dynamics associated with SARS-CoV-2 infection and determine optimal conditions for whole-viral particle antigen production we infected Vero E6 cells with SARS-CoV-2 at two multiplicity of infection (MOI 0.01 and 0.001) and monitored the kinetics of the infection by means of tandem mass spectrometry over several days (Figure 1). Overall, we identified 3220 Vero cell proteins and 6 SARS-CoV-2 proteins with 27388 and 94 unique peptides (FDR below 1%), respectively (Table S1). Among the identified viral proteins, three (N, S, M) out of the four encoded by the viral genome were structural proteins while three were non-structural ones. In particular, out of the sixteen nonstructural proteins we identified peptides corresponding to the ORF1a papain-like protease (PLpro) / 3C-like protease (3CLpro) and to the accessory proteins encoded by ORF3a and ORF7a. These protein sequences were covered with 40 (N), 29 (S), 7 (M), 13 (ORF1ab), 4 (ORF3a), and 1 (ORF7a) distinct peptides. The sequence coverage of these polypeptides depends on their abundance, size, and lysine and arginine residues occurrence, the ORF7a protein with 121 residues being logically poorly detected compared to the others. Based on the sets of peptides shared by the different proteins, 2984 protein groups were identified, for which a total of 457,111 PSMs were assigned. The viral proteins represented 1.4% of these PSMs.

The dynamics of viral protein levels across time points indicated that SARS-CoV-2 protein synthesis increased continuously after infection with a peak registered around day 3 post infection. Viral amplification was slightly delayed in cells infected at a MOI of 0.001 (Figure 2A). SARS-CoV-2 N, S, and M were consistently among the most abundant proteins detected. Their respective ratios were relatively constant along time. Decline in abundance of viral proteins registered at 7 days post infection was in accordance to notable cytopathic effect following intense virus replication observed here for each culture at this time-point, as previously reported [START_REF] Park | Virus Isolation from the First Patient with SARS-CoV-2 in Korea[END_REF].

As expected, a larger variety of peptides was found for overrepresented proteins. Figure 2B shows the distribution of this diversity across the time points for the three most abundant viral proteins.

Interestingly, increase of protein levels relied on increasing abundance of the same set of peptides with only few new sequences registered at the peak of viral production compared with an early time point.

To evaluate to what extent virus profiles obtained by tandem mass spectrometry reflected virus production we measured SARS-CoV-2 RNA molecules by quantitative PCR analysis across the same time points (Figure 2C). Variations in most abundant viral protein yields reflected variation in the number of SARS-CoV-2 RNA molecules confirming that LC-MS/MS with label-free quantitation can be applied to monitor SARS-CoV-2 infection kinetics.

Characterization of host cell protein dynamics upon SARS-CoV-2 infection

Next, we characterized changes in cellular protein networks upon SARS-CoV-2 infection at the level of total protein abundance. Dimension reduction by principal component analysis (PCA) showed that early and late time points were distinctively distributed along principal components 1 and 2, with replicates clustering closely. This was observed for both MOI. Interestingly, a degree of overlap was observed for time points J3 and J4 corresponding to the peak of viral protein abundance implying a smaller evolution of the host proteome between these time points (Figure 3A). Addition of the viral proteins measured at each time point did not affect samples separation. To elucidate the host response during virus amplification, we performed a co-expression cluster analysis to determine host proteins showing similar profiles over time (Figure 3B). Functional enrichment analysis performed on the members of each cluster provided a global overview of biological processes (Figure 4A-C). At MOI 0.01, clusters 2 and 5, with 345 and 198 detected protein members, respectively, showed a close similarity of the statistically enriched terms, with functions and pathways related to the viral life cycle among the most represented ones. Besides, as revealed by functional interaction network analysis even cluster-specific enriched functions like membrane trafficking and protein pre-processing in endoplasmatic reticulum and regulation of mRNA processing/splicing via spliceosome, respectively, were highly interconnected (Figure 4A-B). Also, an overlap of enriched biological functions was observed for clusters 3 and 6, with 16 and 10 representatives, respectively, characterized by a specific increase in the abundance of clustered proteins 3 days post-infection, with proteins involved in cornification and ECM regulators (Extra Cellular Matrix) among the top 20 significantly enriched terms (Figure 4A). Four distinct expression profiles were identified at MOI 0.001 (Figure 3B). Comparative analysis and inference of enriched biological pathways revealed a significant enrichment of functions related to host responses to the viral replication in cluster 2 and 3, with clathrin-mediated endocytosis (R-HSA-8856828) and vesicle-mediated transport (R-HSA-5653656) pathways specific of the former one (Figure 4D) and likely involved in vacuole formation and viral budding. Interestingly, similar to cluster 2 obtained at higher MOI, the expression profiles of the members of these clusters are similar to the dynamics of the viral protein levels across the kinetics. Clusters 4 (MOI 0.01) and 3 (MOI 0.001) were enriched in pathways and biological functions related to the host central metabolism with the metabolism of RNA and translation among the top enriched pathways. Despite the temporal expression profiles of the proteins belonging to these clusters are not characterised by major changes, their remodelling by the virus and a major effect on their abundance at earlier time points (<24 hours) cannot be excluded.

Discussion

In the race to develop a vaccine for fighting the global spreading of SARS-CoV-2, different technology platforms have been evaluated [START_REF] Le | The COVID-19 vaccine development landscape[END_REF]. In this context, the obtainment of vaccines based on inactivated or attenuated whole viral particles could be challenged in the finding of the right conditions for virus amplification. Virus yields from the dedicated cell culture systems could also represent a limitation.

Concomitantly with vaccine development, inactivated virus particles are also of interest for testing real serology or screening neutralizing antibodies. Evidently, production of well-characterized active virus particles is also of interest for fundamental research purposes. Given the requirement for speed, here we evaluate the use of LC-MS/MS as a tool for guiding the optimisation of the conditions for SARS-CoV-2 whole viral particle antigen production.

The results presented here demonstrate the potential of our pipeline to profile virus production across time. In particular, by analysing the proteome of Vero cells infected with SARS-CoV-2 at two different MOI, it was possible to monitor changes in the levels of three SARS-CoV-2 structural proteins and three non-structural ones. Whilst as for other analyses [START_REF] Bojkova | SARS-CoV-2 infected host cell proteomics reveal potential therapy targets[END_REF][START_REF] Davidson | Characterisation of the transcriptome and proteome of SARS-CoV-2 using direct RNA sequencing and tandem mass spectrometry reveals evidence for a cell passage induced in-frame deletion in the spike glycoprotein that removes the furin-like cleavage site[END_REF] we could not detect peptides from protein E like. The lack of detection of other accessory proteins could be imputed to differences in samples processing with the protocol described here favouring simplified steps and speed while maintaining accuracy. Deeper analyses are envisaged for monitoring virus homogeneity during the different steps of viral production once the most permissive conditions will be established.

Remarkably, comparable profiles were obtained at the two tested MOI, with the profiles obtained at lower MOI slightly delayed and hence more insightful regarding the timing of the burst in the abundance of viral levels.

Overrepresented proteins were described by a larger variety of peptides. Interestingly, an increase of protein levels relied on the increasing abundance of the same set of peptides with only a few new sequences registered at late time points suggesting that absolute quantification of the virus could be obtained by targeted approaches by following early detectable peptides. Eventually tandem mass spectrometry proteotyping [START_REF] Grenga | Pathogen proteotyping: a rapidly developing application of mass spectrometry to address clinical concerns[END_REF][START_REF] Karlsson | Proteotyping: Proteomic characterization, classification and identification of microorganisms--A prospectus[END_REF] could be also proposed to detect SARS-CoV-2 viruses.

Besides profiling virus production, our mass spectrometry analysis of the whole cell content provides detailed information is available regarding virus cell entry, increased understanding of the different steps in the SARS-CoV-2 replication cycle are needed [START_REF] Tang | Coronavirus membrane fusion mechanism offers as a potential target for antiviral development[END_REF]. To our knowledge, this is one of the first attempt to characterize cellular response to infection with SARS-CoV-2 in Vero E6 cells. Interestingly, such proteomics data can be acquired on different cell lines from humans and primates in order to define by comparative proteomics the common mechanisms of cell infection and the mechanisms specific of a given cell line.

The analysis of our proteomic data suggested substantial temporal remodelling of the host proteome over the time points. Functional enrichment analysis of clusters of proteins showing similar expression profiles highlight key pathways during virus replication providing a potential target for effective therapeutics against coronaviruses rises. Consistently with the pathways identified by Bojkova et al.

[11] by using the human colon epithelial carcinoma cell line Caco-2 as system for the analysis of SARS-CoV-2 replication, we identified clusters of proteins increased by infection and enriched in RNA modifiers, such as spliceosome components, and carbon metabolism, further supporting the preliminary evidence that classifies splicing as an essential pathway for SARS-CoV-2 replication and potential therapeutic target. Additional clusters of proteins identified in our analysis highlighted the regulation of pathways critical for the virus life cycle like those involved in protein pre-processing in the endoplasmatic reticulum, vacuole formation and viral budding.

Here, we show that our pipeline based on LC-MS/MS analysis is a suitable tool for the characterisation of SARS-CoV-2 production. We, therefore, suggest that it could be of use in the optimisation of the condition for viral amplification to speed up the initial steps in favour of those that later on during the development process will require a more careful evaluation of effectiveness and safety.

Besides, peptide information described here provide sufficient information to enable a targeted analysis, opening the possibility of using mass spectrometry-based targeted approaches for the evaluation of critical aspects (i.e. quality and quantity) during the different steps of the virus purification processes. Furthermore, the characterized changes in cellular protein networks upon SARS-CoV-2 infection provided valuable insights that could be further explored and guide the identification of drug targets to address the pandemic caused by SARS-CoV-2.

We can anticipate that the same workflow could be successfully applied to expedite the characterisation of human organ-on-a-chip (Organ Chip) microfluidic culture devices used to obtain insights on the different steps of the virus life cycle as well as to study human disease pathogenesis [START_REF] Benam | Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro[END_REF] in response to infection by variants of SARS-Cov-2 under or not the addition of existing [START_REF] Weston | FDA approved drugs with broad anticoronaviral activity inhibit SARS-CoV-2 in vitro[END_REF][START_REF] Jeon | Identification of antiviral drug candidates against SARS-CoV-2 from FDA-approved drugs[END_REF][START_REF] Si | Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID19 therapeutics in viral pandemics[END_REF] and novel therapeutics.

LG, FG, OP, LB, and JA conceptualized the stud design; FG, JCG, HB, SR, NB performed the experiments;

LG, OP, DG, FG, LB and JA analysed the data; GM, KC, SD, MAR, GS, CF, AD, BAB, and CB contributed reagents and software development;

LG and JA draft the manuscript. All authors read and approved the final manuscript.

  : nCoV_IP2-12669Fw (ATGAGCTTAGTCCTGTTG) nCoV_IP2-12759Rv (CTCCCTTTGTTGTGTTGT) nCoV_IP2-12696bProbe(+) (AGATGTCTTGTGCTGCCGGTA [5']Hex [3']BHQ-1) and nCoV_IP4-14059Fw (GGTAACTGGTATGATTTCG) nCoV_IP4-14146Rv (CTGGTCAAGGTTAATATAGG) nCoV_IP4-14084Probe(+) (TCATACAAACCACGCCAGG [5']Fam [3']BHQ-1), respectively, using 0.4 µM per reaction.

Figure 1 .

 1 Figure 1. Schematic representation of the experimental design. Vero E6 cells were infected at Day 0 with SARS-CoV-2 at two multiplicity of infection (MOI 0.01 and 0.001). The kinetics of the infection was monitored by means of tandem mass spectrometry over several days. The main steps and the output of the analysis are highlighted.

Figure 2 .

 2 Figure 2. Virus profiling by tandem mass spectrometry. (A) Kinetic of viral production by LC-MS/MS. Relative abundance at each time points represents the mean ± standard deviation of the technical replicates at the two MOI tested. Dashed curves indicate results obtained at MOI 0.01 while solid curves refer to MOI 0.001. (B) Comparison between viral peptides identified at the different time points. Sets of intersections are visualized using the UpSet matrix layout and plotted horizontally. Each column corresponds to an exclusive intersection that contains the elements of the sets represented by the dark circles. Sets are represented by the different peptides assigned to viral proteins at each time point. J2, J3, J4 and J7 refer to the different time points analysed: Day 2, 3, 4 and 7, respectively. (C) Correlation between viral counts (copies/mL) obtained by qRT-PCR and the abundance of viral proteins measured by LC-MS/MS across time points. Dashed curves indicate results obtained at MOI 0.01 while solid curves refer to MOI 0.001.

Figure 3 .

 3 Figure 3. Host response upon SARS-CoV-2 infection. (A) Dimension reduction by principal component analysis (PCA) of the different time points at MOI 0.01 and 0.001, respectively. Normalised abundance of proteins with Spectral Count >3 was used as input. J1, J2, J3, J4 and J7 refer to the different time points analysed, Day 2, 3, 4 and 7, respectively. (B) Clusters of proteins showing similar expression profiles over time. J1, J2, J3, J4 and J7 refer to the different time points analysed, Day 2, 3, 4 and 7, respectively. The number on the top of each plot identifies the cluster.

Figure 4 .

 4 Figure 4. Host pathways dysregulated by the infection. (A-C) Heatmap showing the top enrichment clusters, one row per cluster, using a discrete color scale to represent statistical significance. Gray color indicates a lack of significance. MOI 0.01(A) and 0.001 (C). (B-D) Enrichment network visualization for results from the proteins present in each of the identified clusters. Nodes are represented by pie charts indicating their associations with each cluster. Color code represents the clusters. At MOI 0.01 (B), red represents Cluster 1 while blue, green, purple and orange refer to Cluster 2, 3, 5 and 6, respectively. At MOI 0.001 (D), indicates Cluster 1 while blue and green refer to Cluster 2 and 4, respectively.

Figure 1 Figure 3

 13 Figure 1

  sequences; 10,847,418 residues) containing the UniProt Chlorocebus sequences (downloaded March 2020) and the Italy-INMI1 SARS-CoV-2 protein sequences was queried after a first analysis to remove contaminant spectra against an in-house

	'common contaminants' database (384 sequences; 187,250 residues) encompassing 361 contaminants
	classically observed in proteomics (cRAP + additional contaminants) and 23 Bos taurus sequences
	corresponding to the most abundant proteins from foetal calf serum present in the cell culture medium
	[16]. Peptide-to-MS/MS spectrum assignation was done with the following parameters: full trypsin
	specificity, maximum of two missed cleavages, mass tolerances on the parent ion of 5 ppm and 0.02
	Da on the MS/MS, static modification of carbamidomethylated cysteine (+57.0215), and oxidized
	methionine (+15.9949) and deamidation of asparagine and glutamine (+0.984016) as dynamic

modifications. Mascot DAT files were parsed using the Python version of Matrix Science msparser version 2.5.1 with function ms_peptidesummary. Peptide-to-Spectrum Matches (PSMs) with the expectation values corresponding to 1% False Discovery Rate (FDR) were validated using the MASCOT homology threshold option. Multiple PSMs per MS/MS spectra were allowed in case of ion scores higher than 98% of the top ion score. Proteins were grouped if they shared at least one peptide, and in each group label-free quantification was based on PSM counts for each protein following the principle of parsimony. Proteins identified by one or more specific peptides were retained for the analysis (protein FDR 1%).

Acknowledgements

The authors are indebted to Dr Silvia Meschi (National Institute for Infectious Diseases "Lazzaro Spallanzani" IRCCS, via Portuense 292, 00149 Rome, Italia) for making the Human 2019-nCoV strain 2019-nCoV/Italy-INMI1 (008N-03893) available. The authors thank all their colleagues from Li2D (CEA) for strong support, as well as all those who make the experimental work possible while facing the Covid19 pandemic and lockdown.

Funding

This work was funded in part by the French Alternative Energies and Atomic Energy Commission (CEA), the joint ministerial program of R&D against CBRNE threats, and the ANR program "Phylopeptidomics" (ANR-17-CE18-0023-01). This publication was supported by the European Virus Archive goes Global (EVAg) project that has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement N°653316.

Disclosure statement

No potential conflict interest was reported by the authors.