Angela Parise

Giada Ciardullo

Mario Prejanò

Aurélien De

La Lande

Tiziana Marino

Aurélien De La Lande

On the Recognition of Natural Substrate CTP and Endogenous Inhibitor ddhCTP of SARS-CoV-2 RNA-Dependent RNA Polymerase: A Molecular Dynamics Study

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

Introduction

The world is currently in a state of pandemic emergency due to the spread of a disease called COVID-19 caused by a new viral etiologic agent, SARS CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) [START_REF] Riva | Discovery of SARS-CoV-2 Antiviral Drugs through Large-Scale Compound Repurposing[END_REF] . Today SARS-CoV-2 has become probably the fastest-spreading virus in the human history. In fact, despite the vaccination, the occurring continuous specific mutations hide it from the immune system so that the use of antiviral therapy is mandatory to prevent future outbreaks of rapidly spreading coronaviruses. SARS-CoV-2 is a positive RNA virus, which for its proliferation requires RNA-dependent RNA polymerase (RdRp), a key enzyme that regulates replication and transcription of the viral genome. For this reason, RdRp has become the validated target for the development of drugs against COVID-19 disease that can be hindered by the capacity of human coronavirus to proofread [START_REF] Robson | Coronavirus RNA proofreading: molecular basis and therapeutic targeting[END_REF] and to remove mismatched nucleotides during genome replication and transcription. In fact, incorporating nucleoside analog molecules into viral RNA can lead to increased mutation rates and to the failure of the virus through "lethal mutagenesis". [START_REF] Boehr | Structure, dynamics, and fidelity of RNA-dependent RNA polymerases[END_REF] The RdRp complex, one of the largest proteins in the viral genome (932 residues), has multiple nonstructural protein (nsp) units, and, in detail, is organized in nsp12 core catalytic unit, in nsp7-nsp8 heterodimer, nsp8 subunit, with a stoichiometry of 1:1:2 respectively. [START_REF] Jiang | RNA-Dependent RNA Polymerase: Structure, Mechanism, and Drug Discovery for COVID-19[END_REF] These three non-structural proteins along with the nsp13 helicase are the "core" of the replication transcription complex (RTC).

The category of antiviral drugs targeting the RdRp includes remdesivir, [START_REF] Wang | Remdesivir and Chloroquine Effectively Inhibit the Recently Emerged Novel Coronavirus (2019-NCoV) in Vitro[END_REF][START_REF] Yin | Structural Basis for Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir[END_REF] a nucleotide analogue adenosine mimic that showed efficacy against Ebola infection and has been successfully repurposed for the treatment of SARS-CoV-2 infection, such that it is the only drug approved against SARS-CoV-2 by the US Food and Drug Administration (FDA), on August 10, 2020. [START_REF] Lamb | First Approval[END_REF] Several works have proposed remdesivir to induce delayed chain termination [START_REF] Aranda | Mechanism of reaction of RNA-dependent RNA polymerase from SARS-CoV-2[END_REF][START_REF] Gordon | The Antiviral Compound Remdesivir Potently Inhibits RNA-Dependent RNA Polymerase from Middle East Respiratory Syndrome Coronavirus[END_REF][START_REF] Tchesnokov | Delayed Chain Termination Protects the Anti-Hepatitis B Virus Drug Entecavir from Excision by HIV-1 Reverse Transcriptase[END_REF][START_REF] Salgado-Benvindo | Suramin Inhibits SARS-CoV-2 Infection in Cell Culture by Interfering with Early Steps of the Replication Cycle[END_REF][START_REF] Ko | Arguments in Favour of Remdesivir for Treating SARS-CoV-2 Infections[END_REF] or to cause the pausing and backtracking of polymerase, paving the way to the use of other nucleotide analogs. [START_REF] Seifert | Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective[END_REF] 3'-deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral nucleotide-like compound produced by enzyme viperin as part of the innate immune response, [START_REF] Carlton-Smith | MTAP44, and Protein Kinase R Contribute to the Interferon-Induced Inhibition of Bunyamwera Orthobunyavirus Replication[END_REF][START_REF] Chin | Viperin (Cig5), an IFN-Inducible Antiviral Protein Directly Induced by Human Cytomegalovirus[END_REF] and its easy synthesis of ddhCTP has been recently described. [START_REF] Wood | Chemical Synthesis of the Antiviral Nucleotide Analogue DdhCTP[END_REF] Its chain terminator effect for RNA-dependent polymerases of multiple members of the Flavivirus genus has been further reported [START_REF] Gizzi | A Naturally Occurring Antiviral Ribonucleotide Encoded by the Human Genome[END_REF] and it has been recently demonstrated that the SARS-CoV-2 polymerase can incorporate this cytosine analogue well. [START_REF] Seifert | Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective[END_REF] Drawn inspiration from that evidence on ddhCTP [START_REF] Seifert | Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective[END_REF][START_REF] Shi | A Nucleobase-Binding Pocket in a Viral RNA-Dependent RNA Polymerase Contributes to Elongation Complex Stability[END_REF] and the computational work on Remdesivir, [START_REF] Aranda | Mechanism of reaction of RNA-dependent RNA polymerase from SARS-CoV-2[END_REF][START_REF] Arba | Mechanistic Insight on the Remdesivir Binding to RNA-Dependent RNA Polymerase (RdRp) of SARS-Cov-2[END_REF] a comparative in silico molecular modelling study (ddhCTP versus CTP) has been carried out on RdRp of SARS-CoV-2, to gain more insights on the incorporation of nucleoside triphosphates (see Scheme 1), which is one of the widely supported modes of interfering during viral replication pursuant to the high error rate (or low viral fidelity) of RNA virus replication. [START_REF] Robson | Coronavirus RNA proofreading: molecular basis and therapeutic targeting[END_REF][START_REF] Denison | Coronaviruses: an RNA proofreading machine regulates replication fidelity and diversity[END_REF] The comparison between these two species is interesting because the ddhCTP competes with CTP, [START_REF] Gordon | Remdesivir Is a Direct-Acting Antiviral That Inhibits RNA-Dependent RNA Polymerase from Severe Acute Respiratory Syndrome Coronavirus 2 with High Potency[END_REF] as confirmed from an intracellular concentration of ~100 µM and lower than purine analogues, [START_REF] Recovery | Dexamethasone in Hospitalized Patients with Covid-19[END_REF] despite differ of the -OH group loss in 3' position (see Scheme 2).

However, the absence of 3'-OH precludes further nucleotide incorporation from RdRp enzyme, which is consequently stalled when the RNA chain moves through the polymerase and the ddhCTP is processed. Another advantage of ddhCTP, with respect to other nucleoside analogue inhibitors, is that it does not require any particular metabolic transformations, differing from the case of other prodrugs (remdesivir and its derivatives). [START_REF] Seifert | Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective[END_REF] The choice of the investigation in silico of the endogenous product ddhCTP as antiviral drug represents a good strategy to develop other compounds in more reduced times than those needed to obtain new therapeutic molecules from the scratch. Furthermore, ddhCTP represents an example of how the location of the modification in the ribose differently from other antiviral nucleotide analogs can affect the catalytic pathway used for incorporation. [START_REF] Seifert | Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective[END_REF] In the present study, classical Molecular Dynamics (MD) simulations are used to investigate the early steps of the RdRp working mechanism (recognition, see Scheme 1), including the contribution of possible conformational rearrangements in the substrate selection and binding. Scheme 1. Canonical schematic reaction of RNA-dependent RNA polymerase.

Molecular docking has been used to test the affinity between polymerase active site and the examined ligands. MDs were performed on RdRp:RNA binary complex, including nsp7, nsp8, nsp12, and a partial double stranded RNA represented by a primer nucleotide strand of 6 units and a template strand of 8 nucleotides, and the ternary complexes formed by RdRp:RNA with ddhCTP and CTP.

The comparative analysis of the dynamic behaviour of RdRp in presence of both endogenously produced nucleotide analog and chain terminator, ddhCTP, and the natural nucleotide CTP provides deeper insights useful to better discriminate the factors underlying their incorporation in RdRp. In addition, the identification of amino acid residues important for the nucleotide recognition, and/or discrimination, represents one of the main focus of the present investigation, coupled to the description at atomistic level outer coordination shell of the two cations of the active site, which can result crucial for the viral replication. Scheme 2. 2D representation of a) cytidine triphosphate (CTP) and b) 3'-deoxy-3',4'-didehydrocytidine triphosphate (ddhCTP). P α , P β and P γ refer to phosphate groups.

Computational Methods

In silico engineered RdRp:RNA model

The investigation started from a cryo-EM 23 crystal complex of RNA polymerase, with a resolution of 2.50 Å (PDB code 7AAP) and with a partial bound template-primer RNA and RDV-TP. The crystallographic structure concerns the subunits nsp7, nsp8 and the catalytic nsp12 bound to a templateprimer RNA duplex, in complex with favipiravir ribonucleoside triphosphate (favipiravir-RTP). 23 Due to different pyrimidine-like nature of the ddhCTP, a painstaking process was also required in the selection of the sequence of the RNA fragment.

Our study additionally includes the RNA template-primer similarly to Arba et al. work on the remdesivir, [START_REF] Arba | Mechanistic Insight on the Remdesivir Binding to RNA-Dependent RNA Polymerase (RdRp) of SARS-Cov-2[END_REF] which is crucial for the nucleotide triphosphate binding, differently to other computational investigation on remdesivir -SARS-CoV-2 RdRp interaction. [START_REF] Elfiky | SARS-CoV-2 RNA Dependent RNA Polymerase (RdRp) Targeting: An in Silico Perspective[END_REF][START_REF] Elfiky | Anti-HCV, Nucleotide Inhibitors, Repurposing against COVID-19[END_REF][START_REF] Kato | Intermolecular Interaction among Remdesivir, RNA and RNA-Dependent RNA Polymerase of SARS-CoV-2 Analyzed by Fragment Molecular Orbital Calculation[END_REF][START_REF] Zhang | Structural Basis of the Potential Binding Mechanism of Remdesivir to SARS-CoV-2 RNA-Dependent RNA Polymerase[END_REF] Among the available crystallographic structures our choice fell on the one that presented two magnesium ions essential for the RdRp activity. In fact, it is not always trivial to find out experimentally the number of Mg ions present in the active site as well as the identity and the number of their coordination ligands. [START_REF] Palermo | Catalytic metal ions and enzymatic processing of DNA and RNA[END_REF] The nsp12 structure contains a right-hand RdRp domain (residues Val398 to Thr929) and a specific Nterminal extension domain (residues from Asp60 to Ala250) that adopts a nidovirus RdRp-associated nucleotidyltransferase (NiRAN) architecture (see Figure 1). [START_REF] Lehmann | Discovery of an Essential Nucleotidylating Activity Associated with a Newly Delineated Conserved Domain in the RNA Polymerase-Containing Protein of All Nidoviruses[END_REF] The polymerase domain and NiRAN domain are connected by an interface domain (residues Leu251 to The polymerase domain adopts the conserved structure of the viral polymerase family [START_REF] Mcdonald | RNA Synthetic Mechanisms Employed by Diverse Families of RNA Viruses[END_REF] and consists of three subdomains of a "cupped right hand": a finger subdomain (residues Val398 to Ala581 and Asn628 to Thr687), a palm subdomain (residues Thr582 to Pro627 and Ala688 to Gln815) and a thumb subdomain (residues His816 to Thr929). This right-hand architecture in turn evidences seven conserved structural motifs: motifs A-E located in the palm subdomain and motifs F and G in the fingers subdomain. [START_REF] Geronimo | Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology[END_REF] Most of these motifs are shared with other polymerases indicating the importance of these structural elements in their enzymatic function. [START_REF] Itoh | Dynamic properties of SARS-CoV and SARS-CoV-2 RNA-dependent RNA polymerases studied by molecular dynamics simulations[END_REF] The active site, contained in the palm domain, is formed by residues Ser759, Asp760 and Asp761 of motif C. [START_REF] Gao | Structure of the RNA-Dependent RNA Polymerase from COVID-19 Virus[END_REF] The carboxylate groups of these aspartates anchor a pair of divalent metal ions (Mg 2+ ), which play the major role in catalysis, [START_REF] Mcdonald | RNA Synthetic Mechanisms Employed by Diverse Families of RNA Viruses[END_REF] while two other metal ions (Zn 2+ ) play a structural role to stabilize the enzyme. One of the zinc ions is coordinated to amino acid residues His295, Cys301, Cys306, and Cys310 in the N-terminal domain while the second is bonded to Cys487, His642, Cys645, Cys646 residues, in the finger domain. [START_REF] Kirchdoerfer | Structure of the SARS-CoV Nsp12 Polymerase Bound to Nsp7 and Nsp8 Co-Factors[END_REF] As above-mentioned, the selected RdRp crystal structure incorporates the Favipiravir inhibitor, which is a purine nucleic acid analogue derived from pyrazine carboxamide (6-fluoro3-hydroxy-2-pyrazinecarboxamide). [START_REF] Shannon | Rapid Incorporation of Favipiravir by the Fast and Permissive Viral RNA Polymerase Complex Results in SARS-CoV-2 Lethal Mutagenesis[END_REF] The design of RNA double strand length and the type of bases for cytosine analogue was then required.

The selection of RNA sequence has been based on MALDI TOF experiments on CTP-like RpRd inhibitors. [START_REF] Chien | Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase, a Key Drug Target for COVID-19[END_REF] The spectrometry study showed that the required base sequence for ddhCTP and CTP is UAAAAU (5' -> 3') and AUUUUAGU (3' -> 5'), for primer and template, respectively.

To obtain the desired model, the phosphodiester bond strain has been retained from the starting cryoEM structure, while all the already present nucleobases have been scrupulously replaced in silico, as shown in Figure 2, in agreement with the sequence for pyrimidine-like RdRp inhibitors. tleap tool in Amber16 37 has been used to get the final model (Figure 2C).

MD simulations setting

The parameters of cytidine triphosphate (CTP) were obtained by adopting AMBER 94 and AMBER 99 force field [START_REF] Hornak | Comparison of Multiple Amber Force Fields and Development of Improved Protein Backbone Parameters[END_REF] data of mono-phosphate cytidine and parameters for the two terminal phosphate group of ATP are available in AMBER parameter database. In order to obtain ddhCTP parameters, gas-phase geometry optimization has been carried out using B3LYP/6-311G*. Atomic charges were derived by fitting the electrostatic potential according to the Merz-Singh-Kollman scheme, [START_REF] Bayly | A Well-Behaved Electrostatic Potential Based Method Using Charge Restraints for Deriving Atomic Charges: The RESP Model[END_REF] using the RESP procedure. Antechamber and parmchk modules of Amber16 have been employed for generating preparatory files to perform MM relaxation of the complexes. 20 Na + counter ions were added to neutralize the system for both RdRp:RNA:ddhCTP and RdRp:RNA:CTP cases.

The RdRp-RNA protonation state was prepared at physiological pH by using H ++ server, [START_REF] Anandakrishnan | H++ 3.0: Automating PK Prediction and the Preparation of Biomolecular Structures for Atomistic Molecular Modeling and Simulations[END_REF] with a salt concentration of 0.15 M. 37 The Amber force field FF14SB [START_REF] Maier | ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB[END_REF] and RNA.OL3 [START_REF] Zgarbová | Nucleic Acids Force Field Based on Reference Quantum Chemical Calculations of Glycosidic Torsion Profiles[END_REF] were used for protein and RNA, respectively. ZAFF force field, [START_REF] Yu | Extended Zinc AMBER Force Field (EZAFF)[END_REF] specifically parameterized for zinc-containing systems in its commonly coordination in proteins, was selected for two Zn 2+ ions, each of them coordinated to three Cys and one His as above specified. These ions only play a structural function. The Mg ions were treated with the Li et al. force field, [START_REF] Li | Rational Design of Particle Mesh Ewald Compatible Lennard-Jones Parameters for +2 Metal Cations in Explicit Solvent[END_REF] which allows a good structural description of the coordination sphere in polymerases [START_REF] Genna | The catalytic mechanism of DNA and RNA polymerases[END_REF][START_REF] Geronimo | Local Structural Dynamics at the Metal-Centered Catalytic Site of Polymerases is Critical for Fidelity[END_REF] The system was solvated in a orthorhombic box (12 Å from the protein) of TIP3P water molecules. [START_REF] Vassetti | Assessment of GAFF2 and OPLS-AA General Force Fields in Combination with the Water Models TIP3P, SPCE, and OPC3 for the Solvation Free Energy of Druglike Organic Molecules[END_REF] The solvated structures were first minimized by applying harmonic restraints on all atoms of the enzyme (50 kcal mol-1 Å 2 ), using 5.000 steps of the steepest descent algorithm, followed by 5.000 steps of the conjugate gradient algorithm (CG). In the second minimization step, we released restraint on hydrogen atoms, with third and fourth minimizations being conducted with and without restraints to the protein backbone atoms, respectively. We carried out a progressive heating phase from 0 to 310 K for 200 ps using the Langevin thermostat in NVT ensemble, with a time step of 0.002 ps. The production phase of 300 ns for RdRp:RNA and 300 ns for both RdRp:RNA:ddhCTP and RdRp:RNA:CTP complexes was performed under the following conditions: integration step of 2 fs coupling SHAKE algorithm, NPT ensemble at 1 bar pressure using the barostat. [START_REF] Berendsen | Molecular Dynamics with Coupling to an External Bath[END_REF] The Particle Mesh Ewald summation method [START_REF] Darden | Particle Mesh Ewald: An N ⋅log( N ) Method for Ewald Sums in Large Systems[END_REF] was employed for the electrostatic potential and the long-range electrostatic interactions were calculated with 12 Å cut-off distance. Trajectories for RdRp:RNA and its complexes with ddhCTP and CTP were saved every 0.2 ps and analyzed through the ptraj module. [START_REF] Roe | PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data[END_REF] Overall, 0.9 µs of simulations were conducted (300 ns for each complex).

MD trajectories so obtained can then be used to assess the magnitude of structural changes (root-mean-square deviation, RMSD), the propensity for a given residue or region to move (root-mean-square fluctuation, RMSF), and the evolution of hydrogen bonding (HB) networks. The secondary structures were assigned with DSSP algorithm. [START_REF] Kabsch | Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-Bonded and Geometrical Features[END_REF] RMSD-based clustering of the whole trajectories was performed according to the relaxed complex scheme (RCS) protocol as implemented in Amber16, 37 to provide a sampled and energetically accessible conformational ensemble. After removing overall rotations and translations by RMS-fitting the Cα atoms' positions of the trajectory, the average linkage clustering algorithm was applied as implemented in cpptraj to identify 10 representative conformations clusters of the protein. The four most populated structures were used as a starting point for the docking procedure. The described procedure has been successfully adopted in a number of investigations on different enzymes. [START_REF] Prejanò | Why hydroxy-proline improves the catalytic power of the peptidoglycan N-deacetylase enzyme: insight from theory[END_REF][START_REF] Prejanò | Computational Study Reveals the Role of Water Molecules in the Inhibition Mechanism of LAT1 by 1,2,3-Dithiazoles[END_REF][START_REF] Prejanò | On the Catalytic Activity of the Engineered Coiled-Coil Heptamer Mimicking the Hydrolase Enzymes: Insights from a Computational Study[END_REF] Principal component analysis (PCA) was performed with cpptraj module of Ambertools 16 to extract the large-scale collective motions occurring in MD simulations of RdRp:RNA, RdRp:RNA:CTP and RdRp:RNA:ddhCTP. 37 This procedure usually extracts information on the major conformational changes taking place along the MD trajectories.

Binding free energies between the binary complex RdRp:RNA and ddhCTP or CTP were calculated by solving the linearized Poisson-Boltzman equation using the molecular mechanics-Poisson Boltzman surface area (MM-PBSA) method, as implemented in Amber16 code, 37 selecting per-residue decomposition scheme. The value of igb flag equal to 5 associated to a salt concentration of 0.1 M was used. For the calculations 200 frames from each MD trajectory in the last 100 ns were analyzed.

Results and Discussions

Molecular dynamics of RdRp:RNA complex

As initial part of the investigation, 300 ns of molecular dynamics simulation on the RdRp:RNA complex were performed. The RMSD plot calculated for all the backbone atoms of the amino acid residues shows that the system reached the equilibrium after few ns of simulation, as confirmed by the calculated average value 1.49 ± 0.21 Å (see Figure S1). Such observed behaviour can be further evinced by the visual inspection of the protein structures obtained from the hierarchical clustering procedures (see Figure S2).

In addition, the RMSF of each individual residue of the non-structural proteins and RNA was calculated to characterize the local structural fluctuations in each subunit. (Figure S3) High peaks were observed at Val405, Lys430, Thr644, Val737 and Thr850, which corresponded to loop regions of the protein and at Leu895 and Glu903 in the proximity of carboxyl ends. Altogether, the preliminary analysis of the set of results on structural stability of the RdRp:RNA complex, allowed to consider reasonably stable the in silico-designed model.

PCA applied to MD trajectories to disclose the displacement of nsp7 nsp8 and nsp12 along the simulation has been carried out. The domains nsp7 and nsp8 are those that offset more during the simulation, with respect to the nsp12 one (see Figure 3). However, the present investigation does not consider such event, which is not therefore object of the study. Indeed, although nsp7 and nsp8 were retained in the simulations, we focused our attention on the catalytic subunit nsp12. The structural stability of the RNA chains is not affected by the above-mentioned movement, as confirmed by the well embedded conformation of RNA in the nsp12 pocket (Figure 3). The visual inspection of PCA calculated for the RNA, furthermore, highlights that a slight shift takes place during the simulation for the nucleic acid, mainly in 3' and 5' positions of the T nucleotides (See Figure 3). The structural homogeneity of nucleic acid is ensured by hydrogen bond interactions occurring with the positively charged residues of the domains, such as Arg569, Lys849, and Arg858 (Figure S4, Table S1). Further high interactions occupancies occurred between P:U20 T:G10 T:U12 P:A17 P:A18 and Asp760, Ser682, Arg569, Arg585, Arg836, respectively, of the different subunits with an occupancy range of 77-100, as shown in Table S2 related to native contacts.

In the selected crystal structure, the active site Mg A and Mg B cations are far from putative ligands, like aspartate or glutamate residues, making hardly the establishment of metal first coordination sphere. 23 However, the analysis of trajectory revealed that Mg A and Mg B interacted mainly with Asp618, Asp760 and Asp 761, as will be shown later in detail. In general, both cations resulted in an octahedral geometry further consisting of solvent molecules, as interestingly evidenced by the analysis of radial distribution functions (RDFs) for the Mg A -O W and Mg B -O W pairs. In detail, first and second hydration sphere for the two metal ions were identified, with higher peaks observed in the case of Mg B (see Figure S5). The most frequent presence of waters in proximity of Mg B , with respect to Mg A , can be ascribable to P:U20 nucleotide located in proximity of the latter, which occupies the room for solvent molecules. The presence of H 2 O molecules has been further confirmed by the visual inspection of structures collected from the hierarchical clustering analysis, supplying an average number of 4 and 5 molecules in proximity of Mg A and Mg B , respectively (Table S3).

Molecular dynamics of RdRp:RNA:CTP and RdRp:RNA:ddhCTP complexes

The average binding affinities (in kcal/mol) obtained for the best docking poses of ddhCTP and CTP into the SARS-CoV-2 RdRp of are reported in Table S4. The most energetically favored conformers, -11.4 and -11.3 kcal/mol for of RdRp:RNA:CTP and RdRp:RNA:ddhCTP respectively, confirm that the introduction of ddhCTP can be considered feasible and, thus, can be allowed.

After the docking of the ligands, 300 ns of MDs were further performed on both RdRp:RNA:CTP and RdRp:RNA:ddhCTP ternary complexes. The RMSD plot reveals that the system reached the equilibrium, with average values slightly higher than the respective RdRp:RNA system (2.18 ± 0.28 Å and 2.20 ± 0.17 Å, for CTP and ddhCTP respectively vs 1.49 ± 0.21 Å for the binary, see Figure 4).

Consistently with such behaviour, also the calculated RMSF values of ternary complexes were higher than the binary one (see Figure 4A).

In particular, in the case of the RdRp complexed to CTP, more intense fluctuations were detected in proximity of NiRAN (Asn198 and Pro227), fingers (Ser425) and thumb (Met902) subdomains. In analogy to the observation obtained for the RdRp:RNA complex, PCA revealed that for both ternary complexes nsp12 and nsp8 are the regions of the system involved in more detectable movements during the MDs and that the RNA molecule conformation is not affected by any important structural rearrangement (see Figure 3).

In the binding site, CTP and ddhCTP ligands are stably located during the entire simulation, as can be observed by the analysis of their RMSD plots reported in Figure S6. The orientation of the substrates is mainly ensured by the presence of cation-phosphate interactions, occurring with different combination between the phosphate arms of CTP and ddhCTP with Mg A and Mg B . The distances of each Mg ions with P α , P β and P γ were analyzed to characterize the binding mode of the phosphate moieties of ligands during the simulation and the relative distributions are displayed in Figure S7. In the case of CTP, the plot analysis revealed that oxygens of P α group bridge both cations, while those of P γ coordinate mainly the Mg B (see Figure S8 for further details). A different behaviour of P α group can be observed in ddhCTP as far as the Mg A is concerned. In fact, it is shifted at distances around 5 Å. For Mg B the distribution of P α and P γ results very similar for both ligands, while P β in ddhCTP is longer than that in CTP. In addition to the phosphate groups of CTP and ddhCTP, Mg A and Mg B complete their hexa-coordination (Figure S8) with the side chain of two consecutive Asp residues in motif C, Asp 760 in a µ(1,3) fashion, as confirmed by measured distance of ca 2 Å of metal-O Asp760 distances (see Figure 5), and Asp761. Asp618, at the beginning of motif A, preferentially coordinates the Mg B one.

The above-described orientation of P α , P β and P γ differs from that observed for the remdesivir, in the active site of RdRp. [START_REF] Aranda | Mechanism of reaction of RNA-dependent RNA polymerase from SARS-CoV-2[END_REF] It can be ascribable to a more advanced step of the recognition of remdesivir, considering that the OH of the primer is deprotonated and already coordinated to Mg A . However, the orientation here-described of ddhCTP is encountered in other inhibitors, generally identified as "chain terminators". [START_REF] Seifert | Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective[END_REF] The oxygen atoms of P β interacted during the whole simulation with the positively charged Lys551 and Arg553 residues of motif F for both examined ternary complexes, thus contributing to the binding of CTP and ddhCTP. Our finding is in good agreement with observations on other viral nucleic acid polymerases. [START_REF] Aranda | Mechanism of reaction of RNA-dependent RNA polymerase from SARS-CoV-2[END_REF][START_REF] Shi | A Nucleobase-Binding Pocket in a Viral RNA-Dependent RNA Polymerase Contributes to Elongation Complex Stability[END_REF][START_REF] Kabsch | Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-Bonded and Geometrical Features[END_REF][START_REF] Xu | A novel mechanism of enhanced transcription activity and fidelity for influenza A viral RNA-dependent RNA polymerase[END_REF][START_REF] Wilson | Exploring the identity of the general base for a DNA polymerase catalyzed reaction using QM/MM: the case study of human translesion synthesis polymerase η[END_REF] Very interestingly, the RdRp:RNA:CTP and RdRp:RNA:ddhCTP analysis of the trajectories of saltbridges established by Asp618, revealed that it is frequently implicated in the interaction with Lys798, while the same interaction was not frequently observed in the binary complex (Figure 6). This behavior highlights the role played by Lys798 as "needed counterion" in orienting the P γ towards Mg B . Lys798 is indeed located within interactive hydrogen-bond distance to the P γ of incoming ligands, thereby suggesting that such residue is involved in the binding of phosphorylated substrates and thus in the release of PPi after the chemical reaction has been completed (Figure 6). This residue indeed represents the highly conserved lysine, critical in the control of the activity and fidelity of transcription and in the action act as a general acid to protonate the pyrophosphate leaving group, as widely emphasized in literature. [START_REF] Boehr | Structure, dynamics, and fidelity of RNA-dependent RNA polymerases[END_REF][START_REF] Kabsch | Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-Bonded and Geometrical Features[END_REF] Finally, a lower number of water molecules coordinating Mg A and Mg B was observed during the simulations due to the presence of phosphate groups, as can be noted from the count of solvent molecules coordinating the cations in the clustered geometries (see Table S3).

During the simulations, Mg A and Mg B lie at a distance of 4.48 ± 0.12 Å and 4.96 ± 0.15 Å for CTP and ddhCTP, respectively, (Figure S9). The observed value is consistent with that required by the two metal ions reaction mechanism (< 4 Å). It was suggested that during the reaction a shrinking of this distance may occur, leading O3′ closer to Pα and consequently facilitating the nucleophilic attack. The bit longer distance in the ddhCTP compared with CTP could aff ect the O3′-P α alignment for catalysis during the nucleophilic attack, in the reactive Michaelis-Menten complex. [START_REF] Ng | Structure-function relationships among RNAdependent RNA polymerases[END_REF][START_REF] Sgrignani | The structural role of Mg 2+ ions in a class I RNA polymerase ribozyme: a molecular simulation study[END_REF][START_REF] Boero | Double-metal-ion/single-metal-ion mechanisms of the cleavage reaction of ribozymes: First-principles molecular dynamics simulations of a fully hydrated model system[END_REF][START_REF] Chaudret | Correlation between electron localization and metal ion mutagenicity in DNA synthesis from QM/MM calculations[END_REF][START_REF] Gleghorn | X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides[END_REF] In order to analyze the interaction involving the cytosine nucleobase, which is the same for both ligands, attention was focused on three main geometrical parameters involving the pairing with guanosine nucleobase, like C1' CTP/ddhCTP -C1' G:T , N1 CTP/ddhCTP -N1 G:T and N3 CTP/ddhCTP -C1' G:T .

Outcomes on the analysis of these distances are linked to the fidelity of DNA polymerases [START_REF] Geronimo | Local Structural Dynamics at the Metal-Centered Catalytic Site of Polymerases is Critical for Fidelity[END_REF][START_REF] Yang | Making and breaking nucleic acids: two-Mg 2+ -ion catalysis and substrate specificity[END_REF] and can be extended to RNA polymerase, due to many similarities between the two classes of enzymes [START_REF] Yoon | Simulating the fidelity and the three Mg mechanism of pol η and clarifying the validity of transition state theory in enzyme catalysis[END_REF][START_REF] Otsuka | Similarity relations of DNA and RNA polymerases investigated by the principal component analysis of amino acid sequences[END_REF][START_REF] Yin | The structural mechanism of translocation and helicase activity in T7 RNA polymerase[END_REF] .

In the case of CTP ligand, the analysis of the distribution distances revealed that the nucleobases lie in an unpaired-like conformation, which can be linked to the low fidelity behavior of the RNApolymerases accordingly to the mechanism. Indeed, the calculated average values of 11.73 ± 0.47 Å, 6.72 ± 0.59 Å and 5.98 ± 0.10 Å, for C1' CTP -C1' T:G , N1 CTP -N1 T:G and N3 CTP -N1 T:G fit results described in previous work [START_REF] Geronimo | Local Structural Dynamics at the Metal-Centered Catalytic Site of Polymerases is Critical for Fidelity[END_REF] . More interestingly, the inclusion of ddhCTP, increased of ca 2 Å all the considered distances (see Figure S10), thus suggesting that in presence of the endogenous inhibitor the fidelity of the enzyme is further affected and that errors in the course of the duplication can take place already in this step, thus concurring to the stalling of the enzyme.

Due to the modification present at the 3' position of the ribose ring of ddhCTP, the analysis of radial distribution function and visual inspection of clustered structures were conducted on the interactions involving the ribose group, focusing on the hydroxyl groups at C2' and C3' positions of CTP in comparison with ddhCTP. In the case of CTP molecule, O3' atom established hydrogen bonds with phosphate group of U20 and water molecules, while O2' resulted mainly involved in interactions with solvent molecules. In absence of O3'-H group in the ddhCTP containing system, the interaction with U20 phosphate was observed occurring with the O2' atoms. (Figure S11) Contact analysis effected for both CTP and ddhCTP evinced as residues (618-621) of motif A, 760 of motif C and 551 of motif F are implicated interactions with the ligands (see Table S5, S6, S7) suggesting a similar network of interaction inside the binding site. However, the analysis did not provide any relevant information about the interactions involving ribose hydroxyl groups and the amino acid residues.

The calculation of ΔG bind , carried out according to the MM-PBSA scheme, revealed some differences for the ligands. In detail, the binding free energy of ddhCTP resulted 15 kcal/mol higher than CTP (Table S8). Energy decomposition analysis of every residue involved in non-covalent interaction with CTP and ddhCTP showed that, for the former, a stronger stabilization of tertiary complex is due to the electrostatic interaction of Arg553, Arg555, Lys621 and Asp761 sidechains (see Supporting information for details, Figure S12) with the active site environment. In addition, the higher ΔG bind (lower affinity) can be linked to the absence of hydroxyl group at C3' position, which causes a loss of 3 hydrogen bond interactions from ddhCTP species.For both CTP and ddhCTP, finally, it can be noted a reacting like conformation in the course of the dynamics. This can be evinced, in particular, analyzing the Mg A -O3'P:U20 and the O3'P:U20-P αNTP distances, which are the reaction coordinates involved in the reaction catalyzed by the enzyme.

A very similar distribution of values was observed, see Figure 7, which can be further linked to experimental evidences concerning the incorporation and reactivity in presence of ddhCTP ligand. [START_REF] Seifert | Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective[END_REF] In addition, considering the importance of the distances Mg A -O3' P:U20 , P αNTP -O3' P:U20 and OD1 Asp761 -HO3' P:U20 we run umbrella sampling simulations, to restrain a reaction window corresponding to the transition slot from non-bonded-like to bonded-like conformations (Figure S13). [START_REF] Hub | g_wham-A Free Weighted Histogram Analysis Implementation Including Robust Error and Autocorrelation Estimates[END_REF] The analysis of the obtained potential of mean force (PMF) showed that the approach of the O3' P:U20 to the Mg A generates two almost equal in energy minima, in both CTP and ddhCTP (Figure S13a). The free energy difference is the driving force of the process of unbound/bound, which indicates possible incorporation of a new base into the RNA strand. The analysis of the distance P α NTP -O3' P:U20 (Figure S13b) revealed PMF values reasonably compatible with other theoretically studied polymerases. [START_REF] Geronimo | Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology[END_REF][START_REF] Genna | The catalytic mechanism of DNA and RNA polymerases[END_REF][START_REF] Geronimo | Local Structural Dynamics at the Metal-Centered Catalytic Site of Polymerases is Critical for Fidelity[END_REF] Following the reaction coordinate of the H-transfer from the ribose O3' (last reacting uracil of elongating strand) to the general base Asp761 (Figure S13c), finally, it was possible to establish a slightly favored thermodynamic process in the case of ddhCTP. In general, the presence of different local accessible minima reflects the complexity of the process, confirming anyway the potential of ddhCTP as an inhibitor of RdRp.

Conclusions

Nucleotide analogs represent a powerful tool to fight the viral infections and their consequences. In the present work, the interaction of RdRp protein, involved in the spreading of SARS-CoV-2 disease, and the endogenously synthesized ddhCTP, the product of the viperin antiviral protein, has been investigated. Such molecule differs from CTP, its natural precursor, by the absence of 3'-OH group. However, it is reputed that the missing hydroxyl group can favor the stalling of the enzyme and, consequently, the replication of viral RNA. The study has been conducted in the mean of all-atom MD simulation, on the RdRp:RNA system and on each RdRp:RNA:NTP complex, with NTP equal to CTP or ddhCTP, and focused on the recognition step of RdRp catalytic mechanism.

From the analysis of binary complex, it turned out that the RNA is kept in active site by interaction with a number of residues, such as Ser682 and Arg585, and that, in absence of other substrates, Mg A and Mg B present a hexa-coordination sphere, comprised of Asp618, Asp760, Asp761 and water molecules.

The comparative analysis of conformational behavior of ddhCTP and CTP, overall, indicated high similarity between the two molecules. In detail, phosphate groups P α and P γ of CTP and ddhCTP are directly involved in coordination of Mg A and Mg B , while P β mainly interacted with positively charged Lys551 and Arg553, facilitating the binding of the ligands in the active site. Importantly, Lys789 has been identified as crucial residue bridging Asp618 and P γ group of both ddhCTP and CTP.

The hydroxyls groups of CTP at C2' and C3' positions established hydrogen-bond interaction with solvent molecules and U20 of RNA, respectively, while in the case of ddhCTP the interaction with nucleic acid is ensured by the O2'-H group.

The interactions of the cytidine group of both nucleosides with guanine of RNA chain were evaluated in term of C1' CTP -C1' T:G , N1 CTP -N1 T:G and N3 CTP -N1 T:G distances. The analyzed results are in agreement with low fidelity of RdRp, which resulted slightly more affected by the presence of ddhCTP molecule. Finally, both CTP and ddhCTP showed pro-reactive conformation and umbrella sampling simulations revealed high energy similarity reacting-like and non-reacting like conformations of the ligands.

The results above-described shed light on relevant aspects of the incorporation of CTP and ddhCTP species in the RdRp, laying the foundation for the rational design of the SARS-CoV2 vaccine and antiviral targets and providing further insights that were not accessible to experiments. Altogether, the available results from experiments and the rationalization from computational investigation can thus be very helpful to stimulate and drive future experimental investigations.

  Ser397). A further N-terminal β hairpin (residues Val31 to Lys50) inserts into the cleavage blocked by the NiRAN domain and the palm subdomain in the RdRp domain.

Figure 1 .

 1 Figure 1. A) Structures of SARS-CoV-2 nsp7(2-68)-nsp8(78-191,84-111)-nsp12(4-929) complex. B) Structure of nsp12 domain organized by color for subdomains and in gray for A-G motifs (Motif A: s 612-626; Motif B: residues 678-710; Motif C: residues 753-767; Motif D: residues 771-796; Motif E: residues 810-820; Motif F: residues 544-560; Motif G: residues 499-511).

Figure 1 .

 1 Figure 1. A) Cryo-EM structure of favipiravir-RTP in the catalytic site of the SARS-CoV-2 RdRp, in complex with template:primer RNA and 2D representation of template (T) and primer (P) including T-705 (favipiravir). B) Structure of favipiravir-RTP at the catalytic site of the RdRp in which only the phosphate-ribose scaffold is retained from the pdb 7AAP. C) Computational model used for RdRp in complex with template:primer RNA and 2D representation of template (T) and primer (P) incorporating ddhCTP inhibitor.

Figure 3 .

 3 Figure 3. Principal component analysis of the RdRp:RNA binary (A), RdRp:RNA:CTP (B) and RdRp:RNA:ddhCTP (C) ternary complexes. In every system, RNA binding site is circled.

Figure 4 .

 4 Figure 4. RMSD and RMSF plots of RdRP:RNA:CTP (red) and RdRP:RNA:ddhCTP (green) complexes.

Figure 5 .

 5 Figure 5. A) Active site of RdRp:RNA:NTP complex (NTP=CTP or ddhCTP). Distances calculated between Mg A or Mg B and B) Asp618, C) Asp760 and D) Asp761, for the RdRp:RNA (blue), RdRp:RNA:CTP (red) and RdRp:RNA:ddhCTP (green) system.

Figure 6 .

 6 Figure 6. A) Focus on Lys798 orientation in active site of RdRP:RNA:NTP complex. B) Asp618-Lys798 salt-bridge distance and C) O Pγ -N Lys798 hydrogen bond calculated in during the MDs of RdRP:RNA:NTP complexes (NTP=CTP or ddhCTP) .

Figure 7 .

 7 Figure 7. Distribution of O3'P:U20-P α vs O3'P:U20-Mg A distances calculated for RdRp:RNA:CTP and RdRp:RNA:ddhCTP complexes.

Acknowledgments

We thank the Università degli Studi della Calabria-Dipartimento di Chimica e Tecnologie Chimiche (CTC) for the support. Computer simulations were carried out adopting resources of supercomputer Marconi 100 from ISCRA (Project code: IsC92).

Data and Software Availability

AutoDock version 4.2 was adopted to effect molecular docking calculations. All MD simulations were carried out adopting the AMBER16 package. AmberTools package was further adopted in the preparation of parameters and input for MD simulations. DFT calculations, to parametrize ddhCTP, were carried out using Gaussian 09 ver. D01. Trajectory analysis was done adopting AMBER cpptraj module. The preparation of the structures and the images were carried out using VMD software, version 1.9.3, and Chimera software. The full workflow is reported in the "Computational Methods" section of the manuscript.


Ana-Maria Florescu

Vitari Claudio

Serge Amabile

Amabile Serge

Digital Commons and Management towards Sustainability, Equity and Resilience

Introduction

The initial goal of this study is to make a systematic literature review on different kinds of management activities and processes that take place within the digital commons. In order to advance theories around governing digital commons, the analysis of the literature is held through the lenses of ecological sustainability, equity and resilience. This analysis could be of interest to structures and organisations working on topics related to digital commons, as well as to policy makers that are interested in developing the subject. This exploration is inspired by Elinor Ostrom's works in managing the commons and could be placed within the disciplines of IS, MIS and Management literature.

Discussions around commons are being held more and more often these days. In economics, a common good is defined as non-excludable (you cannot exclude anyone from its use), like a public good, and rivalrous in consumption (its consumption prevents simultaneous consumption by other actors), like a private good. One of the authors that popularised this term was the Nobel prize winning political economist Elinor Ostrom, who described the commons as "long-enduring, self-organised and self governed" (Ostrom, 1990, p. 143) goods. She analysed how people can manage shared resources, such as forests, fisheries, oil fields, irrigation systems in order to solve the tragedy of the commons.

Beyond natural resources and human infrastructures, shared resources are increasing in the digital world. Digital commons are information and communication technologies and information that are accessible to the members of a society, as well as managed and controlled by the community that develops these resources. Yochai Benkler sees "digital commons" through the lens of information production environment, social relationships and collaboration that is capable of producing a common field of action [START_REF] Benkler | The Wealth of Networks: How Social Production Transforms Markets and Freedom[END_REF]. Some evident examples are Wikipedia and Creative Commons. Digital commons are recognised as contributing to several sustainable development goals, hence they merit to be protected, developed and disseminated [START_REF] Bradley | The sharing economy as the commons of the 21st century[END_REF] (Pouri & Hilty, 2018, p. 14). However, the scientific knowledge accumulated on traditional commons is not immediately applicable to digital commons as digital commons have special characteristics that require a dedicated research effort [START_REF] Rozas | Analysis of the Potentials of Blockchain for the Governance of Global Digital Commons[END_REF]. The main difference between traditional commons and digital commons is seen in terms of the fact that "the former are an existing scarce resource that could be destroyed by over-exploitation, the latter are created and improved through contributions from a community, but they are virtual assets that cannot be over-harvested" (Isabel Alonso de [START_REF] Alonso De Magdaleno | Sustainability and social responsibility reporting in open source software[END_REF]. For example a digital common good like Wikipedia is not consumed by its users, as a fishery can be by overfishing.

Hence, the scientific community could play an important role in improving the understanding of the key success factors of the digital commons. These theories and guidelines should include ecological and social issues. The analysis around the term "common good", each in its own way, appeals to the concept of ecological sustainability, as it's directly related to natural resources. Ecological sustainability is defined as ''the ability of one or more entities, either individually or collectively, to exist and thrive (either unchanged or in evolved forms) for lengthy timeframes, in such a manner that the existence and flourishing of other collectivities of entities is permitted at related levels and in related systems'' (Chen et al., 2008). Given the negative environmental consequences all over the information and communication technology life cycle, different measures are needed to ensure the digital commons' ecological viability. In this context, in order to exist and develop, it's supposed that "digital commons" should be ecologically sustainable as well. With an important increase in use of all types of digital technologies, it's crucial to guarantee that this use is not coming up with negative environmental consequences. At the same time, social equity should be pursued Forty-Third International Conference on Information Systems, Copenhagen 2022 2 to assure that the good remains a common, hence not excluding anyone from a fair part of it. Equity is analysed in terms of values, "as the basis of distributive justice", alongside "equality" and "need" (Deutsch, 1975, p. 137). "In cooperative relations in which economic productivity is a primary goal, equity rather than equality or need will be the dominant principle of distributive justice" (Deutsch, 1975, p. 143). Finally, actual turbulence and shocks should be taken into consideration to improve common good resilience. Resilience is seen as "the ability to withstand and survive shocks and disturbances [...] related to the way societies adapt to externally imposed change" (Joseph, 2013, p. 39).

In this manner, the aim of this paper is to examine previous studies regarding the actions and processes that occur in the digital commons, in order to be able to answer the following question: how can the environmental sustainability, equity and resilience of digital commons be improved ?

Research methodology

This systematic literature review is a qualitative analysis of the papers on digital commons and concepts related to their ecological sustainability, equity and resilience. When drafting the systematic review, the main purpose kept in mind was to provide a succinct, but trustworthy, reliable and useful digression into the subject, in order to assist colleagues in navigating the current developments in this field. Three principles guided the data retrieval. Firstly, the search for relevant papers should be as complete as possible. Secondly, it is essential to explicitly set criteria for inclusion and exclusion of articles. Third, we consider critical that the chosen criteria are both accurate and replicable. In order to find articles that could correspond to the proposed research topic, the Web of Science platform was used as the main resource that could provide the database. The initial search query included the main keyword: "digital common" AND "digital commons" (with quotation marks in the search field). Complementary, we retrieved articles citing Elinor Ostrom, with the same combination of "digital common" AND "digital commons" (with quotation marks in the search field), as well as searching separately for 'digital' 'common' and 'digital' 'commons' (without quotation marks in the search field).

We explicitly search for singulars and plurals terms, as preliminary exploratory tentative returns two different types of results between singulars and plurals of words. Papers from different fields are using different words. The singular of the words "digital common" are mostly used by the "Engineering, Electrical and Electronic" field, as well as by the "Computer Science Hardware Architecture", the plural of words "digital commons" is more specific to "Information Science, Library Science" and "Communication" fields. In the case of the use of singular words, the "Communication" field is the least active and "Engineering, Electrical and Electronic" is not even known for using the plural of words "digital commons". When it comes to "Management" or "Information Systems", the fields of our interest, it's mostly using the combination "digital commons".

Considering the fact that the number of articles gathered via this search totaled 260, an analysis of each article's title, abstract and keywords was made in order to retain only those that could be useful for the literature review and closely related to concepts of ecological sustainability, equity and/ or resilience. Consequently, 40 articles were selected for further study and analysis, which were subjected to the chosen criteria.

The inclusion criteria were: Does the article talk about "digital common" (or any other relevant synonyms, for example: open source common) AND "sustainability" OR "equity" OR "resilience"? Do the authors mean "ecological" (or any other relevant synonyms, for example: environmental) when talking about "sustainability"?

The exclusion criteria: Any other types of sustainability (economic, human, social, etc.), but "ecological" (or any other relevant synonyms, for example: environmental). Any other types of commons (for example, natural resources), but "digital" (or any other relevant synonyms, for example: open source common).

Forty-Third International Conference on Information Systems, Copenhagen 2022 3 Finally, 6 articles were satisfying all the criteria. An inductive reasoning was made, arriving at conclusions, through the analysis from the particular to the general. Backward citation searching was made as well in order to analyse one author's previous works and conclusions made.

Findings

5 articles respond directly to the searching criteria related to the topic of ecological sustainability and the concept of digital commons. Nonetheless, the authors of these 5 articles write about sustainability, sometimes clearly referring to ecological sustainability, while sometimes the discourse is more vague and ambiguous and sustainability could be interpreted as an umbrella term including ecological as well as economic sustainability, without any clear-cut way to distinguish between the two intentions.

For example, when it comes to the definition of sustainability in information systems (IS), it "has been looked at in three principal ways: sustaining competitive advantage through the deployment of IS, creating (online) communities and inclusive collaborative efforts, and green IS (that urges us to consider environmental and social concerns alongside economic ones and the role that IS have in increasing, mitigating, or even reducing environmental harm)" (Curto-Millet & Corsin Jimenez, 2022, p. 2).

Nonetheless, a tension emerges from the 5 articles covering digital common goods sustainability. On one side, reasons are advanced insisting on the similarity and proximity between digital common goods and traditional common goods in terms of sustainability [START_REF] Curto-Millet | The sustainability of open source commons[END_REF]. A parallel between traditional commons through the lenses of Elinor Ostrom's work and digital commons was made by Curto-Millet & Corsin Jimenez [START_REF] Curto-Millet | The sustainability of open source commons[END_REF]. Consumers of a certain resource, either traditional or digital, can form a group in order to manage a common good in accordance with a set of rules shared by them. Resources are distributed in a more predictable and efficient manner, the conflict between actors is minimised, and the resource system itself may exist for a long period of time, remaining sustainable. As long as actors are disorganised, they will not be able to achieve the tangible gains that would be available if they worked together [START_REF] Curto-Millet | The sustainability of open source commons[END_REF]. Also the 8 Ostrom's principles or factors that assure the long-term usage of shared resources that make it feasible to secure the long-term usage of shared natural resources, are considered applicable to the sustainability of digital common goods [START_REF] Curto-Millet | The sustainability of open source commons[END_REF].

On the opposite side, sustainability of digital commons is considered as a much more complex notion and different from sustainability related to traditional commons, theorised by Elinor Ostrom (Curto-Millet & Corsin Jimenez, 2022) [START_REF] Rozas | Analysis of the Potentials of Blockchain for the Governance of Global Digital Commons[END_REF]. First of all, a continuous effort seems necessary but not sufficient as far as sustainability may not be achieved (Curto-Millet & Corsin Jimenez, 2022, p. 13). This continuous effort requires assemblages of varied actors (technologies, democracy, quality through diffusion and adoption), unexpected alliances (publishing world), collective efforts, and values" (Curto-Millet, 2013, p. 45). They are considered crucial to the sustainable governing of open source. The efforts and the enactions of these actors are supported by their specific values, regularly questioning the reasons and purposes of the digital commons and the adequateness of its processes, conducting to an never ending evolution of the digital commons and at the end their sustainability (Curto-Millet, 2013, p. 45).

Moreover, the digital commons sustainability can be structured in a typology. Three types of sustainability are distinguished, which can nonetheless coexist: resource-based, infrastructural and interactional [START_REF] Curto-Millet | The sustainability of open source commons[END_REF], but ecological sustainability is not explicitly described. Complementary, five themes are proposed that contribute to sustainability of digital commons [START_REF] Curto-Millet | The sustainability of open source commons[END_REF]: participation, focus on certain actors, time, dimensions and logic.

A solution in order to reinforce the sustainability and the management of digital commons is considered to be the blockchain technology, that could be of help in different ways: "distribute power, facilitate coordination, scale up governance, visibilise traditionally invisible work, monitor and track compliance with rules, define collective arrangements, and enable cooperation across communities" [START_REF] Rozas | Analysis of the Potentials of Blockchain for the Governance of Global Digital Commons[END_REF].

If a scientific debate is open about sustainability of digital common goods, when it comes to digital commons and management activities towards equity and resilience, very poor results were found. One article vaguely analysed the topic of equity when related to digital commons. In this one, digital commons are seen as a new model and a new paradigm that, by default, is seen as a new sense of fairness and justice compared to existing digital content models (Ciesielska & Jemielniak, 2022, p. 908). While zero articles analyse the topic of resilience of digital commons.

Discussion

Out of these 260 articles, only 6 papers comply with our criteria: 5 approaching the topic of ecological sustainability and one article the resilience. In fact, the terms "digital common" or "digital commons" are not extensively used in the academic sphere, given the little volume of results retrieved by Web of Science. Still, the research made is showing poor results in terms of managerial actions that could be applied towards ecological sustainability of digital commons. We are even more surprised to discover that social equity is completely neglected and resilience only superficially evoked once. We conclude that, by one side, little research exists around the topic of ecological sustainability of digital commons and all kinds of related managerial actions.

The mentioned papers have a common reference to Elinor Ostrom's works on the natural resource commons, as well as a clear parallel and applicability of some of Ostrom's principles to the digital commons, which is the basis for their sustainability analysis. Beyond sustainability, it seems that when it comes to digital commons, notions of equity and resilience can be conceptually close (Lew et al., 2016, p. 5) or even equity and resilience can be implied in the notion of sustainability or the very concept and existence of digital commons. Indeed, equity and resilience are becoming fundamental principles for actions. For example, one of the main sustainable development goals of UNICEF is to achieve a more equitable and just world (UNICEF and the Sustainable Development Goals, 2022).

Overall, more than answers, our literature review highlights three avenues of research. The first one is around the extent of applicability of the sustainability principles identified for traditional commons when it comes to sustainability of digital commons. Some similarities exist justifying the replicability of traditional commons knowledge to digital commons. But some differences justify special treatments and hence new theories and models when approaching the sustainability of digital commons. Further exploration seems necessary to clarify the boundary conditions of traditional common knowledge and the specific rules applicable for digital common sustainability.

The second avenue of research concerns the contribution of the blockchain in enhancing digital commons and their sustainability. Blockchain is mentioned as a possible key success factor, but research should more extensively explore conditions and methods to substantiate its potential.

The third avenue of research questions the inextricable inclusion, or not, in the concept of digital commons of sustainability, equity and resilience. Said otherwise, when we talk about digital commons, we inevitably include sustainability, equity and resilience in the concept of common goods, hence making impossible any independent and separate analysis of each concept. This proposition requires further investigation to be able to better understand to what extent the terms of sustainability, equity and resilience are implied to the terms of digital commons.

The lack of research in these directions does not mean that these 3 issues are irrelevant. The lack of attention around these 3 issues opens the ways to new tragedies of the commons, and of the digital commons in particular. If professionals and researchers do not invest this unexplored domain, we will assist to new tragedies. New because they are not related to the overexploitation of the commons it-self, but more on the overexploitation of what is around the digital commons: like the natural resources necessary to produce the digital technologies, the energy sources to run the digital services, the human race lost while fighting for access to these last natural resources and energy sources.

We hypothesize that this inattention could originate in some disputable assumptions. The first assumption is that common goods are ecologically sustainable. If common goods include the issue of ecological sustainability, the ecological sustainability is multidimensional. Maybe the common goods are sustainable only on certain dimensions and not on all dimensions of the ecological sustainability. Neglecting the sustainability for each dimension would make at risk the commons. We invite to unpack the ecological sustainability to make it more comprehensive. In particular, for the digital commons the issue of the exploitation and depletion of the natural resources on which the digital technologies are build and run.

The second assumption is that common goods are resilient. If common goods include the issue of resilience, the resilience can be effective in front of some shocks but less effective in front of some disturbances. The actual uncertainties around the electric provision seems a shock not taken into account so far by digital common goods. Hence, as for ecological sustainability, the resilience should be unpacked and digital common good resilience should be assessed and managed in a more comprehensive matter.

The third assumption is that common goods are socially equitable. If common goods are socially equitable among the members of the community, digital commons restrict the community to the people with a certain access to the digital world and excluding hence large pans of the human population because they lack the instruments (like a mobile phone) and infrastructure (like high speed internet) to access the digital commons. This barrier approaches the digital commons toward digital clubs. An outward looking could be fruitful to explore way to include ever larger pans of the population and in particular the most in need of each digital common good.

Limitations

Our results should be interpreted cautiously given the some limitations of our review. The main one is about the limited set of retrieved and pertinent articles. We suspect that this small retrieved sample could be related to the choice of terms in the queries. Some synonyms of "digital commons" could've been searched as well in order to enrich the sample, such as open source, open data, open access, free software. Similarly, sustainability, equity and resilience could be debated but using other terms as synonyms. Hence searching for their synonyms could be a way to extend the review. More than academic literature, we think that an effort could be directed toward grey literature by the communities leading the digital commons.