Marc Fleurbaey

Marie-Laure Salles-Djelic

Progress is Back

This paper presents the International Panel on Social Progress and expounds key ideas from its first report, Rethinking Society for the 21 st Century (Cambridge U. P., 2018). It emphasizes the importance of three dimensions of progress on which serious challenges need to be addressed: equity, freedom and sustainability. Addressing these challenges primarily requires reforming power and governance structures in the economy, society, and politics.

The linear view of progress promised by the Enlightenment was deeply weakened by the horrors of the 20th Century. It has also, according to Wagner (2016), lost momentum after the abolition of most forms of formal domination (serfdom, slavery, female subordination). And the profound ecological, social and technological disruptions of our contemporary world would only come to reinforce, it seems, a widespread disillusion with the idea of progress. But Wagner identifies three ways in which progress can (and should) be revived and reoriented for the 21 st century: 1) building democratic agency; 2) overcoming new kinds of domination; 3) avoiding hubris (in our interaction with Nature).

The International Panel on Social Progress (IPSP 2018) has adopted a similar stance with respect to the idea of progress. The IPSP emerged as the bottom-up initiative of a group of scholars, with the blessing of an advisory committee chaired by Amartya Sen. We decided to claim the "progress" banner to clearly assert that, when studying the long term evolution of societies, it is worthwhile and important to be guided by normative principles. Other panels, which often refrain from making explicit recommendations (e.g., the IPCC's mantra is to be "policy-relevant but not policy-prescriptive"), are nevertheless inevitably guided by normative considerations (e.g., in the case of the IPCC, the goal of preventing "dangerous anthropogenic interference with the climate", as stipulated in Art. 2 of the UN Framework Convention on Climate Change). The IPSP rejects the old-fashioned linear view of progress and rejects the neo-imperialist idea that there is only one direction for all regions of the world and for all cultures. But the IPSP also rejects a form of lazy relativism that would be oblivious to the important convergence of many parts of the world toward basic values of human flourishing and equal dignity, and it rejects the claims to authenticity made by the oppressive traditions that resist this convergence.

Why a new panel?

The creation of the IPSP has been motivated by the lack of collective effort, in public debates around the world, to define the direction that the social contract should take in this crucial period. The 21 st century will probably be remembered as a major crossroads in the history of humanity. After the tyrannical ideologies of the 20 th century have been crushed, in the streets and the trenches, as well as in the minds of the masses, today the faith in the market and liberal democracy that prevailed at the end of the last century is clearly showing its limits and flaws. While our contemporary form of liberal capitalism generates significant financial, social and political instability, it also appears to lead humanity towards outright ecological disaster. As shown in various protest movements such as Occupy, the Yellow Vests, or the students' climate change protests, the call for rethinking the social contract, for curbing the irresponsible excesses of global elites (e.g., tax evasion), for eradicating deep poverty, for preventing the revival of authoritarianism, and for addressing the environmental threats is becoming everyday more urgent.

Existing initiatives such as the UN Sustainable Development Goals, the Paris Agreement on climate mitigation, the OECD work on inclusive growth, the World Bank reports on improving governance and rethinking the social contract, the T20 and T7 work on social cohesion,2 have all been pushing compelling agendas, even if they do not seem to be powerful enough to curb the most worrisome developments of recent decades. Adding the voice of social scientists to the public debate, in a document that makes available the bulk of academic work on social trends to a wider audience, could help in bringing new ideas and new momentum to the search for solutions.

To put it bluntly, after the traumatic episodes of fascism and Stalinism, and after the failure of liberalism to deliver on its promise of emancipation and effective management of human (and environmental) affairs, we are in great need of finding a new compass. It can no longer be a totalitarian ideology, it can no longer be based on the exclusion or the elimination of a particular category of the population, it can no longer rely on blind faith in automatic mechanisms such as the market or algorithms, and it can no longer be subservient to technocrats or any type of elite.

A report produced by a panel like the IPSP cannot deliver such a vision in a radically innovative way, because it primarily synthesizes existing knowledge, but it can provide the tools and the bricks for the construction of a new vision, or multiple new visions. The small book that accompanies the report was written to reach a wider audience (Fleurbaey et al. 2018) and it is more daring in its search for a new vision. This paper builds upon and draws from both texts.

An alarmed celebration of success

The IPSP report is playing a balancing act between acknowledging the remarkable achievements that have been made since WWII and warning against the looming threats that are emerging.

On the positive side, it is hard to define what is most rejoicing about past achievements: the drastic reduction in deep poverty, the increase in life expectancy and improvements in health in general, the end of colonial domination and the spread of democratic institutions, the economic growth and the surge of a new middle class in emerging economies, broader and cheaper access to information and communication, the end of various forms of segregations and the greater inclusion of women, LGBTQ and ethnic groups, not to forget major inroads in the protection of nature and the fight against certain pollutants like CFCs or HFCs… We live in a world that is much less unequal, much more open-minded and inclusive, in many ways, than 75 years ago.

Yet, while humanity would appear to be at the peak of its possibilities, the report also warns that we are facing an abyss. The benefits of development and globalization have been spread very unequally and the populations left behind live in increasingly unstable settings and the new rise of authoritarianism and identity politics builds upon the anxieties and worries of populations whose lives are being disrupted; technological innovation is unsettling the make-up of the labor market and even our own identity as human beings; corruption and the capture of politics by financial interests pulls the trust of the electorate in political institutions down to an all-time low; the degradation of the environment is not being curbed on some key fronts such as pesticides, antibiotics, plastic or CO2 emissions.

Vicious circles around these worrisome trends can unravel many of our past achievements: economic inequalities generate social unrest that then destabilizes the political system and undermines efforts to protect the environment; the politics of identity exploited by cynical elites rip societies apart and disrupt international cooperation; environmental degradation and climate change threaten vulnerable populations; migrations triggered by conflicts or by poverty jeopardize social peace in recipient areas and drain communities in sending areas. Tipping points are often discussed in relation to climate change, but the most sensitive and dangerous tipping points probably lie in our fragile socio-economic and political systems, which will become dysfunctional before the environment itself unleashes deadly forces.

We are therefore facing the real possibility of a catastrophic collapse, not necessarily of "civilization", but of key elements of the current unstable equilibrium, such as democratic institutions, integrated trade and communications, or ambitious safety nets.

The three pillars of social progress

The report is not pessimistic, though. Possibilities are real for improving our institutions and our norms. We not only have reached high levels of economic and institutional development but we have also developed a fair amount of knowledge about what works and what doesn't. Market failures have been studied in great details, and government failures are also better understood. Even corporate failure is on the radar of many analysts. It is now hard to defend policy recipes which rely on a naïve faith in any one of these institutions, but this is in fact (and should be seen as) a positive development. The death of illusions paves the way for realistic ventures.

Still, we are confronted by a key challenge -the imperative necessity to work on three fronts simultaneously, lest the vicious circles mentioned earlier kick in and divert our path toward failure. We must at the same time promote equity, sustainability, and freedom (including democracy). Our progress imperative, today, has three main pillars.

The first pillar of contemporary progress is equity, and especially the reduction of inequalities. There are three forms of inequalities which need urgent redress. The first is the lack of development in certain parts of the world, especially in Africa, where large pockets of poverty and high fertility offer a grim outlook of the future. The second is the extreme concentration of wealth that has resulted from a combination of decreasing redistribution, expansion of opportunities in globalized markets with winner-take-all patterns, and unhinged rent seeking in the financial sector. The third is the concentration of power in politics and the economy, with an excessive influence of wealth and financial interests, and the appearance of transnational economic giants. These inequalities stunt people's opportunities and in fact not only their own but also that of their children. In countries with high levels of inequalities, such as the USA (IPSP, Chapter 3), social mobility is significantly reduced. Where you are born in the world greatly determines not only where you end up in the global income distribution but also the range of economic and social opportunities you have access to [START_REF] Milanovic | Global Inequality. A New Approach for the Age of Globalization[END_REF].

The second pillar of progress is sustainability, and here also there is great concern that little is being done to curb current worrying trends with regard to the degradation of our natural environment and the disruption of climate equilibria. Some past successes such as the fight against the ozone hole or SO2 might suggest solutions for the gridlock on other fronts such as CO2. It would be indeed promising to combine targets for pollution reduction with advantages in funding, technological transfers, and trade arrangements, as well as enlist business innovation toward greening production and consumption patterns and helping developing countries to leapfrog to a green configuration.

The third pillar of progress is freedom, understood in a comprehensive way and therefore encompassing the protection of human rights, the rule of law, and democratic decisionmaking. According to the dataset produced by Polity, the number of countries defined as democracies has been increasing until 20173 , but the definition of democracy is minimal in this type of statistics. It is hard, for instance, to describe the US political system as a democracy when so much, in the political dynamics of that country, is determined by money and so little by the popular vote [START_REF] Bartels | Unequal Democracy. The Political Economy of the New Gilded Age[END_REF]). The deconsolidation of democracy observed by [START_REF] Foa | The Danger of Deconsolidation: The Democratic Disconnect[END_REF] through the decreasing support for democratic political institutions across generations resonates with many observers' sense of alarm at the rise of illiberal regimes over the world, including at the very center of Europe. This deconsolidation trend is, however, often over-interpreted. A survey done by IPSP in the USA, for instance, shows that the population still supports democracy. Moreover, it believes in a participatory form of democracy in equal proportions as classical forms of electoral competition, would like to give much more power to ordinary citizens, and is favorable to the expansion of democracy to other institutions, especially business companies (Fleurbaey 2018). Therefore, a more accurate reading of current trends might be that people are increasingly dissatisfied with the flaws of the currently corrupt political institutions that usurp the title of democracies, but are not enamored with authoritarianism and actually long for a more authentic, more participatory form of democracy.

The challenge for the current era is that the promotion of progress is bound to fail if any of the three pillars falters. The Western way of life as we know it was environmentally possible only because drastic inequalities in development prevented its geographical spread. But as emerging economies aspire to the same standards of living, the impact of excessive energy use and pollution on our planet becomes unmitigated and devastating. Even moderate inequities in the French tax system have been enough recently to block modest policies toward carbon pricing. Hence, the protection of the environment is impossible without a legitimate and durable social contract and there is no legitimate or durable social contract without social justice.

Likewise, many pundits muse about the Chinese way and the possibility to more effectively impose changes in lifestyles and industrial development, or even population growth, through authoritarian policies. But this largely appears to be an illusion. No authoritarian regime escapes the trap of corruption, and the Chinese is no exception. And no culture in the world, no matter how holistic and collectivist, seems to make authoritarianism and abuse of power viable in the long run. Trying even to have a short period of authoritarianism during the transition to a green economy is more unrealistic than any other transition scenario, and the return path back from dictatorship and abuse is generally very painful. Therefore, there is no credible scenario in which equity and sustainability could be achieved without freedom and democracy.

Finally, there is no sustainable and inclusive social and political system without a stable ecosystem. Environmental degradation and natural catastrophes have social, economic and political consequences that are becoming ever more profound and destabilizing. Everywhere in the world we now see bodies in the streets after floods and wildfires. Conflicts that emerge with climate migration or around the issue of access to vital and fragile resources, such as food or water, will rise to unbearable intensities if nothing serious is done to stabilize our climate and ecosystems. The current human species, with its level of intelligence, has been around for hundreds of millennia, and its development to the current stage has been possible only after the Holocene miraculously stabilized temperature at a moderate level. In spite of our modern knowledge, science and technology, we are neither equipped nor ready to deal with the shocks that are bound to strike our communities as those stable conditions are threatened in the coming decades.

Power, power, power

In front of this daunting challenge, the IPSP does not propose magic wands or easy solutions. In particular, the difficulties of failed states in high-conflict areas are particularly hard to address without strong external support and equally hard to manage with external intrusion, the latter being increasingly difficult to legitimate. But a general thread through the report is that rather than focus on resources, it may be more promising to go a little upstream and focus on the mechanisms by which decisions are made. Power is important in three ways.

First, it is a key dimension of human flourishing. People need agency, they need some degree of control over their lives, and this is strongly correlated to a sense of dignity. Being strongly dependent on others for one's livelihood or for key decisions in one's life is not compatible with full development. This does not mean that an individualistic view of life is promoted in the report. On the contrary, it is argued that the individual and the collective can weave their projects in an intimate way. Everyone's flourishing is in good part requiring to go beyond the self and to develop one's projects through collective endeavors and strong social ties. But this is not the same thing as sacrificing individual flourishing to the authority of the group or the authority of leaders in the group. Dignity is an important value that is often invoked by disadvantaged people, and it captures very well the sense of impoverishment and debasement that accompanies unbalanced relationships with powerful people. Dignity is obtained not just by a decent standard of living, but at least as much by a decent status in the hierarchy of power.

The second way in which power is essential is that it determines how decisions are made in other spheres, in particular with respect to the allocation of resources. Governance structures are crucial features of every organization and system. The low level of trust of contemporary electorates in political systems reflects the beliefs that those systems are rigged and that politicians are not listening to people like them, which is largely accurate. International organizations are still dominated by rich countries and this undermines the possibilities for global cooperation and hinders action on many fronts, from development and trade to climate change. Where power is more equally shared, as in the UN General Assembly and similar bodies, one sees that decisions are characteristically different from what rich countries would favor. The Security Council is paralyzed by the extraordinary power of its permanent members. Similarly, in the economic world, people have low trust in big companies and indeed the concentration of power in corporations paves the way for extensive rent seeking and corruption. It also generates toxic behavior because when organizations such as business companies are captured by small groups (CEOs and active shareholders), these groups orient the organization towards their narrow interests and treat all other stakeholders as costs that need to be minimized or externalized. Hence a pattern of squeezing workers until they quit, de-localizing production or replacing manual labor by machines, which all generate on the one hand higher profits for shareholders but also, on the other hand, significant negative costs and consequences for local communities. Likewise, the environmental consequences of the firm's activities are externalized as much as possible on the rest of society. As these examples show, excessive power concentration is everywhere preventing more progressive and responsible decisions from being made because the narrow interests that dominate decision-making processes reject the negative consequences on other parts of society. In contrast, an inclusive form of governance can at least partially contain and limit this kind of cost externalization logic. It may not be sufficient, of course, because relevant stakeholders, such as future generations or other species, may never have access to the decision process. This therefore requires additional safeguards that can be provided by a well-organized and common-good, long-term oriented central political process.

Finally, power is important in a third way. Protecting its fair distribution among stakeholders prevents the spillovers from other inequities. It is not just unfair and counterproductive to concentrate power in the hands of a few. It is especially egregious if the powerful few happen to be advantaged in terms of wealth, gender, and race, and if they got access to power through these other advantages conferred by their economic success or their social status. Shielding the distribution of power from other inequities is therefore absolutely central. It is sometimes believed that power has to be associated with property, for instance in business firms. The only way to achieve social justice is then to jointly distribute property and power, or to focus on property redistribution and let power follow from this. But this is typically not said in relation to political power. It would indeed be odd to argue that power should go to the rich anyway and that redistributing wealth will ensure that the political system will be less unfair. While it is true that protecting politics from money is hard, the more typical view is that political power should not automatically go to the rich and that the influence of money should be stringently controlled. The same view should prevail everywhere, including in the economy. It is possible to share power more equitably and to disconnect it from wealth. In the case of business firms, what justifies an inclusive governance involving non-wealthy stakeholders is that these stakeholders do have stakes, and often (as is the case with workers) have great competence to help with managing the organization.

This focus on power does not mean that the other dimensions are not important. They are and it is important to prevent spillovers between these dimensions, especially between resources, status and power. But the Gordian knot that needs to be cut in order to make progress on the front of inequalities, freedom and sustainability lies in where and how decisions are made in the economy, in politics and in all settings, including families, for instance.

For all this focus on governance and power, however, the report is not at all envisioning a classical fight for political power as the main way to carve a route toward social progress. In particular, the report highlights the emergence of civil society as a new actor of change. It is through multiple actions and especially through the loose but effective coordination of multiple grassroots initiatives that social progress can be pushed forward. While classical organizations such as trade unions or political parties have been on the decline, others have emerged, in particular NGOs and associations, but also a great variety of coalitions of local actors (interesting in that respect is the global coalition of progressive cities, the c40 cities but there are many other examples). Civil society in the many different forms it takes today may be the key resource to tap when one seeks to set up whistleblower and watchdog platforms and systems for the preservation of commons and vital resources, for the control of private corporations, government, the internet, the media, or for the exercise of participatory democracy, and so on. Today, we are in the position to project ourselves as being at the dawn of a new era -where participatory economic organization should replace the predatory forms of contemporary capitalism, where political democracy should be strengthened through participatory deliberation and representation, and where the exploitation of nature will give way to a more responsible and respectful interaction with our environment and ecosystem. Unfortunately, however, as noted by the late Erik Olin [START_REF] Wright | Envisioning Real Utopias[END_REF], what is feasible and viable may not always be reachable through an easy transition. The report does not tread on the issue of political strategies and leaves it to its readers and to our organized communities of actors to develop and deploy progressive scenarios.

Members of the Steering Committee of the IPSP. MF is professor at Princeton University and MLSD is Professor and Dean of the School of Management and Innovation at Sciences Po, Paris.

See https://www.un.org/sustainabledevelopment/sustainable-development-goals/, http://www.oecd.org/inclusive-growth/, http://www.worldbank.org/en/publication/wdr2017, http://www.worldbank.org/en/region/eca/publication/eca-social-contract, https://www.g20-insights.org, http://www.g8.utoronto.ca/T7/T7-2018-Recommendations-Realized.html.

See http://www.systemicpeace.org/polity/polity1.htm.


Philippe Colson

Lucile Pinault

Said Azza

Nicholas Armstrong

Eric Chabriere

La Bernard

Pierre Scola

Didier Pontarotti

Prof. Didier Raoult

Bernard La Scola

Pierre Pontarotti

Didier Raoult
email: didier.raoult@gmail.com

A metallo-beta-lactamase with both beta-lactamase and ribonuclease

Keywords: Giant virus, mimivirus, tupanvirus, beta-lactamase, ribonuclease

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

INTRODUCTION

The metallo-beta-lactamase (MBL) superfamily encompasses a large set of enzymes, including MBL and ribonuclease (RNase) Z enzymes [START_REF] Alderson | One origin for metallo-beta-lactamase activity, or two? An investigation assessing a diverse set of reconstructed ancestral sequences based on a sample of phylogenetic trees[END_REF] . These enzymes are pleitropic proteins that can hydrolyze a wide range of substrates, among which beta-lactams, and DNA or RNA [START_REF] Aravind | An evolutionary classification of the metallo-beta-lactamase fold proteins[END_REF][START_REF] Bebrone | Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily[END_REF] . Such capabilities rely on an ancient and highly conserved fold, which represents a stable scaffold that has evolved to perform a broad range of chemical reactions and on which various catalytic, regulatory and structural activities are based [START_REF] Aravind | An evolutionary classification of the metallo-beta-lactamase fold proteins[END_REF][START_REF] Bebrone | Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily[END_REF][START_REF] Caetano-Anolles | An evolutionarily structured universe of protein architecture[END_REF] . This wide array of activities is enabled by variations in the composition and size of loops located near the enzyme active site [START_REF] Bebrone | Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily[END_REF] . A well-known catalytic activity of MBLs consists in breaking beta-lactam rings, which was primarily identified in bacteria [START_REF] Abraham | An enzyme from bacteria able to destroy penicillin[END_REF] . Nevertheless, this hydrolase activity is suspected to have evolved in response to the environmental beta-lactams from an ancestral protein whose function was not related to beta-lactams and which may have been devoid of such hydrolase capability [START_REF] Bebrone | Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily[END_REF] . Concurrently to their capability to interact with various substrates that likely emerged through adaptive evolution, members of the MBLs superfamily have been identified in a broad range of cellular organisms, including bacteria, but also eukaryotes and archaea with a beta-lactamase activity [START_REF] Aravind | An evolutionary classification of the metallo-beta-lactamase fold proteins[END_REF][START_REF] Diene | Paradoxical ß-Lactamase activity of archaeal encoding enzymes[END_REF] .

Giant viruses are bona fide microbes as their virions are visible under a light microscope and they display a complexity similar to that of small cellular microorganisms [START_REF] Colson | Mimivirus: leading the way in the discovery of giant viruses of amoebae[END_REF][START_REF] Scola | A giant virus in amoebae[END_REF] .

Since their discovery in 2003, their diversity has increased considerably, with nine families and more than 100 isolates cultured. Their classification alongside cellular microorganisms is still debated, but their characteristics clearly distinguish them from conventional viruses [START_REF] Raoult | Redefining viruses: lessons from Mimivirus[END_REF][START_REF] Raoult | TRUC or the need for a new microbial classification[END_REF] .

We have investigated whether genes encoding members of the MBLs superfamily may also be present in giant viruses. We found one in Tupanvirus deep ocean, a giant mimivirus 4 isolated from Brazilian Atlantic ocean sediments, and confirmed that its product harbored a biologically active MBL fold with both beta-lactamase and RNase activities.

RESULTS

While annotating the genome of Tupanvirus deep ocean, the second isolate of a new mimivirus genus, Tupanvirus [START_REF] Rodrigues | Tupanvirus", a new genus in the family Mimiviridae[END_REF] , a gene (GenBank: AUL78925.1) that encodes a metallohydrolase-like MBL fold was identified (Conserved Protein Domain Family Accession no. cl23716) [START_REF] Abrahao | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF] . This gene has a homolog in the other tupanvirus isolate (Soda Lake) (AUL77644.1). Beyond, best BLASTp hits against cellular organisms included MBL fold harboring proteins from an unclassified deltaproteobacterium whose genome was assembled from a marine water metagenome (evalue, 5e-38; identity, 33.0; coverage, 83%), from an actinobacteria (Nonnomuraea spp.) (1e-36; 30.0; 86%), from Microscilla marina (6e-34; 28.5%; 89%) and from Acanthamoeba castellanii (4e-33; 29.8%; 81%) (Fig. 1, Supplementary Fig. S1). Significant BLASTp hits (evalues ranging from 1e-41 to 8e-6) against viruses were also obtained with genes from putative giant viruses whose genomes was assembled from metagenomes obtained from environmental samples [START_REF] Schulz | Giant viruses with an expanded complement of translation system Components[END_REF][START_REF] Schulz | Hidden diversity of soil giant viruses[END_REF][START_REF] Backstrom | Virus Genomes from deep sea sediments expand the ocean megavirome and support independent origins of viral gigantism[END_REF] and from Cafeteria roenbergensis virus, a distant Mimivirus relative [START_REF] Fischer | Giant virus with a remarkable complement of genes infects marine zooplankton[END_REF] . The 322 amino acid long tupanvirus protein exhibits the conserved MBL motif "HxHxDH" in amino acid positions 60-65. A search for domains using the NCBI conserved domain search (CD Search) tool [START_REF] Marchler-Bauer | CDD: conserved domains and protein three-dimensional structure[END_REF] identified a MBL fold belonging to a ribonuclease Z (RNase_Z_T_toga, TIGR02650, interval= amino acids 24-273, E-value= 1.81e-14; RNaseZ_ZiPD-like_MBL-fold, cd07717, interval= amino acids 56-282, E-value= 1.63e-04), which is a transfer RNA (tRNA)-processing endonuclease. This Tupanvirus deep ocean protein was further analyzed by three-dimensional comparison against the Phyre2 web portal for protein modeling, prediction and analysis [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF] . This analysis reported a best match with 100% confidence and 85% coverage (273 amino acid residues) with the crystal structure of a long form ribonuclease Z (RNase Z) from yeast (template c5mtzA) (Supplementary Fig. S2). Proteome analysis conducted for Tupanvirus Soda Lake, and Tupanvirus deep ocean, as previously described [START_REF] Abrahao | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF] , did not allow the detection of these proteins with a MBL fold in the virions. In addition, the dramatic RNA shutdown observed during the replication of this giant virus hindered the achievement of transcriptomic analyses.

Interestingly, the genomes of 20 of the 21 (95%) giant viruses found to encode a MBL fold protein concurrently encode tRNAs, whereas this is only the case for 46 of the 122 (38%) giant viruses devoid of a MBL fold protein (p<10 -3 ; Yates-corrected chi-square test) (Supplementary Fig. S3 and4 and Table S2). The presence of a MBL fold protein among Megavirales members was correlated with the size of the gene repertoire and the number of translation-associated components (p<10 -3 ; Anova test). Putative proteins with a MBL fold from giant viruses comprised two related phylogenetic clusters (Fig. 1). These clusters appeared deeply rooted in the phylogenetic tree, which suggests an ancient origin for these genes. In addition, one of the clusters of giant virus genes encoding MBL fold proteins appeared closely related to two genes from Acanthamoeba castellanii, an amoebal mimivirus hosts, suggesting a transfer from these giant viruses to A. castellanii.

The recombinant protein AUL78925.1 of Tupanvirus deep ocean (named TupBlac) was expressed in Escherichia coli and was then purified, as described previously [START_REF] Diene | Paradoxical ß-Lactamase activity of archaeal encoding enzymes[END_REF] . Based on the phylogenetic analysis and as MBL folds can hydrolyse nucleic acids 2 , both beta-lactamase and nuclease activities of this purified protein were thereafter tested. We first evaluated the beta-lactamase activity of a pure solution of TupBlac used at a concentration of 1 µg/mL by incubating it with nitrocefin, a chromogenic beta-lactam used to test the beta-lactamase activity [START_REF] Diene | Human metallo-ß-lactamase enzymes degrade penicillin[END_REF] . A significant hydrolysis activity was observed (Fig. 2). A concentrate of protein extract (50 mg/mL) obtained from tupanvirus virions also degraded, albeit slightly, nitrocefin.

Thereafter, we monitored by liquid chromatography-mass spectrometry the effect of TupBlac on penicillin G (10 µg/mL) and observed a significant hydrolysis activity of this coumpound within 48h (Fig. 3). We also detected, in the presence of the tupanvirus protein, benzylpenilloic acid, the metabolite resulting from the enzymatic hydrolysis of penicillin G [START_REF] Deshpande | Degradation of beta-lactam antibiotics[END_REF] .

Finally, we confirmed that these observations were related to a beta-lactamase activity as both penicillin G degradation and benzylpenilloic acid appearance were inhibited by sulbactam, a beta-lactamase inhibitor (Fig. 3). We further tested if pre-treatment with sulbactam had an impact on the duration of the giant virus replication cycle and replication intensity. After replication on A. castellanii strain Neff in the presence of a high concentration (10 µg/mL) of sulbactam, the virions produced (10 6 /mL) were inoculated on fresh amoebae at different concentrations. No differences were observed regarding viral growth in the absence or presence of pre-treatment with sulbactam as assessed using high content screening (Supplementary Fig. S5).

Finally, as some proteins with a MBL fold can hydrolyse DNA and RNA 2 , we tested the capability of tupanvirus enzyme TupBLac to degrade synthetic single-and doublestranded DNAs and bacterial RNAs. We found no effect on both DNA types. In contrast, we observed a strong RNase activity (Fig. 4). Another set of experiments was conducted using E. coli RNA as a substrate with an assessment of RNA size distribution on a bioanalyzer (Agilent Technologies, Palo Alto, CA) after incubation with TupBlac. It showed a dramatic degradation of RNAs by the tupanvirus enzyme (Fig. 5a). In contrast with the beta-lactamase activity, this was not inhibited, neither by sulbactam (Fig. 5a and Supplementary Fig. S6), nor by ceftriaxone (Fig. 5b), a cephalosporin that inhibits human SNM1A and SNM1B, that are DNA repair nucleases with a MBL fold [START_REF] Lee | Cephalosporins inhibit human metallo beta-lactamase fold DNA repair nucleases SNM1A and SNM1B/apollo[END_REF] . In addition, a RNase activity of the Tupanvirus protein was further observed on A. castellanii RNA, and not inhibited either by sulbactam or ceftriaxone (Fig. 5c). TupBLac also degraded RNA extracted from bacteria with genomes with different G+C contents ranging between 41.8% and 66.6% (Fig. 5d), suggesting an absence of influence of the G+C richness on the RNase activity. Finally, TupBlac RNase activity was estimated to be 0.451±0.153 mU/mg using a fluorescence-based assay, without difference in the presence of sulbactam or ceftriaxone (0.520±0.003 and 0.551±0.024 mU/mg, respectively) (Supplementary Fig. S7).

DISCUSSION

Hence, we found herein by several bioinformatic approaches that a gene of Tupanvirus deep ocean, a recently discovered giant virus classified in family Mimiviridae [START_REF] Rodrigues | Tupanvirus", a new genus in the family Mimiviridae[END_REF][START_REF] Abrahao | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF] , encodes for a protein with a MBL fold. We further observed that this protein exhibited dual beta-lactamase and RNase activities. This is the first evidence of the presence of a biologically-active protein with a MBL fold in a virus. In addition, this work parallels the one on a protein detected by functional screening of a metagenomic library from the deep-seep sediments [START_REF] Lee | Dual activity of PNGM-1, a metallo-beta-lactamase and tRNase Z, pinpoints the evolutionary origin of subclass B3 metallo-beta-lactamases[END_REF] , showing that the same enzyme has both beta-lactamase and RNase activities. Indeed, MBL fold proteins were previously biologically-tested for either activity, but not for both. It is noteworthy that the beta-lactamase activity of the MBL fold protein of Tupanvirus was inhibited by a betalactamase inhibitor but this was not the case for the RNase activity [START_REF] Baier | Connectivity between catalytic landscapes of the metallobeta-lactamase superfamily[END_REF] . The phylogenetic study of this beta-lactamase shows the presence in several other giant viruses of phylogeneticallyclustered counterparts, the origin of which seems very old. Interestingly, it also appears that there may have been a gene transfer between these giant viruses and Acanthamoeba sp., the amoebal host of many giant viruses. Such potential for horizontal transfer of these MBL fold proteins is well-recognized [START_REF] Bebrone | Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily[END_REF] .

Beta-lactamases are a priori useless for giant viruses, which are grown in the presence of various antibiotics, including beta-lactams [START_REF] Khalil | Updating strategies for isolating and discovering giant viruses[END_REF] , but our findings enhance the recent reconsideration of the function of MBL fold proteins. Thus, the recent description of penicillin secretion by arthropods [START_REF] Suring | Evolutionary ecology of beta-lactam gene clusters in animals[END_REF] and the demonstration of active beta-lactamase in vertebrates including humans [START_REF] Diene | Human metallo-ß-lactamase enzymes degrade penicillin[END_REF] , as well as in archaea [START_REF] Diene | Paradoxical ß-Lactamase activity of archaeal encoding enzymes[END_REF] and fungi [START_REF] Gao | Fungal lactamases: their occurrence and function[END_REF] show that MBL fold proteins have a dramatically broad distribution. In humans, 18 genes were annotated as betalactamases, whose activity had not been biologically-tested until recently [START_REF] Diene | Human metallo-ß-lactamase enzymes degrade penicillin[END_REF] . In addition, MBL fold proteins were highlighted to digest DNA or RNA [START_REF] Aravind | An evolutionary classification of the metallo-beta-lactamase fold proteins[END_REF][START_REF] Diene | Human metallo-ß-lactamase enzymes degrade penicillin[END_REF] . Thus, a class of enzymes, that were named beta-lactamases because of their original discovery in bacteria resistant to betalactamines, are in fact potentially versatile proteins. This differs from the drasticallysimplified paradigm consisting in enzymes with a beta-lactamase activity being secreted by bacteria under the selective pressure of natural or prescribed antibiotics. The RNase activity observed here for the Tupanvirus MBL fold protein could be related to the host ribosomal shutdown observed in the presence of Tupanvirus deep ocean with various protists, the mechanism of which has not been elucidated [START_REF] Abrahao | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF] . This activity could allow these viruses to take over on their cellular hosts by degrading cellular messenger RNAs and shutting down cellular gene expression. The giant virus mRNAs should be protected from such a degradation, which may be explained by the encapsidation of RNA transcripts into giant virions that was detected for some of these viruses [START_REF] Raoult | The 1.2-megabase genome sequence of Mimivirus[END_REF] . Bioinformatic analyses suggested that the tupanvirus MBL fold protein may belong to the RNase Z group that was proposed to be one of the two main groups of the MBL superfamily with that encompassing MBLs 1 .

RNase Z enzymes perform tRNA maturation by catalyzing the endoribonucleolytic removal of the 3' extension of tRNA precursors that do not contain a chromosomally-encoded CCA determinant [START_REF] Ma | The crystal structure of Trz1, the long form RNase Z from yeast[END_REF][START_REF] Perwez | RNase Z in Escherichia coli plays a significant role in mRNA decay[END_REF][START_REF] Schierling | tRNA 3' end maturation in archaea has eukaryotic features: the RNase Z from Haloferax volcanii[END_REF] . The presence in giant viruses of RNases showing the greatest homology to tRNases suggests a specific activity on tRNAs, which seems consistent with the presence of a large set of translation components in these viruses, first and foremost Tupanvirus deep ocean that is the current record holder of the number of translation components (including 70 tRNAs targeting all 20 canonical amino acids). The presence of a putative tRNase in the virus that currently has the most complete set of translation components of the whole virosphere is likely not fortuitous. Furthermore, it was described for Escherichia coli that its RNase Z had endoribonucleasic activity on messager RNAs, being responsible for their decay in in vitro experiments [START_REF] Perwez | RNase Z in Escherichia coli plays a significant role in mRNA decay[END_REF] . This further argues that MBL fold proteins may contain a wide range of activities. PNGM-1, a MBL fold protein whose sequence was recently described from a deepsea sediment metagenome by detection of its beta-lactamase activity [START_REF] Park | PNGM-1, a novel subclass B3 metallo-beta-lactamase from a deep-sea sediment metagenome[END_REF] , was also found to harbor dual beta-lactamase and RNase activities [START_REF] Lee | Dual activity of PNGM-1, a metallo-beta-lactamase and tRNase Z, pinpoints the evolutionary origin of subclass B3 metallo-beta-lactamases[END_REF] . MBL fold proteins from giant viruses are clustered with this protein in the phylogenetic analysis. Interestingly, PNGM-1 was suspected to have evolved from a tRNase Z [START_REF] Lee | Dual activity of PNGM-1, a metallo-beta-lactamase and tRNase Z, pinpoints the evolutionary origin of subclass B3 metallo-beta-lactamases[END_REF] . In conclusion, our data still broaden the range of biological hosts of MBL fold proteins and demonstrate that such proteins can display dual beta-lactamase and nuclease activities. Therefore, we reannotated the tupanvirus MBL fold protein as a beta-lactamase/nuclease.

MATERIALS AND METHODS

Bioinformatics

Searches for Tupanvirus deep ocean protein AUL78925.1 homologs were performed using the BLAST tool [START_REF] Altschul | Basic local alignment search tool[END_REF] . Phylogeny reconstruction was performed after amino acid sequence alignment with the Muscle program [START_REF] Edgar | MUSCLE: a multiple sequence alignment method with reduced time and space complexity[END_REF] and the Maximum-Likelihood method using FastTree [START_REF] Price | FastTree 2--approximately maximum-likelihood trees for large alignments[END_REF] , and tree visualization used MEGA 6 software [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis version 6.0[END_REF] . The amino acid sequences analyzed are Tupanvirus deep ocean protein AUL78925.1 and its homologs with the greatest BLASTp scores from the NCBI GenBank protein sequence database, our sequence database of giant virus genomes, and previously described draft genome sequences from 14 Acanthamoeba species [START_REF] Chelkha | A Phylogenomic Study of Acanthamoeba polyphaga draft genome sequences suggests genetic exchanges with giant viruses[END_REF] ; a set of previously described MBL fold proteins [START_REF] Diene | Human metallo-ß-lactamase enzymes degrade penicillin[END_REF] ; and a set of sequences from the UniProtKB database 1 previously used for phylogeny reconstructions. Three-dimensional comparisons for protein modeling, prediction and analysis were carried out against the Phyre2 web portal [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF] . The set of translation components from each representative of the proposed order Megavirales 37 was obtained through a BLASTp search [START_REF] Altschul | Basic local alignment search tool[END_REF] with their repertoire of predicted proteins against clusters of orthologous groups of proteins (COGs) involved in translation (category J) [START_REF] Tatusov | The COG database: an updated version includes eukaryotes[END_REF] , using 10 -4 and 50 amino acids as thresholds for e-values and sequence alignment lengths, respectively. The set of tRNAs from each virus was collected using the ARAGORN online tool (http://130.235.244.92/ARAGORN/) [START_REF] Laslett | a program to detect tRNA genes and tmRNA genes in nucleotide sequences[END_REF] . Hierarchical clustering was performed using the MultiExperiment Viewer software [START_REF] Saeed | TM4: a free, open-source system for microarray data management and analysis[END_REF] based on the patterns of presence/absence of MBL fold protein, numbers of translation-associated components (number of tRNAs, aminoacyl tRNA-synthetases, other tRNA-associated proteins, other translation-associated proteins) and size of the gene repertoires for Megavirales members (Supplementary Table S2). For each item, the maximum value was determined, and values for each virus were considered relatively to these maximum values, being therefore comprised between 0 and 100%.

Cloning, expression and purification

The Tupanvirus deep ocean gene bioinformatically predicted to encode a beta-lactamase superfamily domain (AUL78925.1 [START_REF] Abrahao | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF] ) was designed to include a Strep-tag at the N-terminus and optimized for its expression by Escherichia coli. It was synthetized by GenScript (Piscataway, NJ, USA) and ligated between the NdeI and NotI restriction sites of a pET24a(+) plasmid. E. coli BL21(DE3)-pGro7/GroEL (Takara Shuzo Co., Kyoto, Japan) grown in ZYP-5052 media were used for the expression of the recombinant protein. When the culture reached an O.D. 600 nm = 0.6 at 37°C, the temperature was lowered to 20°C and L-arabinose (0.2% m/v) was added in order to induce the expression of chaperones. After 20 hours, cells were harvested by centrifugation (5,000 g, 30 min, 4°C) and the pellet was resupended in washing buffer (50 mM Tris pH 8, 300 mM NaCl) and then stored at -80°C overnight. Frozen E. coli were thawed and incubated on ice for 1 hour after having added lysozyme, DNAse I and PMSF (phenylmethylsulfonyl fluoride) to final concentrations of 0.25 mg/mL, 10 µg/mL and 0.1 mM, respectively. Partially lysed cells were then disrupted by 3 consecutive cycles of sonication (30 seconds, amplitude 45) performed on a Q700 sonicator system (QSonica). Cellular debris were discarded following a centrifugation step (10,000 g, 20 min, 4°C). The Tupanvirus protein was purified with an ÄKTA avant system (GE Healthcare, Bucks, UK) using Strep-tag affinity chromatography (wash buffer: 50 mM Tris pH 8, 300 mM NaCl, and elution buffer: 50 mM Tris pH 8, 300 mM NaCl, 2.5 mM desthiobiotin) on a 5 mL StrepTrap HP column (GE Healthcare). Fractions containing the protein of interest were pooled. Protein purity was assessed using 12.5% SDS-PAGE analysis (Coomassie staining). Protein expression was confirmed by performing MALDI-TOF MS analysis on gel bands previously obtained by SDS-PAGE. Protein concentrations were measured using a Nanodrop 2000c spectrophotometer (Thermo Scientific, Madison, WI, USA).

Spectrophotometry assay for the detection of beta-lactamase activity in Tupanvirus virions

Tupanvirus purified virions in solution were centrifuged at 5,000 RPM in order to collect 1g of humid matter. Virions were then suspended into 2 mL of a phosphate-buffered saline (PBS) solution at pH 7.4 prepared in water from a commercial salt mixture (bioMerieux, Marcy-l'Etoile, France). Virions were broken after five freeze-thaw cycles followed by 10 minutes of sonication (Q700 sonicator with a Cup Horn, QSonica, Newtown, Connecticut, USA). Integrity of virions was checked by scanning electron microscopy (TM 4000, Hitachi High-Technologie Corporation, Tokyo, Japan). Debris were discarded following a centrifugation step (15,000 g, 10 minutes). The clear supernatant was lyophilized and then reconstituted in 100 μL of PBS (corresponding to a final concentration of 50 mg/mL of soluble proteins). A pure solution of Tupanvirus protein was buffer-exchanged in PBS and the concentration was adjusted to 1 mg/ml. The degradation of nitrocefin (1 mM in PBS), a chromogenic cephalosporin substrate, was monitored as previously described after the addition of virion protein extract or Tupanvirus protein to the solution [START_REF] Diene | Paradoxical ß-Lactamase activity of archaeal encoding enzymes[END_REF] .

Beta-lactam antibiotic degradation monitoring by liquid chromatography-mass spectrometry (LC-MS)

Penicillin G and sulbactam stock solutions at 10 mg/mL were freshly prepared in water from the corresponding high purity salts (Sigma Aldrich). A total of 30 μL of tupanvirus protein solution at 1 mg/mL was spiked with penicillin G and sulbactam at a final concentration of 10 μg/mL, before incubation at room temperature. Each time point corresponded to triplicate sample preparations. Negative controls consisted of PBS spiked with penicillin G and sulbactam. Then, 70 μL of acetonitrile were added to each sample, and tubes were vortexed 10 minutes at 16,000 g to precipitate the proteins. The clear supernatant was collected for analysis using an Acquity I-Class UPLC chromatography system connected to a Vion IMS Qtof ion mobility-quadrupole-time of flight mass spectrometer, as previously described [START_REF] Diene | Paradoxical ß-Lactamase activity of archaeal encoding enzymes[END_REF] .

Assessment of the effect of a beta-lactamase inhibitor on Tupanvirus growth

To evaluate the effect of a beta-lactamase inhibitor sulbactam on Tupanvirus growth, we tested Tupanvirus replication on A. castellanii pre-incubated with a high dose of sulbactam.

Tests were performed in triplicate and amoebae cultivated in trypticase soy medium [START_REF] Abrahao | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF] . Four 1 mL culture wells containing 5.10 5 A. castellanii were incubated at 32°C, one of which contained 500 mg/L of sulbactam. After 24 hours, Tupanvirus was added at a multiplicity of infection (MOI) of 1 in the well with sulbactam. Two other wells were inoculated with Tupanvirus, including one in which 500 mg/L of sulbactam was added. The last well was used as control of amoeba survival. After 24h, amoebae were counted and Tupanvirus was titrated by qPCR as previously described [START_REF] Abrahao | Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere[END_REF] . In order to assess whether sulbactam could have affected newly formed virions, tupanviruses produced on amoebae incubated with sulbactam were inoculated on fresh amoebae at different concentrations. Their growth was monitored using high content screening microscopy every 8h for 48h [START_REF] Francis | High-throughput isolation of giant viruses using high-content screening[END_REF] . Viral replication was compared to that of tupanviruses produced on amoebae non-treated with sulbactam at the same MOIs.

Nuclease activity assessment

Nuclease activity was assessed using double-stranded DNA, (+) and (-) single-stranded DNAs, and single-stranded RNAs as substrates. Single-stranded DNAs were synthetic polynucleotides (Supplementary Table S3); double-stranded DNA was obtained by annealing (+) and (-) single-stranded DNAs in a thermocycler at temperatures decreasing from 95°C to 25°C over 1h. RNAs used as substrate were from Escherichia coli, from different bacteria that differ by the G+C content of their genomes (Streptococcus parasanguinis (41.8%), Vibrio parahaemolyticus (45.4%), Vitreoscilla massiliensis (49.4%), Aeromonas salmonicida (58.5%), Aeromonas hydrophila (61.5%) and Pseudomonas aeruginosa (66.6%)), and from Acanthamoeba castellanii. RNAs were purified using RNeasy columns (Invitrogen, Carlsbad, CA, USA). Enzymatic reactions were performed by incubating each polynucleotide (2 µg) with 15 µg of the expressed Tupanvirus protein TupBlac in Tris-HCl buffer 50 mM, pH 8.0, sodium chloride 0.3 M, using a final volume of 20 µL at 30°C for 2 h. After incubation, the material was loaded onto denaturing polyacrylamide gel electrophoresis (dPAGE) at 12% or analysed using the Agilent RNA 6000 Pico LabChip kit on an Agilent 2100 Bioanalyzer (Agilent Technology, Palo Alto, CA, USA). Controls were carried out under the same conditions. The action of TupBlac on RNAs was also assayed in the presence of ceftriaxone, an inhibitor of human metallo β-lactamase fold DNA repair nucleases SNM1A and SNM1B [START_REF] Lee | Cephalosporins inhibit human metallo beta-lactamase fold DNA repair nucleases SNM1A and SNM1B/apollo[END_REF] .

To do this, enzymatic reactions were conducted at 30°C by incubating E. coli RNA (1 µg) with TupBlac (15 µg) in the presence of ceftriaxone at 200 µM. At different times, reactions were stopped by addition of proteinase K (10 µg) and incubated 1h at 37°C. For a quantitative assessment of the RNase activity of the TupBlac enzyme, we used the RNaseAlert QC System kit (Fisher Scientific, Illkirch, France) according to the manufacturer's protocol. This assay uses as substrate a fluorescence-quenched oligonucleotide probe that emits a fluorescent signal in the presence of RNase activity. RNase activities were assayed in the absence or presence of sulbactam (10 µg/mL) or ceftriaxone (200 µM). Negative controls were made with all the reagents used (RNase free water, enzyme buffer, sulbactam and ceftriaxone).

Fluorescence was monitored continuously at 37°C for 1h by a Synergy HT plate reader (BioTek Instruments SAS, Colmar, France) with a 485/528 nm filter set. RNase activities of TupBlac were estimated using supplied RNase A used as a standard (10 mU/mL). Two independent experiments were conducted.

FIGURES Figure 1  Phylogeny reconstruction based on metallo-beta-lactamase (MBL) fold proteins

Phylogeny reconstruction was performed after amino acid sequence alignment with the Muscle program [START_REF] Edgar | MUSCLE: a multiple sequence alignment method with reduced time and space complexity[END_REF] with the Maximum-Likelihood method using FastTree [START_REF] Price | FastTree 2--approximately maximum-likelihood trees for large alignments[END_REF] , and tree was visualized with the MEGA 6 software [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis version 6.0[END_REF] . The amino acid sequences analyzed are Tupanvirus deep ocean protein AUL78925.1 and its homologs with the greatest BLASTp scores from the NCBI GenBank protein sequence database (nr) (see Supplementary Table S1), our sequence database of giant virus genomes, and previously described draft genome sequences from 14 Acanthamoeba species 36 ; a set of previously described MBL fold proteins [START_REF] Diene | Human metallo-ß-lactamase enzymes degrade penicillin[END_REF] ; and a set of sequences from the UniProtKB database 1 , previously used for phylogeny reconstructions.

Extended tree is available in Supplementary Figure S1. S3), or Escherichia coli RNA (b).

No treatment (A); buffer (B); succinate dehydrogenase enzyme produced and purified by the same process and collected in the same fractions as Tupanvirus beta-lactamase TupBlac, used as negative control (C); Tupanvirus beta-lactamase TupBlac (D).

Figure 2 -

 2 Figure 2  Effect on nitrocefin of expressed Tupanvirus protein (TupBlac).

Figure 3 Figure 4 

 34 Figure 3  Effect on penicillin G of expressed Tupanvirus protein (TupBlac).

Acknowledgments

This work was supported by the French Government under the "Investments for the Future" program managed by the National Agency for Research (ANR), Méditerranée-Infection 10-IAHU-03, and was also supported by Région Provence-Alpes-Côte d'Azur and European funding FEDER PRIMMI (Fonds Européen de Développement Régional -Plateformes de Recherche et d'Innovation Mutualisées Méditerranée Infection).

We are thankful to Rania Francis for her technical help.

Competing interest

The authors declare no competing interests. Funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.

Author contributions