Jože Pungerčar
email: joze.pungercar@ijs.si

Franck Bihl

Gérard Lambeau
email: lambeau@ipmc.cnrs.fr

Igor Križaj
email: igor.krizaj@ijs.si.

What do secreted phospholipases A2 have to offer in combat against different viruses up to SARS-CoV-2?

Keywords: Secreted phospholipase A2, Antiviral action, Host defence, Viruses, SARS-CoV-2, Covid-19

Secreted phospholipases A2 (sPLA2s) form a widespread group of structurally-related enzymes that catalyse the hydrolysis of the sn-2 ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. In humans, nine catalytically active and two inactive sPLA2 proteins have been identified. These enzymes play diverse biological roles, including host defence against bacteria, parasites and viruses. Several of these endogenous sPLA2s may play a defensive role in viral infections, as they display in vitro antiviral activity by both direct and indirect mechanisms. However, endogenous sPLA2s may also exert an offensive and negative role, dampening the antiviral response or promoting inflammation in animal models of viral infection. Similarly, several exogenous sPLA2s, most of them from snake venoms and other animal venoms, possess in vitro antiviral activities. Thus, both endogenous and exogenous sPLA2s may be exploited for the development of new antiviral substances or as therapeutic targets for antagonistic drugs that may promote a more robust antiviral response. In this review, the antiviral versus proviral role of both endogenous and exogenous sPLA2s against various viruses including coronaviruses is presented. Based on the highlighted developments in this area of research, possible directions of future investigation are envisaged. One of them is also a possibility of exploiting sPLA 2 s as biological markers of the severity of the Covid-19 pandemic caused by SARS-CoV-2 infection.

Introduction

Phospholipases A2 (PLA2s) comprise a superfamily of intracellular and secreted enzymes that catalyse the hydrolysis of glycerophospholipids at the sn-2 position, releasing free fatty acids and lysophospholipids [START_REF] Dennis | Phospholipase A2 enzymes: Physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention[END_REF][START_REF] Murakami | Recent progress in phospholipase A₂ research: From cells to animals to humans[END_REF]. Secreted PLA2s (sPLA2s) completely differ from intracellular PLA2 enzymes in structure, enzymatic properties, tissue distribution and biological functions. sPLA2s constitute a family of structurally-related enzymes which are widespread in nature and exert highly diverse biological functions [START_REF] Lambeau | Biochemistry and physiology of mammalian secreted phospholipases A2[END_REF][START_REF] Valentin | Increasing molecular diversity of secreted phospholipases A2 and their receptors and binding proteins[END_REF][START_REF] Valentin | What can venom phospholipases A2 tell us about the functional diversity of mammalian secreted phospholipases A2?[END_REF][START_REF] Pungerčar | Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2[END_REF][START_REF] Lomonte | Snake Venom Phospholipase A2 Toxins, In: Handbook of Venoms and Toxins of Reptiles[END_REF]. Nine catalytically-active sPLA2s and two catalytically-inactive sPLA2-like proteins (classified into groups and subgroups) are present in humans and other mammals [START_REF] Lambeau | Biochemistry and physiology of mammalian secreted phospholipases A2[END_REF]. These human sPLA2s are small proteins of 14-18 kDa with 6-8 disulphide bonds and a highly conserved His-Asp catalytic dyad at the active site using Ca 2+ as a key cofactor for catalysis. They are also termed endogenous sPLA2s and are involved in a myriad of physiological and pathological processes, from digestion, lipid metabolism, skin homeostasis, spermatogenesis or host defence to regulation of the immune response and inflammation in disease conditions such as asthma, sepsis and cardiovascular diseases. sPLA2s participate to such processes by various mechanisms, for instance by modifying the lipid composition of cellular membranes or by producing pro-and/or antiinflammatory lipids from both cellular and noncellular lipid components such as cell membranes, lipoproteins, microvesicles, mitochondria or foreign phospholipids from microbes and food [START_REF] Lambeau | Biochemistry and physiology of mammalian secreted phospholipases A2[END_REF][START_REF] Murakami | Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice[END_REF][START_REF] Murakami | A new era of secreted phospholipase A₂[END_REF]. sPLA2s are also found in large amount in animal venoms, especially in those from snakes, scorpions and bees [START_REF] Valentin | What can venom phospholipases A2 tell us about the functional diversity of mammalian secreted phospholipases A2?[END_REF][START_REF] Pungerčar | Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2[END_REF][START_REF] Lomonte | Snake Venom Phospholipase A2 Toxins, In: Handbook of Venoms and Toxins of Reptiles[END_REF]. Venom sPLA2s are structurally similar to endogenous mammalian sPLA2s but display toxic effects including neurotoxicity, myotoxicity, anticoagulant activity, cardiotoxicity and haemolytic activity. They can be considered as exogenous sPLA2s when injected to mammals, acting as weapons to kill preys. However, they are also viewed as pharmacological agents to possibly cure diseases [START_REF] Gutierrez | Phospholipases A2, Unveiling the secrets of a functionally versatile group of snake venom toxins[END_REF][START_REF] Carpena | Bee venom: An updating review of its bioactive molecules and its health applications[END_REF][START_REF] Šribar | Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2: An update[END_REF]. As such, they are valuable research tools for analysing physiological and pathophysiological conditions in humans as well as promising molecules for the development of new therapeutic agents [START_REF] Sobrinho | Antitumoral potential of snake venom phospholipases A2 and synthetic peptides[END_REF][START_REF] Lee | Bee venom phospholipase A2: Yesterday's enemy becomes today's friend[END_REF][START_REF] Zambelli | Secreted phospholipases A₂ from animal venoms in pain and analgesia[END_REF][START_REF] Almeida | Harnessing snake venom phospholipases A2 to novel approaches for overcoming antibiotic resistance[END_REF].

Both endogenous and exogenous sPLA2s play direct and indirect roles in host defence against various types of bacteria, parasites and viruses [START_REF] Nevalainen | Antibacterial actions of secreted phospholipases A2. Review[END_REF][START_REF] Weiss | Molecular determinants of bacterial sensitivity and resistance to mammalian group IIA phospholipase A2[END_REF][START_REF] Guillaume | In vitro anti-Plasmodium falciparum properties of the full set of human secreted phospholipases A2[END_REF][START_REF] Teixeira | Insights into the antiviral activity of phospholipases A2 (PLA2s) from snake venoms[END_REF][START_REF] Fenard | Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells[END_REF]. For example, an early study showed that several snake and bee venom sPLA2s exhibit direct antiviral activity by blocking HIV-1 virus entry into host cells [START_REF] Fenard | Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells[END_REF]. Later, it was shown that group III, V and X (GIII, GV and GX) endogenous sPLA2s play antiviral roles against adenoviruses and HIV-1 by different mechanisms [START_REF] Mitsuishi | Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells[END_REF][START_REF] Mitsuishi | Human group III phospholipase A2 suppresses adenovirus infection into host cells. Evidence that group III, V and X phospholipase A2s act on distinct cellular phospholipid molecular species[END_REF][START_REF] Kim | Lysis of human immunodeficiency virus type 1 by a specific secreted human phospholipase A2[END_REF]. Other studies have shown that the levels of endogenous sPLA2s are upregulated after infection by dengue virus, H1N1 or coronavirus [START_REF] Nevalainen | Serum phospholipase A2 in dengue[END_REF][START_REF] Ito | Distribution of type V secretory phospholipase A2 expression in human hepatocytes damaged by liver disease[END_REF][START_REF] Kelvin | Lack of group X secreted phospholipase A2 increases survival following pandemic H1N1 influenza infection[END_REF][START_REF] Vijay | Critical role of phospholipase A2 group IID in age-related susceptibility to severe acute respiratory syndrome-CoV infection[END_REF] and can modulate the antiviral response by indirect immune mechanisms [START_REF] Kelvin | Lack of group X secreted phospholipase A2 increases survival following pandemic H1N1 influenza infection[END_REF][START_REF] Vijay | Critical role of phospholipase A2 group IID in age-related susceptibility to severe acute respiratory syndrome-CoV infection[END_REF].

In this review, we present the different antiviral activities of both endogenous and exogenous sPLA2s but also their antagonistic role in worsening viral infection. It appears that sPLA2s interfere with the life cycle of various viruses at different steps and also act indirectly by immunomodulation of the antiviral immune response. Some of their modes of antiviral action may be used to formulate strategies for the development of diagnostic and therapeutic procedures, perhaps even agents to treat viral infections.

Human sPLA2s

In mammals, mainly based on information obtained from mice (m) and humans (h), 11 to 12 sPLA2 members have been identified: GIB, GIIA, GIIC (pseudogene in humans), GIID, GIIE, GIIF, GIII, GV, GX, GXIIA, GXIIB and otoconin-90. These sPLA2s are usually classified into three structural collections, i.e. the collection of I/II/V/X sPLA2s, and the collections of III and XII sPLA2s [START_REF] Lambeau | Biochemistry and physiology of mammalian secreted phospholipases A2[END_REF][START_REF] Valentin | Increasing molecular diversity of secreted phospholipases A2 and their receptors and binding proteins[END_REF][START_REF] Rouault | Novel mammalian group XII secreted phospholipase A2 lacking enzymatic activity[END_REF]. The corresponding human sPLA2 gene names are PLA2G1B, PLA2G2A, PLA2G2C, PLA2G2D, PLA2G2E, PLA2G2F, PLA2G3, PLA2G5, PLA2G10, PLA2G12A, PLA2G12B and OC90.

Mammalian sPLA2s are differentially expressed in tissues and cell types, and play distinct, mostly non-redundant, roles that may be dependent or independent of their enzymatic activity. All but two mammalian sPLA2s are enzymatically active, with a wide range of specific activities depending on the nature of glycerophospholipids, and in vitro versus in vivo settings [START_REF] Singer | Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2[END_REF][START_REF] Yamamoto | Secreted phospholipase A2 specificity on natural membrane phospholipids[END_REF]. GXIIB sPLA2 is a catalytically-inactive sPLA2-like protein due to substitution of the highly conserved histidine by a leucine residue in the active site [START_REF] Rouault | Novel mammalian group XII secreted phospholipase A2 lacking enzymatic activity[END_REF]. Despite its obvious lack of enzymatic activity, the protein has been shown to play a role in hepatic triglyceride metabolism by an unknown molecular mechanism [START_REF] Liu | Activation of farnesoid X receptor promotes triglycerides lowering by suppressing phospholipase A2 G12B expression[END_REF]. Otoconin-90 is another sPLA2-like protein found in the inner ear and involved in balance regulation [START_REF] Zhao | Otoconin-90 deletion leads to imbalance but normal hearing: a comparison with other otoconia mutants[END_REF][START_REF] Wang | Otoconin-90, the mammalian otoconial matrix protein, contains two domains of homology to secretory phospholipase A2[END_REF][START_REF] Verpy | Characterization of otoconin-95, the major protein of murine otoconia, provides insights into the formation of these inner ear biominerals[END_REF]. It possesses a tandem repeat of two sPLA2-like domains [START_REF] Zhao | Otoconin-90 deletion leads to imbalance but normal hearing: a comparison with other otoconia mutants[END_REF] with substitutions in key amino acids involved in enzymatic activity, rendering it catalytically inactive. It can however still bind Ca 2+ ion, which is important for its binding properties within the inner ear extracellular matrix called otoconia [START_REF] Zhao | Otoconin-90 deletion leads to imbalance but normal hearing: a comparison with other otoconia mutants[END_REF][START_REF] Wang | Otoconin-90, the mammalian otoconial matrix protein, contains two domains of homology to secretory phospholipase A2[END_REF][START_REF] Verpy | Characterization of otoconin-95, the major protein of murine otoconia, provides insights into the formation of these inner ear biominerals[END_REF]. The proposed biological functions of endogenous sPLA2s are largely based on observations from transgenic and/or knock-out mouse lines [START_REF] Murakami | Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice[END_REF][START_REF] Murakami | Emerging roles of secreted phospholipase A2 enzymes: The 3rd edition[END_REF][START_REF] Murakami | Updating phospholipase A2 biology[END_REF]. Although a large number of important sPLA2 functions have been revealed by using this approach, it should be emphasized that the results obtained in mice and humans are not always comparable. For example, GIIC sPLA2, which is abundantly expressed in meiotic cells in the mouse testis, is found as a pseudogene in the human genome [START_REF] Tischfield | Low-molecular-weight, calcium-dependent phospholipase A2 genes are linked and map to homologous chromosome regions in mouse and human[END_REF][START_REF] Chen | Localization of group IIc low molecular weight phospholipase A2 mRNA to meiotic cells in the mouse[END_REF]. Similarly, GIIE sPLA2 may play various roles related to inflammation and obesity in the mouse [START_REF] Murakami | Group IID, IIE, IIF and III secreted phospholipase A2s[END_REF] but is barely expressed in most human cells [START_REF] Valentin | On the diversity of secreted phospholipases A2. Cloning, tissue distribution, and functional expression of two novel mouse group II enzymes[END_REF][START_REF] Suzuki | Structures, enzymatic properties, and expression of novel human and mouse secretory phospholipases A2[END_REF]. GX sPLA2 is present in the acrosome of mouse spermatozoa while it seems that GIID sPLA2 and/or other sPLA2s are present in human spermatozoa [START_REF] Escoffier | Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice[END_REF][START_REF] Li | Secretory phospholipase A2 group IID is involved in progesterone-induced acrosomal exocytosis of human spermatozoa[END_REF][START_REF] Anfuso | Asthenozoospermia and membrane remodeling enzymes: A new role for phospholipase A2[END_REF].

Furthermore, most studies were performed with sPLA2 knock-out mouse lines in the C57Bl/6 background, where the GIIA sPLA2 is naturally absent due to a frameshift mutation [START_REF] Kennedy | A natural disruption of the secretory group II phospholipase A2 gene in inbred mouse strains[END_REF], while this sPLA2 is highly expressed in other mouse strains like Balb/C [START_REF] Kennedy | A natural disruption of the secretory group II phospholipase A2 gene in inbred mouse strains[END_REF] or in human tissues where it participates to inflammation and associated diseases, cancer and antibacterial defence among other functions [START_REF] Lambeau | Biochemistry and physiology of mammalian secreted phospholipases A2[END_REF][START_REF] Dore | Roles of secreted phospholipase A2 group IIA in inflammation and host defense[END_REF][START_REF] Brglez | Secreted phospholipases A2 in cancer: Diverse mechanisms of action[END_REF][START_REF] Schewe | Secreted phospholipases A2 are intestinal stem cell niche factors with distinct roles in homeostasis, inflammation and cancer[END_REF]. This raises issues for possible redundancy, overlooked or overestimated roles of certain sPLA2s, and the translational impact of the findings from mice to humans. Therefore, with the aim to better understand the biological roles of the different human sPLA2s in diseases, which may be useful for diagnosis and treatment, it is reasonable to combine the key observations obtained from mouse models with those directly derived from human studies, from tissue distribution and function of human sPLA2s in healthy and disease conditions up to analyses of sPLA2 gene polymorphisms and mutations [START_REF] Masuda | Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells[END_REF][START_REF] Masuda | Diverse cellular localizations of secretory phospholipase A2 enzymes in several human tissues[END_REF][START_REF] Limou | Exploration of associations between phospholipase A2 gene family polymorphisms and AIDS progression using the SNPlex method[END_REF][53][START_REF] Guardiola | PLA2G10 Gene variants, sPLA2 activity, and coronary heart disease risk[END_REF][START_REF] Akinkuolie | Group IIA secretory phospholipase A2, vascular inflammation, and incident cardiovascular disease[END_REF][START_REF] Pothlichet | PLA2G1B is involved in CD4 anergy and CD4 lymphopenia in HIV-infected patients[END_REF].

Finally, it should be mentioned that endogenous sPLA2s exhibit complex enzymatic properties depending on the lipid membranes that they will encounter once secreted. Indeed, they can act on a large variety of lipid substrates to produce multiple lipid mediators or modify the biochemical or biophysical properties of cell membranes or extracellular microvesicles, making their action both complex and unique to the microenvironment in which each of the different sPLA2s operate. sPLA2s act primarily on different extracellular or noncellular phospholipids such as those of the plasma membrane, lipoproteins, lipid microparticles, lung surfactant, skin lipid bodies, food and bacterial membranes, mitochondria, but also on intracellular membranes during their secretion [START_REF] Murakami | Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice[END_REF][START_REF] Guillaume | In vitro anti-Plasmodium falciparum properties of the full set of human secreted phospholipases A2[END_REF][START_REF] Singer | Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2[END_REF][START_REF] Yamamoto | Secreted phospholipase A2 specificity on natural membrane phospholipids[END_REF][START_REF] Masuda | Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells[END_REF][START_REF] Fourcade | Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells[END_REF][START_REF] Murakami | Distinct arachidonatereleasing functions of mammalian secreted phospholipases A2 in fibroblastic and mastocytoma cells through heparan sulfate shuttling and external plasma membrane membrane mechanisms[END_REF][START_REF] Mounier | Arachidonic acid release from mammalian cells transfected with human groups IIA and X secreted phospholipase A2 occurs predominantly during the secretory process and with the involvement of cytosolic phospholipase A2-alpha[END_REF][START_REF] Pruzanski | Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2[END_REF][START_REF] Boudreau | Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation[END_REF][START_REF] Jemel | Group X secreted phospholipase A2 proenzyme is matured by a furin-like proprotein convertase and releases arachidonic acid inside of human HEK293 cells[END_REF][START_REF] Sato | Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice[END_REF]. Products of sPLA2 phospholipolysis, free fatty acids and lysophospholipids, are involved in a number of cellular pathways by generation of multiple bioactive lipid mediators, promotion of membrane remodelling, modification of extracellular noncellular lipid components, such as microparticles and lipoproteins, lung surfactant, or degradation of foreign phospholipids from microbes, microbiota and dietary components in response to given microenvironmental cues. Some of the major biological effects of endogenous sPLA2s and their potential involvement in various human disorders are listed in Table 1.

Antiviral Activity of Endogenous Mammalian sPLA2s

Mammalian sPLA2s, especially GIIA sPLA2, are involved in direct and indirect host defence against various pathogens, including bacteria, parasites and viruses, and as such represent an important component of the innate immune system [START_REF] Nevalainen | Antibacterial actions of secreted phospholipases A2. Review[END_REF][START_REF] Weiss | Molecular determinants of bacterial sensitivity and resistance to mammalian group IIA phospholipase A2[END_REF][START_REF] Kim | Lysis of human immunodeficiency virus type 1 by a specific secreted human phospholipase A2[END_REF][START_REF] Kelvin | Lack of group X secreted phospholipase A2 increases survival following pandemic H1N1 influenza infection[END_REF][START_REF] Vijay | Critical role of phospholipase A2 group IID in age-related susceptibility to severe acute respiratory syndrome-CoV infection[END_REF][START_REF] Dore | Roles of secreted phospholipase A2 group IIA in inflammation and host defense[END_REF][START_REF] Pothlichet | PLA2G1B is involved in CD4 anergy and CD4 lymphopenia in HIV-infected patients[END_REF][START_REF] Entwistle | Epithelial-cell-derived phospholipase A2 group 1B is an endogenous anthelmintic[END_REF][START_REF] Wu | Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host[END_REF][START_REF] Koduri | Bactericidal properties of human and murine groups I, II, V, X, and XII secreted phospholipases A2[END_REF][START_REF] Dacheux | Antimalarial activity of human group IIA secreted phospholipase A2 in relation to enzymatic hydrolysis of oxidized lipoproteins[END_REF][START_REF] Balestrieri | Group V secretory phospholipase A2 modulates phagosome maturation and regulates the innate immune response against Candida albicans[END_REF]. For example, studies as early as back in 1979 have shown that GIIA sPLA2 can exert direct killing of Gram-positive bacteria [START_REF] Weinrauch | The potent anti-Staphylococcus aureus activity of a sterile rabbit inflammatory fluid is due to a 14-kD phospholipase A2[END_REF][START_REF] Elsbach | Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship[END_REF]. It was a result of the ability of this enzyme, due to its highly positive net charge, to penetrate the bacterial peptidoglycan envelope, to reach the bacterial plasma membrane and to hydrolyse it [START_REF] Weiss | Molecular determinants of bacterial sensitivity and resistance to mammalian group IIA phospholipase A2[END_REF][START_REF] Dore | Roles of secreted phospholipase A2 group IIA in inflammation and host defense[END_REF]. In addition, the same sPLA2 is responsible for an indirect host response triggering innate immune response, possibly via hydrolysis of lipid microvesicles or mitochondria released by activated platelets (or other cells) to release potent lipid mediators, or when certain bacterial strains manipulate the host defence activity within a bacterial niche [START_REF] Fourcade | Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells[END_REF][START_REF] Boudreau | Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation[END_REF][START_REF] Pernet | Pseudomonas aeruginosa eradicates Staphylococcus aureus by manipulating the host immunity[END_REF]. Multiple studies have shown that GIIA sPLA2 has in vitro and in vivo antibacterial activity against various bacterial strains [START_REF] Nevalainen | Antibacterial actions of secreted phospholipases A2. Review[END_REF][START_REF] Weiss | Molecular determinants of bacterial sensitivity and resistance to mammalian group IIA phospholipase A2[END_REF][START_REF] Dore | Roles of secreted phospholipase A2 group IIA in inflammation and host defense[END_REF][START_REF] Wu | Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host[END_REF][START_REF] Koduri | Bactericidal properties of human and murine groups I, II, V, X, and XII secreted phospholipases A2[END_REF].

The antiviral activity of endogenous mammalian sPLA2s is much less documented than their antibacterial roles. It was first reported in vitro for two potent plasma membranehydrolysing sPLA2s, hGV and hGX sPLA2s, which could prevent host cells from being infected with adenoviruses [START_REF] Mitsuishi | Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells[END_REF][START_REF] Mitsuishi | Human group III phospholipase A2 suppresses adenovirus infection into host cells. Evidence that group III, V and X phospholipase A2s act on distinct cellular phospholipid molecular species[END_REF] (Table 1). These two sPLA2s are expressed in human airway epithelium and macrophages, and the expression of hGV sPLA2 is upregulated by virus-related stimuli of these cells. Adenovirus particles were insensitive to exogenously added hGV and hGX sPLA2s. However, addition of recombinant hGV and hGX sPLA2s, but not hGIIA, to HEK293 cells suppressed the number and size of adenovirus plaque formation. In contrast to the main antibacterial action of sPLA2s, which depends on their direct hydrolytic action on bacterial membranes [START_REF] Koduri | Bactericidal properties of human and murine groups I, II, V, X, and XII secreted phospholipases A2[END_REF], the antiviral action of GV and GX sPLA2s was indirect and dependent on their ability to act on host cell membranes (Figure 1), leading to a decrease in adenovirus entry by clathrin-mediated endocytosis. The observation that suppression of adenoviral infection by hGV and hGX sPLA2s depends on catalytic activity was confirmed with catalytically-inactive sPLA2 mutants and supported by the finding that lysophosphatidylcholine, an sPLA2-hydrolytic product of phosphatidylcholine enriched in the outer leaflet of the plasma membrane, also suppresses adenoviral infection. It was also suggested that the innate immune action of these sPLA2s may be directed not only against adenoviruses but also against other respiratory viruses [START_REF] Mitsuishi | Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells[END_REF]. Besides endogenous GV and GX sPLA2s, hGIII sPLA2 was also able to inhibit adenovirus entry into host cells [START_REF] Mitsuishi | Human group III phospholipase A2 suppresses adenovirus infection into host cells. Evidence that group III, V and X phospholipase A2s act on distinct cellular phospholipid molecular species[END_REF]. It was shown that, in addition to the catalytically-active sPLA2 domain, the N-terminal domain unique to this enzyme is required for the anti-adenoviral effect observed in human bronchial epithelial cells. This suggests a slightly different antiviral mechanism in the case of hGIII sPLA2. By lipidomics analysis, the authors provided evidence that hGIII, hGV and hGX sPLA2s target different phospholipids in host cell membranes, all leading to production of lysophosphatidylcholine, which was proposed to be responsible for the antiviral action. However, the in vivo role of these sPLA2s against adenovirus infection remains to be demonstrated.

In a subsequent study, it was found that endogenous hGX sPLA2 displays antiviral activity against different enveloped lentiviruses including HIV-1, while the enzyme was found unable to prevent adenovirus infection, which contrasts with the above study [START_REF] Kim | Lysis of human immunodeficiency virus type 1 by a specific secreted human phospholipase A2[END_REF]. The enzymatic activity of hGX sPLA2 was necessary for the observed antiviral effect (Figure 1).

Other endogenous sPLA2s such as hGIIA, hGIID, hGIII, hGV and hGXIIA appeared to be ineffective, yet this was not analysed using pure recombinant enzymes. The antiviral activity was proposed to be due to a direct virucidal effect, i.e. direct hydrolysis and degradation of the viral membrane [START_REF] Kim | Lysis of human immunodeficiency virus type 1 by a specific secreted human phospholipase A2[END_REF]. It was assumed that the lipid envelope of HIV-1 may be susceptible to the enzymatic action of hGX sPLA2, since it originates from the host cell membranes and is rich in its outer leaflet in zwitterionic phospholipids including phosphatidylcholine [START_REF] Aloia | Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes[END_REF], while hGX sPLA2 has been shown to hydrolyse phosphatidylcholine from the plasma membrane of live cells [START_REF] Bezzine | Exogenously added human group X secreted phospholipase A2 but not the group IB, IIA, and V enzymes efficiently release arachidonic acid from adherent mammalian cells[END_REF][START_REF] Hanasaki | Purified group X secretory phospholipase A2 induced prominent release of arachidonic acid from human myeloid leukemia cells[END_REF]. However, more detailed lipidomics studies have shown that the lipid composition of the HIV virus particles is unique and distinct from the cell plasma membrane [START_REF] Brügger | Human immunodeficiency virus type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains[END_REF][START_REF] Brügger | The HIV lipidome: a raft with an unusual composition[END_REF][START_REF] Lorizate | Comparative lipidomics analysis of HIV-1 particles and their producer cell membrane in different cell lines[END_REF], leaving unknown the enzymatic properties of this sPLA2 on the surface of HIV particles and whether it has a real capacity to directly hydrolyse and kill the virus (i.e. via a virucidal effect). Furthermore, the concentration of recombinant hGX sPLA2 that inhibits HIV-1 infection in cellular assays versus the one that would produce the direct virucidal activity were not determined. Interestingly, the antiviral activity was observed despite the resistance of virus preparations to lysis by antibody-mediated complement activation, indicating that the sPLA2 may act independently of the adaptive immune response or when the complement pathway is ineffective. As hGX sPLA2 is highly expressed in the intestinal mucosa [START_REF] Mounier | Distinct expression pattern of the full set of secreted phospholipases A2 in human colorectal adenocarcinomas: sPLA2-III as a biomarker candidate[END_REF][START_REF] Surrel | Group X phospholipase A2 stimulates the proliferation of colon cancer cells by producing various lipid mediators[END_REF], the primary site of HIV-1 replication on natural infection, the authors suggested further studies to explore the role of hGX sPLA2 in the innate immunity against HIV-1 infection in the gastrointestinal system. Application of the recombinant protein and/or upregulation of hGX sPLA2 expression may thus limit viral replication and reduce the incidence of productive replication at the primary infection sites. However, as above for other sPLA2s, the in vivo role of hGX sPLA2 against HIV-1 infection remains to be demonstrated.

In a mouse model of infection by H1N1 influenza virus, mGX sPLA2 (Pla2g10 gene) has been involved in host antiviral response, but in a negative and likely indirect manner [START_REF] Kelvin | Lack of group X secreted phospholipase A2 increases survival following pandemic H1N1 influenza infection[END_REF].

Survival after infection was greater in Pla2g10-deficient mice than in wild-type littermates. mGX sPLA2 was induced by H1N1 infection in bronchial epithelial cells and inflammatory cells, and was involved in the production of potent proinflammatory lipid mediators, likely contributing to acute lung injury. On the other hand, B and T cell antiviral responses were stronger in Pla2g10-deficient mice, suggesting that Pla2g10 promotes excessive lung inflammation and inhibits an efficient adaptive immune response, leading to poor survival. Collectively, these in vivo findings suggest that GX sPLA2 is a "proviral sPLA2" which contrasts with its above in vitro antiviral activity (Table 1). Based on data in this mouse model, GX sPLA2 may be a potential therapeutic target that should be inhibited during influenza infection. However, its in vivo antiviral or proviral role after infection with adenovirus or HIV-1 (with mouse adapted strains and related lentiviruses) remains unknown and should be tested in relevant animal models. Soon after, it has been shown that mGIID sPLA2 (Pla2g2d gene) is negatively involved in the adaptive immune response in a mouse model of severe acute respiratory syndrome coronavirus (SARS-CoV) infection [START_REF] Vijay | Critical role of phospholipase A2 group IID in age-related susceptibility to severe acute respiratory syndrome-CoV infection[END_REF]. mGIID sPLA2 is a basic protein which was cloned from spleen and other lymphoid tissues and show a modest in vitro enzymatic activity as compared to its close paralog GIIA sPLA2 [START_REF] Singer | Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2[END_REF][START_REF] Valentin | Cloning and recombinant expression of a novel mouse secreted phospholipase A2[END_REF]. However, in vivo, mGIID sPLA2 was shown to preferentially cleave phosphatidylethanolamine esterified with various polyunsaturated fatty acids at the sn-2 position [START_REF] Miki | Lymphoid tissue phospholipase A2 group IID resolves contact hypersensitivity by driving antiinflammatory lipid mediators[END_REF], releasing not only omega-6 arachidonic acid but also omega-3 eicosapentaenoic and docosahexaenoic acids, which are precursors of potent antiinflammatory lipid mediators (resolvins) involved in resolution of inflammation during infection [START_REF] Serhan | Treating inflammation and infection in the 21st century: New hints from decoding resolution mediators and mechanisms[END_REF]. In mice and humans, GIID sPLA2 is expressed mainly in dendritic cells and M2 macrophages of secondary lymphoid organs, such as spleen and lymph nodes, is downregulated by pro-inflammatory stimuli, and is likely playing an anti-inflammatory and proresolving role [START_REF] Murakami | Group IID, IIE, IIF and III secreted phospholipase A2s[END_REF]. For these reasons, it was termed a "resolving sPLA2" that ameliorates inflammation through mobilisation of pro-resolving lipid mediators [START_REF] Murakami | Group IID, IIE, IIF and III secreted phospholipase A2s[END_REF][START_REF] Miki | Lymphoid tissue phospholipase A2 group IID resolves contact hypersensitivity by driving antiinflammatory lipid mediators[END_REF]. A dual role for GIID sPLA2 as an immunosuppressive endogenous sPLA2 in inflammation and cancer was proposed in which it exerts an anti-inflammatory effect but decreases the anti-tumour effect, thereby promoting tumour development [START_REF] Miki | Dual roles of group IID phospholipase A2 in inflammation and cancer[END_REF]. An immunosuppressive function of GIID sPLA2 was proposed even earlier, when it was cloned from lymphoid tissues of lymphotoxin-deficient mice [START_REF] Shakhov | SPLASH (PLA2IID), a novel member of phospholipase A2 family, is associated with lymphotoxin deficiency[END_REF], and shown to be highly expressed in regulatory T cells and to mediate the immunosuppressive function of these cells [START_REF] Von Allmen | Secretory phospholipase A2-IID is an effector molecule of CD4 + CD25 + regulatory T cells[END_REF]. Regarding its role during viral infection, a critical role of mGIID sPLA2 in age-related susceptibility to SARS-CoV infection was observed [START_REF] Vijay | Critical role of phospholipase A2 group IID in age-related susceptibility to severe acute respiratory syndrome-CoV infection[END_REF], revealing a proviral role of this sPLA2. The authors carried out most of the experiments in mice, but the conclusions they made may be applicable to humans. In agreement with this, oxidative stress was found to induce GIID sPLA2 expression in mice and in human monocytederived macrophages. The study showed that GIID sPLA2, whose expression in lung dendritic cells increased with age in response to oxidative stress and also during coronavirus infection, contributed to worse outcome in mice infected with SARS-CoV. These results were supported by the observation that GIID sPLA2-deficient mice, in comparison with wild-type mice, showed a much higher rate of survival after infection with not only SARS-CoV but also influenza A viruses. On the contrary, the attenuated antiviral immunity in transgenic mice overexpressing GIID sPLA2 leads to severe lung inflammation and early death. The proposed molecular mechanism is that GIID sPLA2, expressed in lung dendritic cells, produces prostaglandin D2 and other lipid mediators that prevent the migration of dendritic cells to lymph nodes, thereby suppressing T-cell activation. This results in a decreased antiviral immunity and, hence, increased viral infection [START_REF] Murakami | The roles of the secreted phospholipase A2 gene family in immunology[END_REF]. Accordingly, the specific inhibition of GIID sPLA2 represents a potentially therapeutic approach for the treatment of patients with severe respiratory infection due to influenza viruses or coronaviruses [START_REF] Murakami | Group IID, IIE, IIF and III secreted phospholipase A2s[END_REF]. It has recently been reported that GIID sPLA2 deficiency in myeloid, probably dendritic cells of middle-aged mice may play a critical role in the induction of humoral immune memory against respiratory coronavirus infection [START_REF] Zheng | Phospholipase A2 group IID is indispensable for coronavirusspecific humoral immune memory in middle aged mice[END_REF].

Together, these findings on a proviral role of GIID sPLA2 during infection with coronavirus and influenza viruses are similar to those observed for GX sPLA2 and H1N1 infection, suggesting that both sPLA2s may share some common molecular mechanisms of action and may be both targeted by specific inhibitors for the treatment of infections with respiratory viruses.

Finally, recent studies suggest that hGIB sPLA2 may negatively contribute to infection by HIV-1 by an indirect mechanism leading to anergy of CD4+ T cells, which may be at the basis of the chronic immunodeficiency syndrome observed in HIV patients [START_REF] Pothlichet | PLA2G1B is involved in CD4 anergy and CD4 lymphopenia in HIV-infected patients[END_REF]. This sPLA2 may act catalytically at the surface of CD4 + T cells in synergy with enzymatic cofactors such as the HIV gp41 envelope protein or degradation products. Importantly, the in vitro effects of the sPLA2 on CD4 + T cell anergy is dependent on its enzymatic activity and can be blocked by a specific neutralizing antibody, which may represent a therapeutic molecule. However, the effect of GIB sPLA2 on T cell anergy should be further demonstrated in in vivo models of HIV infection or T cell immune dysfunction. The cellular source of hGIB sPLA2 that is found in the plasma of HIV patients also remains to be determined.

Antiviral Activity of Exogenous sPLA2s

More than two decades ago, before studies with endogenous sPLA2s on viruses, it was shown that several venom sPLA2s exhibit a potent antiviral activity against HIV-1 in vitro [START_REF] Fenard | Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells[END_REF] (Table 2 and Figure 2). In this study, the bee venom sPLA2 (bvPLA2) and three snake venom sPLA2s were found to be potent inhibitors of HIV-1 replication (IC50 < 1 nM). hGIB and hGIIA sPLA2s did not show such inhibitory effects while other human sPLA2s could not be tested in those days, because they were either unknown or not available as recombinant pure proteins [START_REF] Valentin | Increasing molecular diversity of secreted phospholipases A2 and their receptors and binding proteins[END_REF][START_REF] Valentin | What can venom phospholipases A2 tell us about the functional diversity of mammalian secreted phospholipases A2?[END_REF]. Four venom sPLA2s among 11 enzymes -taipoxin from the Australian coastal taipan (Oxyuranus scutellatus scutellatus), the basic sPLA2 (CM-III) from the Mozambique spitting cobra (Naja mossambica mossambica), nigexine from the black-necked spitting cobra (Naja nigricollis), and bvPLA2 from honey bee (Apis mellifera) -were able to protect human primary blood leukocytes from infection by various HIV-1 strains [START_REF] Fenard | Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells[END_REF]. The observed inhibition resulted neither from a direct virucidal effect nor from a cytotoxic effect on host cells. Time-of-addition experiments monitoring the antiviral effect of sPLA2 after the virus was added to host cells showed that the inhibitory sPLA2s did not interfere with physical binding of the virus to the cell surface but blocked virus entry, possibly by interfering with the complex fusion step between viral envelope and plasma membrane. Accordingly, the inhibitory sPLA2s were effective when added to cells before the virion uncoating step and dissociation of the reverse transcriptase complex from host cell membranes, but were ineffective after these steps (Figure 2). In a follow-up study, a variant of HIV-1 was selected from a mixture of HIV-1 particles present in primary HIV isolates for its resistance to bvPLA2 inhibition [103]. It turned out that this variant, HIVRBV-3, could escape from inhibition of cell entry to increasing doses of bvPLA2 by evolving or selecting a particular envelope glycoprotein mutated in the N-terminal region and the V1-V2 loop extensions [103]. These mutations allowed the HIV-1 variant to infect cells by a completely different clathrin endosomal-dependent pathway and to become resistant to inhibition by bvPLA2 by about 100-fold, when compared to the original virus. In an effort to identify the region of bvPLA2 involved in its inhibitory effect, a series of bvPLA2 peptides were screened, and an N-terminal 15-amino acid peptide was found to block entry of T-tropic HIV-1 strains into host cells, obviously without requiring enzyme catalytic activity. However, surprisingly, the mechanism of action of this peptide was different from that of the whole bvPLA2 molecule, as the peptide selectively inhibited T-but not M-tropic HIV-1 viruses by interfering with binding of HIV-1 to the CXCR4 chemokine coreceptor on target cells [START_REF] Fenard | A peptide derived from bee venom-secreted phospholipase A2 inhibits replication of T-cell tropic HIV-1 strains via interaction with the CXCR4 chemokine receptor[END_REF].

Conversely, bvPLA2 was active on both T-and M-tropic HIV-1 strains and did not bind to the CXCR4 coreceptor. Finally, additional structure-function studies using the neurotoxic snake venom sPLA2 OS2 (from Oxyuranus scutellatus scutellatus venom), which was also effective at inhibiting HIV-1, showed that the N-terminal region of OS2 and its enzymatic activity were both important to inhibit HIV-1 infection [START_REF] Rouault | Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: The N-terminal region is more important than enzymatic activity[END_REF].

During the last decade, several other studies have been carried out, showing a broad antiviral activity of different snake venom sPLA2s (Table 2 and Figure 2). From these reports, it is difficult to unequivocally conclude that enzymatic activity of an sPLA2 is absolutely necessary for the antiviral effect observed. For example, both catalytically-active (D49) and catalytically-inactive (K49) sPLA2s, isolated from the venom of the Bahia lancehead (Bothrops leucurus), seemed to show similar antiviral activity in a cellular model of infection by the dengue virus [START_REF] Cecilio | Molecular characterization of Lys49 and Asp49 phospholipases A₂ from snake venom and their antiviral activities against Dengue virus[END_REF]. However, since no dose-response curves to measure the relative IC50 values between D49 and K49 sPLA2s were performed, it is difficult to compare the relative activity of the two types of sPLA2s and exclude the possibility of a minor contamination of the K49 sPLA2 preparation with the D49 active sPLA2 purified from the same venom. Furthermore, some reports claimed that sPLA2s exert a direct virucidal activity but this was not conclusively demonstrated by either measuring the direct hydrolytic activity of the sPLA2 on viral particles or performing lipidomics analyses, or by washing out the sPLA2 after the incubation of viruses with this latter, or by addition of an sPLA2 inhibitor that would prevent the subsequent action of the sPLA2 on cells at the time of addition of the virus-sPLA2 mixture to host cells.

It was reported that crotoxin from the South American rattlesnake (Crotalus durissus terrificus) exhibits antiviral activity against dengue and yellow fever viruses [START_REF] Muller | Crotoxin and phospholipases A₂ from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses[END_REF]. In addition to preventing the cells from viral infections, the authors observed a pronounced effect of crotoxin when preincubated with the virus particles, suggesting a virucidal effect. However, the sPLA2virus mixture was not separated before addition to cells, leaving open the possibility that the sPLA2 acts in a subsequent step of infection, for instance by interfering with the cell machinery during virus binding or entry. Crotoxin B (CB), the catalytically-active subunit of heterodimeric crotoxin, appeared to lose antiviral activity when its enzymatic activity is inhibited [START_REF] Russo | Phospholipase A2 crotoxin B isolated from the venom of Crotalus durissus terrificus exert antiviral effect against dengue virus and yellow fever virus through its catalytic activity[END_REF][START_REF] Muller | Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope[END_REF]. The virucidal action of crotoxin may be the result of hydrolysis of glycerophospholipids on the virus envelope, leading to a disruption of the lipid bilayer and destabilization of envelope (E) proteins on the viral surface. Molecular dynamics simulations suggested that crotoxin may gain access to the dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices in the E protein shell on the virus surface [START_REF] Muller | Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope[END_REF]. However, definitive experimental evidence supporting this scenario is lacking. Crotoxin and its CB subunit had antiviral activity not only against Flaviviridae viruses (such as dengue, yellow fever and Zika) but also against Peribunyaviridae and Togaviridae viruses whose envelopes are different, originating from Golgi and plasma membranes, respectively. For example, two recombinant enzymatically active CB variants, CB1 and CB2, showed antiviral effects, not only against dengue, yellow fever and Zika viruses, but also against chikungunya virus (Togaviridae) [START_REF] Russo | Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom[END_REF]. In a separate study [100], crotoxin and its CB subunit were effective against in vitro infection with hepatitis C virus (Flaviviridae). The results indicated that CB blocks viral infection by action on the virus particle and host cells [100]. Thus, crotoxin and its CB subunit exert antiviral effects by possibly acting at different stages of the virus life cycle.

Mt-I, a myotoxic sPLA2 from terciopelo (Bothrops asper) snake venom was shown to exert antiviral activity against the enveloped viruses of Flaviviridae family, similar to that of crotoxin and CB [START_REF] Brenes | Potent virucidal activity against Flaviviridae of a group IIA phospholipase A2 isolated from the venom of Bothrops asper[END_REF]. Interestingly, the results showed that Mt-I, which is catalytically active (D49), was approximately 1,000-fold more potent than its catalytically inactive variant Mt-II (K49), suggesting a critical role of the sPLA2 enzymatic activity in the antiviral effect of Mt-I sPLA2.

An interesting finding resulted from the study of the antiviral action of the CM-II sPLA2 isolated from the venom of the Mozambique spitting cobra (Naja mossambica mossambica) against different types of viruses [101]. CM-II possesses potent antiviral activity (IC50 values of 0.03 to 1.3 ng/ml, i.e. less than 1 nM) against hepatitis C, dengue and Japanese encephalitis viruses, all from the Flaviviridae family, whose viral particles bud from the endoplasmic reticulum. In contrast, CM-II was virtually ineffective against viruses that bud either from the plasma membrane (Sindbis, influenza and Sendai viruses) or the trans-Golgi network (herpes simplex virus). One exception was HIV-1, which was inhibited by CM-II with an IC50 value of 5.4 ng/ml, a finding in line with the IC50 value measured for the analogous CM-III sPLA2 in an earlier study [START_REF] Fenard | Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells[END_REF]. The potent antiviral activity of CM-II against hepatitis C and dengue viruses was inhibited by the broadly specific sPLA2 inhibitor manoalide, suggesting that enzymatic activity is necessary. Thus, based on differences in the physicochemical properties of the phospholipid bilayers of viruses and host cells, broad-spectrum antiviral sPLA2s may be developed to specifically target viral envelope lipid bilayers derived from the endoplasmic reticulum without targeting the lipid bilayers of host cell membranes.

Most recently, AP-sPLA2, isolated from the crown-of-thorns starfish (Acanthaster planci) was shown to exert in vitro activity against HIV infection of peripheral blood mononuclear cells [START_REF] Wijanarko | Antiviral activity of Acanthaster planci phospholipase A2 against human immunodeficiency virus[END_REF]. The authors hypothesized but did not demonstrate that AP-sPLA2, like endogenous GX sPLA2 [START_REF] Kim | Lysis of human immunodeficiency virus type 1 by a specific secreted human phospholipase A2[END_REF], directly hydrolyses the HIV phospholipid bilayer envelope which causes the virus to become inactive.

Overall, these studies have shown that several but not all venom exogenous sPLA2s can exert potent and broadly specific antiviral effects against different viruses by multiple mechanisms, from a possible direct virucidal action to more complex interplay between sPLA2, virus and host cells, from primary binding to the plasma membrane and specific receptors to physical virus entry, membrane fusion, internalization, multiplication and budding steps. In most cases, the exact mechanism of action, which may even be miscellaneous for a single sPLA2 acting on different viruses, remain to be discovered or at least ascertained by welldesigned experiments. Furthermore, a major limitation is that all of these studies were performed only in vitro in cellular models of viral infection. Their therapeutic potential should be confirmed by performing in vivo experiments to provide a pathophysiological relevance of the findings.

Conclusion and Future Perspectives

sPLA2s constitute an important family of both enzymatically active and inactive proteins with a wide spectrum of activities whose biological functions, including antiviral action, have not been completely elucidated. Endogenous sPLA2s play an important but complex role in the innate immune system, sometimes defensive, sometimes offensive [START_REF] Murakami | Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice[END_REF][START_REF] Murakami | A new era of secreted phospholipase A₂[END_REF][START_REF] Menschikowski | Secretory phospholipase A2 of group IIA: Is it an offensive or a defensive player during atherosclerosis and other inflammatory diseases?[END_REF]. These yin-yang roles, for instance the proviral or antiviral effects of sPLA2s, sometimes observed when a particular biological system is analysed in in vitro versus in vivo conditions, and as such is difficult to understand as a whole. Furthermore, it is difficult to definitely conclude from the current studies whether a certain sPLA2 is directly or indirectly influencing the virus life cycle, from direct virucidal effect to indirect effect on the binding, entry, or even budding steps of viruses to host cells (Figures 1 and2). As a great deal of studies presented above has reported on the antiviral effects of sPLA2s by using in vitro cellular models of viral infections, there is a need to perform the corresponding in vivo experiments. Only in vivo results will expose the real antiviral potential of sPLA2s, acting either as therapeutic molecules or as "offensive" molecules whose activity would need to be hampered by active site-specific inhibitors or inhibitory antibodies. Most intensively, efforts should be concentrated on the most potent sPLA2 representatives acting on the virus life cycle, such as GIII, GV and GX sPLA2s, which may act on both host cell membranes and the viral envelope that has a lipid membrane derived from host cells, yet with subtle differences in their membrane lipid composition and biophysics. sPLA2s may also play an indirect role in the antiviral adaptive immune response where, interestingly, some sPLA2s like GX but also GIID sPLA2 have a negative (proviral) effect on the viral infection by H1N1 and SARS-coronavirus adapted to mouse strains [START_REF] Kelvin | Lack of group X secreted phospholipase A2 increases survival following pandemic H1N1 influenza infection[END_REF][START_REF] Vijay | Critical role of phospholipase A2 group IID in age-related susceptibility to severe acute respiratory syndrome-CoV infection[END_REF]. In addition, it has been recently shown that GIB sPLA2 is a new player in lentiviral infections, where it may exert a proviral role by inducing CD4+ T cell anergy and thereby participate to the pathophysiology of HIV infection [START_REF] Pothlichet | PLA2G1B is involved in CD4 anergy and CD4 lymphopenia in HIV-infected patients[END_REF]. Here, the specific or combined inhibition of involved sPLA2s may be beneficial to prevent viral infection. Exogenous sPLA2s, mostly but not exclusively of snake venom origin, also display antiviral effect that may be mediated by their enzymatic activity to either directly attack the host-derived membranes of certain enveloped viruses or interfere with the viral cell cycle at different steps. Whether these sPLA2s may be used as antiviral agents in vivo remains to be demonstrated, considering of course their toxicities.

Future research on the antiviral potential of sPLA2s may thus proceed in four major directions. ( 1 Table 1. Key biological roles of endogenous mammalian sPLA2s, including their potential antiviral and/or proviral biological roles (underlined). The table is largely summarized from [START_REF] Murakami | A new era of secreted phospholipase A₂[END_REF][START_REF] Murakami | Group IID, IIE, IIF and III secreted phospholipase A2s[END_REF]; with additional information on GIB [START_REF] Pothlichet | PLA2G1B is involved in CD4 anergy and CD4 lymphopenia in HIV-infected patients[END_REF][START_REF] Entwistle | Epithelial-cell-derived phospholipase A2 group 1B is an endogenous anthelmintic[END_REF], GIII [START_REF] Mitsuishi | Human group III phospholipase A2 suppresses adenovirus infection into host cells. Evidence that group III, V and X phospholipase A2s act on distinct cellular phospholipid molecular species[END_REF], GV [START_REF] Mitsuishi | Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells[END_REF][START_REF] Mitsuishi | Human group III phospholipase A2 suppresses adenovirus infection into host cells. Evidence that group III, V and X phospholipase A2s act on distinct cellular phospholipid molecular species[END_REF], GX [START_REF] Mitsuishi | Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells[END_REF][START_REF] Mitsuishi | Human group III phospholipase A2 suppresses adenovirus infection into host cells. Evidence that group III, V and X phospholipase A2s act on distinct cellular phospholipid molecular species[END_REF][START_REF] Kelvin | Lack of group X secreted phospholipase A2 increases survival following pandemic H1N1 influenza infection[END_REF], GXIIA [START_REF] Muñoz-Sanjuan | Induction of ectopic olfactory structures and bone morphogenetic protein inhibition by Rossy, a group XII secreted phospholipase A2[END_REF][START_REF] Huhtinen | Antibacterial effects of human group IIA and group XIIA phospholipase A2 against Helicobacter pylori in vitro[END_REF][START_REF] Ee | Distribution of secretory phospholipase A2 XIIA in the brain and its role in lipid metabolism and cognition[END_REF][START_REF] Peuravuori | Expression of group XIIA phospholipase A2 in human digestive organs[END_REF] and otoconin-90 [START_REF] Zhao | Otoconin-90 deletion leads to imbalance but normal hearing: a comparison with other otoconia mutants[END_REF][START_REF] Wang | Otoconin-90, the mammalian otoconial matrix protein, contains two domains of homology to secretory phospholipase A2[END_REF][START_REF] Verpy | Characterization of otoconin-95, the major protein of murine otoconia, provides insights into the formation of these inner ear biominerals[END_REF]. [START_REF] Dennis | Phospholipase A2 enzymes: Physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention[END_REF]. Heterodimeric crotoxin is composed of a basic GIIA sPLA2 (CB) and an acidic (CA) enzymatically-inactive subunit of three peptides derived from a GIIA sPLA2 precursor. Taipoxin is a heterotrimeric protein whose α and β subunits are GIA sPLA2s, and with the γ subunit similar to a GIB pro-sPLA2. D49 and K49 denotes enzymatically-active and -inactive sPLA2 variants, respectively, due to a substitution of the conserved aspartic acid residue position 49 in the Calcium-binding loop (binding of the Ca 2+ cofactor is needed for catalytic activity) of enzymatically-active sPLA2s with a lysine residue. Legend to Figures from Bothrops asper snake venom. Other abbreviations used are the same as those in Figure 1 and Table 2.

Endogenous

  ) Confirmation or disproval of the promising in vitro antiviral effects of certain venom and endogenous sPLA2s by conducting appropriate in vivo studies. (2) Description of the exact molecular mechanism(s) of direct or indirect antiviral activity of the various sPLA2s, as a starting point for development of new antiviral sPLA2-based therapies. This would also include unravelling of the indirect mechanisms by which GX and GIID sPLA2s (or possibly other isoforms) exacerbate H1N1 and coronavirus infections to pave the way for new therapeutic avenues. (3) Further description of the broadly specific antiviral role of endogenous and exogenous sPLA2s against various types of viruses, up to infection by SARS-CoV-2 virus, the etiological agent of the Covid-19 pandemic. (4) Exploration of the possibility that various sPLA2s such as GX or GIID may represent new biomarkers of severity in viral infections, as recently shown for the inflammatory-type GIIA sPLA2 in Covid-19 patients with severe disease [105].

Figure 1 .

 1 Figure 1. The antiviral action of endogenous sPLA2s in in vitro cell systems. Some sPLA2s, for example human, may act on viruses either directly, by degrading their lipid envelope (virucidal effect?), such as in the case of HIV-1, or indirectly, by acting on cell lipid membranes to prevent effective production of viral particles, such as in the case of non-enveloped adenoviruses. Via enzymatic activity, sPLA2s hydrolyze phospholipids (PL) from the virus or infected cellular membranes to produce lysophospholipids (LPL) and free fatty acids (FA) that can disturb some key steps of the viral life cycle or lipid metabolism of the host cell. The enveloped virus (e.g. HIV-1), with a lipid membrane derived from host plasma membrane, and the non-enveloped (no lipid) virus (e.g. adenovirus) are shown in violet and blue, respectively. The dashed line separates direct versus indirect sPLA2 actions on cells. CAR, coxsackievirus and adenovirus receptor.

Figure 2 .

 2 Figure 2. The antiviral action of exogenous sPLA2s in in vitro cell systems. Direct action: certain venom sPLA2s may exert an antiviral activity by direct hydrolysis of the lipid viral envelope (virucidal effect?). The surface of various enveloped viruses, with the lipid membrane budding from different host cell membranes, is shown by different colours (orange, originating from the endoplasmic reticulum; blue-green, from the Golgi apparatus; green, from the plasma membrane; violet, from the plasma membrane (e.g. HIV-1) or endoplasmic reticulum). Indirect action: venom sPLA2s may exert antiviral activity by binding and hydrolysis of host cell membranes, thereby inhibiting one or several of the first steps of viral infection: from binding of viruses to entry or virus-driven lipid metabolism. The dashed line separates both direct and indirect sPLA2 actions on cells. A red curved arrow denotes the unsuccessful attack of a virus on the host cell as a result of the sPLA2 action. AP-sPLA2, a catalytically-active sPLA2 from Acanthaster planci starfish; CB, crotoxin B subunit; CM-II and CM-III, catalytically-active sPLA2s from Naja mossambica mossambica snake venom; Mt-I, a catalytically-active sPLA2

Table 2 .

 2 Antiviral activity of exogenous venom sPLA2s. Most of them are monomeric sPLA2s homologous to mammalian endogenous sPLA2s. They belong to groups IA, IB, IIA or III according to the historical classification of sPLA2s

		Tissue and cell distribution	Physiological role	Pathophysiological role
	mammalian
	sPLA2
	GIB	pancreatic acinar cells, duodenum,	digestion of dietary phospholipids in the	obesity, lung inflammation, anergy of
		ileum, stomach, lung, blood,	small intestine, immune response,	T cells in HIV infection (i.e. proviral
		spermatozoa		role), anthelmintic
	GIIA	intestine, Paneth cells, platelets,	regulation of intestinal microflora,	amplification of inflammation in many
		leukocytes, synoviocytes, skin,	inflammation and lipid metabolism, role in	inflammatory diseases and
		biological fluids (blood, tears,	host defence and in innate and immune	cardiovascular diseases, role in
		seminal and synovial fluid, lung	responses, direct killing of Gram-positive	sepsis and various bacterial and viral
		alveolar space)	bacteria and of commensal bacteria	infections, role in cancer and
				metastasis
	GIID	immune system, lymphoid and	anti-inflammatory, resolution of Th1	decrease of immune response, anti-
		dendritic cells, M2 macrophages,	immunity, candidate as an immune	tumour response, antiviral immunity in
		regulatory T cells	checkpoint	H1N1 and SARS-CoV infections (i.e.
				proviral role)
	GIIE	adipocytes, hair follicles, uterus	fat deposition in adipose tissue and liver,	obesity (age-induced anti-obesity?),
			hair follicle homeostasis, lipid metabolism	skin disorders
	GIIF	skin, suprabasal keratinocytes	skin homeostasis, lipid metabolism	skin disorders such as cancer and
				psoriasis
	GIII	intestine, liver, epididymal epithelial	male fertility, mast cell maturation	anaphylaxis, colon cancer and colitis,
		cells, mast cells, colonic epithelial		atherosclerosis, skin inflammation,
		cells, aorta, skin, brain		antiviral (adenovirus) role
	GV	bronchial epithelial cells, endothelial	host defence, surfactant degradation, M2	airway lung injury, asthma,
		cells, macrophages, dendritic cells,	macrophage polarization, Th2 immunity,	atherosclerosis, aortic inflammation,
		hematopoietic cells,	phagocytosis of microorganisms,	myocardial infarction, aneurysm,
		cardiomyocytes, aorta, hypertrophic	phagocytosis of immune complexes,	antiviral (adenovirus) role
		adipocytes, pancreatic β-cells, gut	apoptosis of injured myocardial cells,
		epithelium, brain	reduced adipose tissue inflammation by
			unsaturated fatty acids, anti-arthritis, anti-
			obesity
	GX	airway epithelium, intestinal	enhanced lipid accumulation and TLR4	airway inflammation, asthma,
		mucosa, stomach, immune system,	signalling in macrophages, reduced	decreased tissue damage by
		macrophages, adrenal glands,	corticosteroid synthesis by downregulating	neutrophils, hypercorticosteronemia,
		dorsal root ganglia, hematopoietic	adrenal steroidogenic acute regulatory	pain, aneurysm, myocardial infarction,
		cells, neutrophils, adipocytes, sperm	protein, neuritogenesis and pain	diabetes, adiposity, alopecia, antiviral
		acrosome, hair follicles	transmission, macrophage function,	(adenovirus) role, antiviral immunity in
			reduced Th1 immunity and atherosclerotic	H1N1 infection (i.e. proviral role)
			plaque formation, suppression of insulin
			secretion, phospholipid digestion in the
			gastrointestinal lumen, repression of
			adipogenesis by inhibiting liver X receptor
			activation, anti-atherosclerosis, anti-obesity,
			sperm fertility, hair homeostasis
	GXIIA	ubiquitous, neurons, epithelial cells	neurogenesis and cognition, host defence,	lipid metabolism disorders?
		of gastrointestinal tract	killing of Gram-positive bacteria in vitro,
			poorly understood molecular mechanisms
	GXIIB	liver, intestine	triglyceride metabolism by unknown	lipid metabolism disorders?
			molecular mechanisms, anti-steatosis effect
	otoconin-90	inner ear	maintainance of inner ear balance system	imbalance

Acknowledgments

The authors sincerely thank Franck Aguila for expertise in graphic art and help in designing figures, and Dr. Roger H. Pain for critical reading of the manuscript draft. This work was supported by the Slovenian Research Agency grant P1-0207 to I.K., and by funding from the Centre National de la Recherche Scientifique (CNRS) to G.L.