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International audience; A naturally inspired chemical library of 25 molecules was synthesised guided by 3-D dimensionality and natural product likeness proved to have antiviral activity against SARS-CoV-2.

International audience; Highlights • Viral RNA genomes are modified by epitranscriptomic marks, such as 2′-Omethylation. • 2′-O-methylations are self-markers hijacked by viruses to avoid immune sensing. • Modified viral RNA counteract restriction by interferon-stimulated genes. • New insights suggest that 2′-O-methylations also impair viral replication.

International audience; The COVID-19 pandemic reveals the urgent need to develop new therapeutics targeting the SARS-CoV-2 replication machinery. The first antiviral drugs were nucleoside analogues targeting RdRp and protease inhibitors active on nsp5 Mpro. In addition to these common antiviral targets, SARS-CoV-2 codes for the highly conserved protein nsp14 harbouring N7methyltransferase (MTase) activity. Nsp14 is involved in cap N7-methylation of viral RNA and its inhibition impairs viral RNA translation and immune evasion, making it an attractive new antiviral target. In this work, we followed a structure-guided drug design approach to design bisubstrates mimicking the S-adenosylmethionine methyl donor and RNA cap. We developed adenosine mimetics with an N-arylsulfonamide moiety in the 5'-position, recently described as a guanine mimicking the cap structure in a potent adenosine-derived nsp14 inhibitor. Here, the adenine moiety was replaced by hypoxanthine, N 6-methyladenine, or C7-substituted 7-deazaadenine. 26 novel adenosine mimetics were synthesized, one of which selectively inhibits nsp14 N7-MTase activity with a subnanomolar IC50 (and seven with a single-digit nanomolar IC50). In the most potent inhibitors, adenine was replaced by two different 7-deaza-adenines bearing either a phenyl or a 3-quinoline group at the C7-position via an ethynyl linker. These more complex compounds are barely active on the cognate human N7-MTase and docking experiments reveal that their selectivity of inhibition might result from the positioning of their C7 substitution in a SAM entry tunnel present in the nsp14 structure and absent in the hN7-MTase. These compounds show moderate antiviral activity against SARS-CoV-2 replication in cell culture, suggesting delivery or stability issue.

International audience; N-Acylsulfonamides possess an additional carbonyl function compared to their sulfonamide analogues. Due to their unique physico-chemical properties, interest in molecules containing the N-acylsulfonamide moiety and especially nucleoside derivatives is growing in the field of medicinal chemistry. The recent renewal of interest in antiviral drugs derived from nucleosides containing a sulfonamide function has led us to evaluate the therapeutic potential of N-acylsulfonamide analogues. While these compounds are usually obtained by a difficult acylation of sulfonamides, we report here the easy and efficient synthesis of 20 4′-(N-acylsulfonamide) adenosine derivatives via the sulfo-click reaction. The target compounds were obtained from thioacid and sulfonyl azide synthons in excellent yields and were evaluated as potential inhibitors of the SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14.

International audience; Viral exoribonucleases are uncommon in the world of RNA viruses. To date, they have only been identified in the Arenaviridae and the Coronaviridae families. The exoribonucleases of these viruses play a crucial role in the pathogenicity and interplay with host innate immune response. Moreover, coronaviruses exoribonuclease is also involved in a proofreading mechanism ensuring the genetic stability of the viral genome. Because of their key roles in virus life cycle, they constitute attractive target for drug design.Here we developed a sensitive, robust and reliable fluorescence polarization assay to measure the exoribonuclease activity and its inhibition in vitro. The effectiveness of the method was validated on three different viral exoribonucleases, including SARS-CoV-2, Lymphocytic Choriomeningitis and Machupo viruses. We performed a screening of a focused library consisting of 113 metal chelators. Hit compounds were recovered with an IC50 at micromolar level. We confirmed 3 hits in SARS-CoV-2 infected Vero-E6 cells.

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (P

International audience; AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2 '-methyl-2 '-fluoro guanosine 5 '-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2 '-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 angstrom resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2 '-O but not N7-methylation activity. AT-9010 is discriminated-10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibi-tion through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 approximate to 0.50 mu M), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

International audience

International audience; Enzymes involved in RNA capping of SARS-CoV-2 are essential for the stability of viral RNA, translation of mRNAs, and virus evasion from innate immunity, making them attractive targets for antiviral agents. In this work, we focused on the design and synthesis of nucleoside-derived inhibitors against the SARS-CoV-2 nsp14 (N7-guanine)-methyltransferase (N7-MTase) that catalyzes the transfer of the methyl group from the S-adenosyl-L-methionine (SAM) cofactor to the N7-guanosine cap. Seven compounds out of 39 SAM analogues showed remarkable double-digit nanomolar inhibitory activity against the N7-MTase nsp14. Molecular docking supported the structure−activity relationships of these inhibitors and a bisubstrate-based mechanism of action. The three most potent inhibitors significantly stabilized nsp14 (ΔT m ≈ 11 °C), and the best inhibitor demonstrated high selectivity for nsp14 over human RNA N7-MTase.

International audience; The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. The “priming” of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2′ as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2′ cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

The worldwide circulation of different viruses coupled with the increased frequency and diversity of new outbreaks, strongly highlight the need for new antiviral drugs to quickly react against potential pandemic pathogens. Broad-spectrum antiviral agents (BSAAs) represent the ideal option for a prompt response against multiple viruses, new and re-emerging. Starting from previously identified anti-flavivirus hits, we report herein the identification of promising BSAAs by submitting the multi-target 2,6-diaminopurine chemotype to a system-oriented optimization based on phenotypic screening on cell cultures infected with different viruses. Among the synthesized compounds, 6i showed low micromolar potency against Dengue, Zika, West Nile and Influenza A viruses (IC50 = 0.5–5.3 μM) with high selectivity index. Interestingly, 6i also inhibited SARS-CoV-2 replication in different cell lines, with higher potency on Calu-3 cells that better mimic the SARS-CoV-2 infection in vivo (IC50 = 0.5 μM, SI = 240). The multi-target effect of 6i on flavivirus replication was also analyzed in whole cell studies (in vitro selection and immunofluorescence) and against isolated host/viral targets., Graphical abstract Image 1

The enterovirus genus encompasses many clinically important human pathogens such as poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. These viruses are the etiological agents of several human diseases, including hand-foot-and-mouth disease, neonatal sepsis, encephalitis, meningitis, paralysis and respiratory infections. There is an unmet need for antivirals to treat these diseases. The non-structural protein 2C is a AAA+ helicase and plays a key role in viral replication. As such, it is an attractive target for antiviral drug development. Several repurposing screens with FDA-approved drugs have identified 2C-targeting compounds such as fluoxetine and dibucaine, but the molecular basis of 2C inhibition has remained enigmatic. Here we present the 1.5 Å resolution crystal structure of the soluble fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), which reveals a conserved, hydrophobic drug-binding pocket which is distal to the ATP binding site. To decipher the molecular mechanism of inhibition by fluoxetine and other 2C-targeting compounds, we engineered a soluble, hexameric and ATPase competent 2C protein. Using this system, we show that SFX, dibucaine, HBB and guanidine hydrochloride inhibit 2C ATPase activity in a dose-dependent manner. Moreover, using cryo-EM analysis, we demonstrate that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition and allowing us to generate the first reconstruction of the oligomeric complex. Taken together, these results provide important structural and mechanistic insights into 2C inhibition and provide a robust engineering strategy which can be used for structural, functional and drug-screening analysis of 2C proteins from current or future enteroviruses.

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their mRNAs, protect them from degradation by cellular 5’ exoribonucleases, and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bi-functional replicase subunit harboring an N-terminal 3′-to-5′ exoribonuclease (ExoN) domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14’s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform anin silicocomparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome-CoV, site-directed mutagenesis of selected residues was used to assess their importance forin vitroenzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potentialpan-coronaviral activity spectrum.Significance StatementThe ongoing SARS-CoV-2 pandemic emphasizes the urgent need to develop efficient broad-spectrum anti-CoV drugs. The structure-function characterization of conserved CoV replicative enzymes is key to identifying the most suitable drug targets. Using a multidisciplinary comparative approach and different betacoronaviruses, we characterized the key conserved residues of the nsp14 (N7-guanine)-methyltransferase, a poorly defined subunit of the CoV mRNA-synthesizing machinery. Our study highlights the unique structural features of this enzyme and establishes its essential role in betacoronavirus replication, while identifying two residues that are critical for the replication of the four betacoronaviruses tested, including SARS-CoV-2.

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, this activity has been identified only in the Arenaviridae and the Coronaviridae families. These exoribonucleases play important but different roles in both families: for mammarenaviruses the exoribonuclease is involved in the suppression of the host immune response whereas for coronaviruses, exoribonuclease is both involved in a proofreading mechanism ensuring the genetic stability of viral genomes and participating to evasion of the host innate immunity. Because of their key roles, they constitute attractive targets for drug development. Here we present a high-throughput assay using fluorescence polarization to assess the viral exoribonuclease activity and its inhibition. We validate the assay using three different viral enzymes from SARS-CoV-2, lymphocytic choriomeningitis and Machupo viruses. The method is sensitive, robust, amenable to miniaturization (384 well plates) and allowed us to validate the proof-of-concept of the assay by screening a small focused compounds library (23 metal chelators). We also determined the IC50 of one inhibitor common to the three viruses.HighlightsArenaviridae and Coronaviridae viral families share an exoribonuclease activity of common evolutionary originArenaviridae and Coronaviridae exoribonuclease is an attractive target for drug developmentWe present a high-throughput assay in 384 well-plates for the screening of inhibitors using fluorescence polarizationWe validated the assay by screening of a focused library of 23 metal chelators against SARS-CoV-2, Lymphocytic Choriomeningitis virus and Machupo virus exoribonucleasesWe determined the IC50 by fluorescence polarization of one inhibitor common to the three viruses.

A worldwide effort is ongoing to discover drugs against the Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2), which has so far caused >3.5 million fatalities (https://covid19.who.int/). The virus essential RNA-dependent RNA polymerase complex is targeted by several nucleoside/tide analogues whose mechanisms of action and clinical potential are currently evaluated. The guanosine analogue AT-527, a double prodrug of its 5'-triphosphate AT-9010, is currently in phase III clinical trials as a COVID19 treatment. Here we report the cryo-EM structure at 2.98 Å resolution of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 complex with RNA showing AT-9010 bound at three sites of nsp12. At the RdRp active-site, one AT-9010 is incorporated into the RNA product. Its 2'-methyl group prevents correct alignment of a second AT-9010 occupying the incoming NTP pocket. The 2'-F, 2'-methyl 3'-OH ribose scaffold explains the non-obligate RNA chain-termination potency of this NA series for both HCV NS5 and SARS-CoV RTCs. A third AT-9010 molecule 5'-diphosphate binds to a coronavirus-specific pocket in the nsp12 N-terminus NiRAN domain, a SelO pseudo-kinase structural and functional homologue. This unique binding mode impedes NiRAN-mediated UMPylation of SARS-CoV-2 nsp8 and nsp9 proteins. Our results suggest a mechanism of action for AT-527 in line with a therapeutic use for COVID19.

Venezuelan equine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5′ end. The capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The capping reaction starts with the methylation of GTP. The generated m(7)GTP is complexed to the enzyme to form an m(7)GMP-nsP1 covalent intermediate. The m(7)GMP is then transferred onto the 5′-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m(7)GMP to yield m(7)GpppA, m(7)GpppC, or m(7)GpppU. We show that capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the capping process. IMPORTANCE The emergence, reemergence, and expansion of alphaviruses (genus of the family Togaviridae) are a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date, no efficient vaccine or safe antivirals are available for human use. VEEV nonstructural protein 1 (nsP1) is the viral capping enzyme characteristic of the Alphavirus genus. nsP1 catalyzes methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.

The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic "priming" at PRRAR685{downarrow} into S1 and S2 (cleavage at S1/S2), and "fusion-activation" at KPSKR815{downarrow} (cleavage at S2) for viral entry. Both cleavages occur at Furin-like motifs suggesting that proprotein convertases might promote virus entry. In vitro Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than S2, whereas TMPRSS2 cleaved at both sites. In HeLa cells endogenous Furin-like enzymes cleave mainly at S1/S2 during intracellular protein trafficking, as confirmed by mutagenesis. We also mapped the S2 cleavage site by proteomics and further showed that S2-processing by Furin, while limited, was strongly enhanced in the presence of ACE2. In contrast, the S2 KRRKR815{downarrow} mutant (S2) was considerably better cleaved by Furin, whereas individual/double KR815AA mutants are retained in the endoplasmic reticulum (ER). Pharmacological inhibitors of convertases (Boston Pharmaceuticals - BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. However, under co-expression the S-protein was prematurely cleaved by TMPRSS2 into ER-retained, non-O-glycosylated S2 and S2 products. Quantitative analysis of cell-to-cell fusion and Spike processing using Hela cells revealed the key importance of the Furin sites for syncytia formation and unveiled the enhanced fusogenic potential of the - and {delta}-variants of the S-protein of SARS-CoV-2. Our fusion assay indicated that TMPRSS2 enhances S2 formation, especially in the absence of Furin cleavage, as well as ACE2 shedding. Furthermore, we provide evidence using pseudoparticles that while entry by a "pH-dependent" endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a "pH-independent" viral entry in lung-derived Calu-3 cells was sensitive to inhibitors of Furin and TMPRSS2. Consistently, in Calu-3 cells BOS- inhibitors or Camostat potently reduce infectious viral titer and cytopathic effects and this outcome was enhanced when both compounds were combined. Overall, our results show that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and promote viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. Author SummarySARS-CoV-2 is the etiological agent of the COVID-19 pandemic that spread around the world in 2020 resulting in [~]4 million deaths. The surface spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S-protein needs to be cleaved at two sites: S1/S2 and S2. Cleavage at S1/S2 induces a conformational change in the spike protein favouring the recognition of the ACE2 receptor. The S2 cleavage is critical for the exposure of the fusion domain of the S-protein needed for virus entry into host cells. Several new S-variants contain mutations in the S1/S2 cleavage site possibly enhancing the S-protein processing, especially for the {delta}-variant, which may explain the higher transmissibility of this virus worldwide. Our study contributes to a better understanding of the dynamics of interaction between Furin and TMPRSS2 in SARS-CoV-2 entry and suggest that the combination of a non-toxic Furin inhibitor with that of TMPRSS2 could significantly reduce viral entry in lung cells, as evidenced by our results showing an average synergistic [~]95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

International audience; With sizes

Viral RNA 2'-O-methyltransferases play a crucial role for luring the host cell innate antiviral response during a viral infection by catalyzing either the methylation of the 5'-end RNA cap-structure at 2'-OH of nucleoside N1 or by inducing internal 2'-O-methylation of adenosines within RNA sequence using S-adenosyl-L-methionine (SAM) as the methyl donor. Our goal is to synthetized bisubstrate SAM analogues mimicking the transition state of the 2'-O-methylation of the RNA in order to block viral 2'-O-methyltransferases and struggle against emerging viruses. Here we designed and synthesized five dinucleosides by connecting a 5'-thioadenosine representing the SAM to the 2'-OH of another adenosine unit mimicking the RNA substrate, via various sized sulfur-containing linkers such as alkylthioether linkers, sulfoxide or sulfone derivatives, or a disulfide bond.

SummaryHow viruses from the Coronaviridae family initiate viral RNA synthesis is unknown. Here we show that the SARS-CoV-1 and −2 Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domain on nsp12 uridylates the viral cofactor nsp8, forming a UMP-Nsp8 covalent intermediate that subsequently primes RNA synthesis from a poly(A) template; a protein-priming mechanism reminiscent of Picornaviridae enzymes. In parallel, the RdRp active site of nsp12 synthesizes a pppGpU primer, which primes (-)ssRNA synthesis at the precise genome-poly(A) junction. The guanosine analogue 5’-triphosphate AT-9010 (prodrug: AT-527) tightly binds to the NiRAN and inhibits both nsp8-labeling and the initiation of RNA synthesis. A 2.98 Å resolution Cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 /RNA/NTP quaternary complex shows AT-9010 simultaneously binds to both NiRAN and RdRp active site of nsp12, blocking their respective activities. AT-527 is currently in phase II clinical trials, and is a potent inhibitor of SARS-CoV-1 and −2, representing a promising drug for COVID-19 treatment.

International audience; Eukaryotic nuclear and mitochondrial RNA polymerases (RNAPs) can cap RNAs by initiating with the non-canonical initiating nucleotide (NCIN) nicotinamide adenine dinucleotide (both NAD+ and NADH). Similarly, recently Dengue viral RNAs (vRNA) have been observed with NCIN caps, but how these vRNAs are capped is unknown. Here we show directly that Dengue virus's (DENV) RNAP NS5 can utilize both NAD+ and NADH, as well as flavin adenine dinucleotide and dephospho-coenzyme A, as initiating nucleotides during RNA transcription in place of DENV's well-conserved initiating adenine. In addition, we demonstrate that NCIN-capped dsRNAs can bind to the innate immune receptor RIG-I with KD's between 3 and 6 nM. RIG-I can also use these NCIN-capped dsRNAs to initiate robust interferon production. Taken with the previously published results that NAD+-capped mRNAs cannot be translated, we propose that NCIN-capped DENV vRNA represents a newly discovered mechanism of metabolite-mediated immunity that generates translation-deficient, highly immunogenic vRNAs.

En accès libre; International audience; Studies have shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can appear in feces within three days of infection, which is much sooner than the time taken for people to develop symptoms and get an official diagnosis (Mallapaty 2020). Therefore, earlier identification in wastewater of the virus’s arrival in a community might limit the health and economic damage caused by the coronavirus disease 2019 (COVID-19). Despite centuries of technological advances, the COVID-19 pandemic is highlighting the limitations of our ability to control viral spread and infections worldwide. This knowledge gap has induced highly variable and sometimes contradictory government decisions in many countries, such as whether to wear a mask or not, and various strategies for testing, contact tracing and isolation of infected patients. The failure of control strategies has led many countries to lockdowns. In particular, powerful and rapid analytical techniques are actually still missing because virus detection is a key step in controlling viral transmission. The biomarker approach, which was initially developed in geochemistry to measure the maturity of dead organic matterand to link molecular fossils with biological homologs, has been applied to modern samples such as soils, sediments, wastewaters and plants (Albrecht and Ourisson 1971; Lichtfouse et al. 1994 1997; Bryselbout et al. 1998; Payet et al. 1999; Dsikowitzky and Schwarzbauer 2014; Choi et al. 2020). Here, we discuss the emergence of wastewater epidemiology based on ribonucleic acid (RNA) biomarkers, a discipline linking biomedical and environmental sciences for the detection of pathogens, with focus on analytical challenges.

SARS-CoV-2 is a new human coronavirus (CoV), which emerged in China in late 2019 and is responsible for the global COVID-19 pandemic that caused more than 97 million infections and 2 million deaths in 12 months. Understanding the origin of this virus is an important issue, and it is necessary to determine the mechanisms of viral dissemination in order to contain future epidemics. Based on phylogenetic inferences, sequence analysis and structure–function relationships of coronavirus proteins, informed by the knowledge currently available on the virus, we discuss the different scenarios on the origin—natural or synthetic—of the virus. The data currently available are not sufficient to firmly assert whether SARS-CoV2 results from a zoonotic emergence or from an accidental escape of a laboratory strain. This question needs to be solved because it has important consequences on the risk/benefit balance of our interactions with ecosystems, on intensive breeding of wild and domestic animals, on some laboratory practices and on scientific policy and biosafety regulations. Regardless of COVID-19 origin, studying the evolution of the molecular mechanisms involved in the emergence of pandemic viruses is essential to develop therapeutic and vaccine strategies and to prevent future zoonoses. This article is a translation and update of a French article published in Médecine/Sciences, August/September 2020 (10.1051/medsci/2020123).

The Ebola virus is a deadly human pathogen responsible for several outbreaks in Africa. Its genome encodes the ‘large’ L protein, an essential enzyme that has polymerase, capping and methyltransferase activities. The methyltransferase activity leads to RNA co-transcriptional modifications at the N7 position of the cap structure and at the 2′-O position of the first transcribed nucleotide. Unlike other Mononegavirales viruses, the Ebola virus methyltransferase also catalyses 2′-O-methylation of adenosines located within the RNA sequences. Herein, we report the crystal structure at 1.8 Å resolution of the Ebola virus methyltransferase domain bound to a fragment of a camelid single-chain antibody. We identified structural determinants and key amino acids specifically involved in the internal adenosine-2′-O-methylation from cap-related methylations. These results provide the first high resolution structure of an ebolavirus L protein domain, and the framework to investigate the effects of epitranscriptomic modifications and to design possible antiviral drugs against the Filoviridae family.

The spîke (S)-protein of SARS-CoV-2 binds ACE2 and requires proteolytic “priming” at PRRAR685↓ into S1 and S2 (cleavage at S1/S2), and “fusion-activation” at a S2’ site for viral entry. In vitro, Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than at the putative S2’, whereas TMPRSS2 inefficiently cleaved both sites. In HeLa cells Furin-like enzymes mainly cleaved at S1/S2 during intracellular protein trafficking, and S2’ processing by Furin at KPSKR815↓ was strongly enhanced by ACE2, but not for the optimized S2’ KRRKR815↓ mutant (μS2’), whereas individual/double KR815AA mutants were retained in the endoplasmic reticulum. Pharmacological Furin-inhibitors (Boston Pharmaceuticals, BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. Furthermore, we show using pseudotyped viruses that while entry by a “pH-dependent” endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a “pH-independent” viral entry in lung-derived Calu-3 cells was sensitive to inhibitors of Furin (BOS) and TMPRSS2 (Camostat). Consistently, these inhibitors potently reduce infectious viral titer and cytopathic effects, an outcome enhanced when both compounds were combined. Quantitative analyses of cell-to-cell fusion and spîke processing revealed the key importance of the Furin sites for syncytia formation. Our assays showed that TMPRSS2 enhances fusion and proteolysis at S2’ in the absence of cleavage at S1/S2, an effect that is linked to ACE2 shedding by TMPRSS2. Overall, our results indicate that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and in promoting viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.IMPORTANCESARS-CoV-2 is the etiological agent of COVID-19 that resulted in >5 million deaths. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S-protein is cleaved at two sites: S1/S2 and S2’. Cleavage at S1/S2, induces a conformational change favoring the recognition of ACE2. The S2’ cleavage is critical for cell-to-cell fusion and virus entry into host cells. Our study contributes to a better understanding of the dynamics of interaction between Furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a non-toxic Furin inhibitor with a TMPRSS2 inhibitor could significantly reduce viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. Here we show that Favipiravir exerts an antiviral effect as a nucleotide analogue through a combination of chain termination, slowed RNA synthesis and lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.

In December 2019, a new coronavirus was identified in the Hubei province of central china and named SRAS-CoV-2. This new virus induces COVID-19, a severe respiratory disease with high death rate. The spike protein (S) of SARS-CoV-2 contains furin-like cleavage sites absent the other SARS-like viruses. The viral infection requires the priming or cleavage of the S protein and such processing seems essential for virus entry into the host cells. Furin is highly expressed in the lung tissue and the expression is further increased in lung cancer, suggesting the exploitation of this mechanism by the virus to mediate enhanced virulence as shown by the higher risk of COVID-19 in these patients. In this study, we used structure- based virtual screening and a collection of about 8,000 unique approved and investigational drugs suitable for docking to search for molecules that could inhibits furin activity. Sulconazole, a broad-spectrum anti-fungal agent, was found to be of potential interest. Using Western blot analysis, Sulconazole was found to inhibit the cleavage of the cell surface furin substrate MT1-MMP that contains two furin cleavage sites similar to those of the SARS- CoV-2 spike protein. Sulconazole and analogs could be interesting for repurposing studies and to probe the yet not fully understood molecular mechanisms involved in cell entry.

International audience; Alphaviruses are arthropod-borne viruses of public health concern. To date no efficient vaccine nor antivirals are available for safe human use. During viral replication the nonstructural protein 1 (nsP1) catalyzes capping of genomic and subgenomic RNAs. The capping reaction is unique to the Alphavirus genus. The whole three-step process follows a particular order: (i) transfer of a methyl group from S-adenosyl methionine (SAM) onto a GTP forming m7GTP; (ii) guanylylation of the enzyme to form a m7GMP-nsP1adduct; (iii) transfer of m7GMP onto 5′-diphosphate RNA to yield capped RNA. Specificities of these reactions designate nsP1 as a promising target for antiviral drug development. In the current study we performed a mutational analysis on two nsP1 positions associated with Sindbis virus (SINV) ribavirin resistance in the Venezuelan equine encephalitis virus (VEEV) context through reverse genetics correlated to enzyme assays using purified recombinant VEEV nsP1 proteins. The results demonstrate that the targeted positions are strongly associated to the regulation of the capping reaction by increasing the affinity between GTP and nsP1. Data also show that in VEEV the S21A substitution, naturally occurring in Chikungunya virus (CHIKV), is a hallmark of ribavirin susceptibility. These findings uncover the specific mechanistic contributions of these residues to nsp1-mediated methyl-transfer and guanylylation reactions.

The spreading of new viruses is known to provoke global human health threat. The current COVID-19 pandemic caused by the recently emerged coronavirus SARS-CoV-2 is one significant and unfortunate example of what the world will have to face in the future with emerging viruses in absence of appropriate treatment. The discovery of potent and specific antiviral inhibitors and/or vaccines to fight these massive outbreaks is an urgent research priority. Enzymes involved in the capping pathway of viruses and more specifically RNA N7- or 2′O-methyltransferases (MTases) are now admitted as potential targets for antiviral chemotherapy. We designed bisubstrate inhibitors by mimicking the transition state of the 2′-O-methylation of the cap RNA in order to block viral 2′-O MTases. This work resulted in the synthesis of 16 adenine dinucleosides with both adenosines connected by various nitrogen-containing linkers. Unexpectedly, all the bisubstrate compounds were barely active against 2′-O MTases of several flaviviruses or SARS-CoV but surprisingly, seven of them showed efficient and specific inhibition against SARS-CoV N7-MTase (nsp14) in the micromolar to submicromolar range. The most active nsp14 inhibitor identified is as potent as but particularly more specific than the broad-spectrum MTase inhibitor, sinefungin. Molecular docking suggests that the inhibitor binds to a pocket formed by the S-adenosyl methionine (SAM) and cap RNA binding sites, conserved among SARS-CoV nsp14. These dinucleoside SAM analogs will serve as starting points for the development of next inhibitors for SARS-CoV-2 nsp14 N7-MTase., Graphical abstract Image 1, Highlights • Bisubstrates as mimics of RNA 2′-O-methylation transition state with SAM. • 16 dinucleosides with two adenosines joined by various nitrogen-containing linkers. • Bisubstrate compounds were barely active against viral 2′-O-methyltransferases. • Discovery of a potent and specific inhibitor of SARS-CoV nsp14 N7-methyltransferase. • Molecular docking and biophysical assays corroborated observed SAR.

National audience; SARS-CoV-2 is a new human coronavirus (CoV), which emerged in China at the end of 2019 and is responsible for the global COVID-19 pandemic that caused more than 500,000 deaths in five months. Understanding the origin of this virus is an important issue and it is necessary to determine the mechanisms of its dissemination in order to be able to contain new epidemics. Based on phylogenetic inferences, sequence analysis and structure-function relationships of coronavirus proteins, informed by the knowledge currently available, we discuss the different scenarios evoked to account for the origin - natural or synthetic - of the virus. On the basis of currently available data, it is impossible to determine whether SARS-CoV2 is the result of a natural zoonotic emergence or an accidental escape from experimental strains. Regardless of its origin, the study of the evolution of the molecular mechanisms involved in the emergence of this pandemic virus is essential to develop therapeutic and vaccine strategies. The full article (in French) is available at Médecine/Sciences : https://doi.org/10.1051/medsci/2020123. The full English translation available at https://hal.archives-ouvertes.fr/hal-02891455.; Le SARS-CoV-2 est un nouveau coronavirus (CoV) humain. Il a émergé en Chine fin 2019 et est responsable de la pandémie mondiale de COVID-19 qui a causé plus de 500 000 décès en cinq mois. La compréhension de l’origine de ce virus est une question importante et il est nécessaire de déterminer les mécanismes de sa dissémination afin de pouvoir se prémunir de nouvelles épidémies. En nous fondant sur des inférences phylogénétiques, l’analyse des séquences et les relations structure-fonction des protéines de coronavirus, éclairées par les connaissances actuellement disponibles sur le virus, nous discutons les différents scénarios évoqués pour rendre compte de l'origine – naturelle ou synthétique – du virus. L'article complet est désormais librement accessible sur le site de Médecine/Sciences : https://doi.org/10.1051/medsci/2020123. Une traduction en anglais est disponible ici : https://hal.archives-ouvertes.fr/hal-02891455

Molecular Simulations Group, Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Kalina, Santacruz East, Mumbai-400 098, India Aix-Marseille Univ, CNRS, AFMB UMR 7257, Marseille, France Ambernath Organics Pvt. Ltd., 222, The Summit Business Bay, Andheri (E), Mumbai-400 093, India E-mail: evans.coutinho@bcp.edu.in, santosh.nandan@ambernathorganic.com Manuscript received online 30 June 2020, accepted 20 July 2020 Various studies have shown that NS5 RNA methyltransferase (MTase) is a key enzyme involved in mRNA capping, a step crucial for flaviviral replication. Therefore, it has been identified as a potential target for therapeutic intervention in infections arising due to flaviviruses. In this paper, we report computer-assisted design of 2,3-dioxopyrrolidines, which were synthesised as guided by molecular docking studies on DENV and ZIKA MTase. Their chemical structures and geometric configuration were characterized by FT-IR, NMR (13C and 1H), MS and small molecule X-ray crystallography. Subsequently, their inhibitory potential was evaluated using an enzyme-based assay in DENV MTase (N7 and 2'O-MTase) and Zika virus MTase (N7 and 2'O-MTase). Furthermore, these molecules were also screened against RNMT (human N7 MTase). The most potent lead (compound W07) is seen to inhibit MTase from DENV with IC50 = 24.6±3.8 µM and ZIKA with IC50 = 9.0±1.7 µM. However, it also inhibits human N7 MTase, indicating plausible toxicity in humans. There is scope to further optimize these molecules to achieve selectivity towards flaviviral MTases.

Graphical abstract, Flaviviruses, such as Dengue (DENV) and Zika (ZIKV) viruses, represent a severe health burden. There are currently no FDA-approved treatments, and vaccines against most flaviviruses are still lacking. We have developed several flexible analogues (“fleximers”) of the FDA-approved nucleoside Acyclovir that exhibit activity against various RNA viruses, demonstrating their broad-spectrum potential. The current study reports activity against DENV and YFV, particularly for compound 1. Studies to elucidate the mechanism of action suggest the flex-analogue triphosphates, especially 1-TP, inhibit DENV and ZIKV methyltransferases. The results of these studies are reported herein.

International audience; Despite the worldwide reemergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we aimed to identify the target of a novel class of CHIKV inhibitors, i.e., the CHVB series. CHVB compounds inhibit the in vitro replication of CHIKV isolates with 50% effective concentrations in the low-micromolar range. A CHVB-resistant variant (CHVBres) was selected that carried two mutations in the gene encoding nsP1 (responsible for viral RNA capping), one mutation in nsP2, and one mutation in nsP3. Reverse genetics studies demonstrated that both nsP1 mutations were necessary and sufficient to achieve similar to 18-fold resistance, suggesting that CHVB targets viral mRNA capping. Interestingly, CHVBres was cross-resistant to the previously described CHIKV capping inhibitors from the MADTP series, suggesting they share a similar mechanism of action. In enzymatic assays, CHVB inhibited the methyltransferase and guanylyltransferase activities of alphavirus nsP1 proteins. To conclude, we identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity makes this chemical scaffold a potential candidate for CHIKV drug development.