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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.

Arenaviruses are emerging enveloped negative-sense RNA viruses that cause neurological and hemorrhagic diseases in humans. Currently, no FDA-approved vaccine or therapeutic agent is available except for ribavirin, which must be administered early during infection for optimum efficacy. A hallmark of arenavirus infection is rapid and efficient immune suppression mediated by the exonuclease domain encoded by the nucleoprotein. This exonuclease is therefore an attractive target for the design of novel antiviral drugs since exonuclease inhibitors might not only have a direct effect on the enzyme but could also boost viral clearance through stimulation of the innate immune system of the host cell. Here

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.

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir not only inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp) but also modulates host immune responses. Our results thus reveal the possible anti-SARS-CoV-2 mechanism of simeprevir and highlight the translational potential of optimizing simeprevir as a therapeutic agent for managing COVID-19 and future outbreaks of CoV., Simeprevir, an HCV drug, suppresses SARS-CoV-2 replication by inhibiting two viral proteins and modulating host immune responses, thus providing novel insights in anti-CoV drug design.

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.

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.

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; The rapid global emergence of SARS-CoV-2 has been the cause of significant health concern, highlighting the immediate need for antivirals. Viral RNA-dependent RNA polymerases (RdRp) play essential roles in viral RNA synthesis, and thus remains the target of choice for the prophylactic or curative treatment of several viral diseases, due to high sequence and structural conservation. To date, the most promising broad-spectrum class of viral RdRp inhibitors are nucleoside analogues (NAs), with over 25 approved for the treatment of several medically important viral diseases. However, Coronaviruses stand out as a particularly challenging case for NA drug design due to the presence of an exonuclease (ExoN) domain capable of excising incorporated NAs and thus providing resistance to many of these available antivirals. Here we use the available structures of the SARS-CoV RdRp and ExoN proteins, as well as Lassa virus N exonuclease to derive models of catalytically competent SARS-CoV-2 enzymes. We then map a promising NA candidate, GS-441524 (the active metabolite of Remdesivir) to the nucleoside active site of both proteins, identifying the residues important for nucleotide recognition, discrimination, and excision. Interestingly, GS-441524 addresses both enzyme active sites in a manner consistent with significant incorporation, delayed chain termination, and altered excision due to the ribose 1'-CN group, which may account for the increased antiviral effect compared to other available analogues. Additionally, we propose structural and function implications of two previously identified RdRp resistance mutations in relation to resistance against Remdesivir. This study highlights the importance of considering the balance between incorporation and excision properties of NAs between the RdRp and ExoN.

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp). Our results thus reveal the viral protein targets of simeprevir, and provide preclinical rationale for the combination of simeprevir and remdesivir for the pharmacological management of COVID-19 patients.One Sentence SummaryDiscovery of simeprevir as a potent suppressor of SARS-CoV-2 viral replication that synergizes with remdesivir.

Flaviviruses constitute a public health concern because of their global burden and the lack of specific antiviral treatment. Here we investigated the antiviral activity of the alkaloid anisomycin against dengue (DENV) and Zika (ZIKV) viruses. At non-cytotoxic concentrations, anisomycin strongly inhibited the replication of reference strains and clinical isolates of all DENV serotypes and Asian and African strains of ZIKV in Vero cells. Anisomycin also prevented DENV and ZIKV multiplication in human cell lines. While initial steps of DENV and ZIKV replicative cycle were unaffected, a high inhibition of viral protein expression was demonstrated after treatment with anisomycin. DENV RNA synthesis was strongly reduced in anisomycin treated cultures, but the compounddid not exert a direct inhibitory effect on 2′ O-methyltransferase or RNA polymerase activities of DENV NS5 protein. Furthermore, anisomycin-mediated activation of p38 signaling was not related to the antiviral action ofthe compound. The evaluation of anisomycin efficacy in a mouse model of ZIKV morbidity and mortality revealed that animals treated with a low dose of anisomycin exhibited a significant reduction in viremia levels anddied significantly later than the control group. This protective effect was lost at higher doses, though. In conclusion, anisomycin is a potent and selective in vitro inhibitor of DENV and ZIKV that impairs a post-entry step of viral replication; and a low-dose anisomycin treatment may provide some minimal benefit in a mouse model. Fil: Quintana, Verónica Mara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Selisko, B.. Centre National de la Recherche Scientifique; Francia Fil: Brunetti, Jesús Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Eydoux, C.. Centre National de la Recherche Scientifique; Francia Fil: Guillemot, J.C.. Centre National de la Recherche Scientifique; Francia Fil: Canard, B.. Centre National de la Recherche Scientifique; Francia Fil: Damonte, Elsa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Julander, J.. State University of Utah; Estados Unidos Fil: Castilla, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

International audience; Viral RNA-dependent RNA polymerases (RdRps) play a central role not only in viral replication, but also in the genetic evolution of viral RNAs. After binding to an RNA template and selecting 5'-triphosphate ribonucleosides, viral RdRps synthesize an RNA copy according to Watson-Crick base-pairing rules. The copy process sometimes deviates from both the base-pairing rules specified by the template and the natural ribose selectivity and, thus, the process is error-prone due to the intrinsic (in)fidelity of viral RdRps. These enzymes share a number of conserved amino-acid sequence strings, called motifs A-G, which can be defined from a structural and functional point-of-view. A co-relation is gradually emerging between mutations in these motifs and viral genome evolution or observed mutation rates. Here, we review our current knowledge on these motifs and their role on the structural and mechanistic basis of the fidelity of nucleotide selection and RNA synthesis by Flavivirus RdRps.

RNA synthesis and replication of the members of the Flavivirus genus (including dengue, West Nile and Japanese encephalitis viruses) is regulated by a wide variety of mechanisms and actors. These include the sequestration of the RNA-dependent RNA polymerase (RdRp) for functions other than RNA synthesis, regulatory interactions with other viral and host proteins within the replication complex (RC), and regulatory elements within the RNA genome itself. In this review, we discuss our current knowledge of the multiple levels at which Flavivirus RNA synthesis is controlled. We aim to bring together two active research fields: the structural and functional biology of individual proteins of the RC and the impressive wealth of knowledge acquired regarding the viral genomic RNA.

International audience; In targeting the essential viral RNA-dependent RNA-polymerase (RdRp), nucleotide analogues play a major role in antiviral therapies. In the Flaviviridae family, the hepatitis C virus (HCV) can be eradicated from chronically infected patients using a combination of drugs which generally include the 2'-modified uridine analogue Sofosbuvir, delivered as nucleotide prodrug. Dengue and Zika viruses are emerging flaviviruses whose RdRp is closely related to that of HCV, yet no nucleoside drug has been clinically approved for these acute infections. We have purified dengue and Zika virus full-length NS5, the viral RdRps, and used them to assemble a stable binary complex made of NS5 and virus-specific RNA primer/templates. The complex was used to assess the selectivity of NS5 towards nucleotide analogues bearing modifications at the 2'-position. We show that dengue and Zika virus RdRps exhibit the same discrimination pattern: 2'-O-Me > 2'-C-Me-2'-F > 2'-C-Me nucleoside analogues, unlike HCV RdRp for which the presence of the 2'-F is beneficial rendering the discrimination pattern 2'-O-Me > 2'-C-Me ≥ 2'-C-Me-2'-F. Both 2'-C-Me and 2'-C-Me-2'-F analogues act as non-obligate RNA chain terminators. The dengue and Zika NS5 nucleotide selectivity towards 2'-modified NTPs mirrors potency of the corresponding analogues in infected cell cultures.

The Flavivirus Zika virus (ZIKV) is the causal agent of neurological disorders like microcephaly in newborns or Guillain-Barre syndrome. Its NS5 protein embeds a methyltransferase (MTase) domain involved in the formation of the viral mRNA cap. We investigated the structural and functional properties of the ZIKV MTase. We show that the ZIKV MTase can methylate RNA cap structures at the N-7 position of the cap, and at the 2′-O position on the ribose of the first nucleotide, yielding a cap-1 structure. In addition, the ZIKV MTase methylates the ribose 2′-O position of internal adenosines of RNA substrates. The crystal structure of the ZIKV MTase determined at a 2.01-Å resolution reveals a crystallographic homodimer. One chain is bound to the methyl donor ( S -adenosyl- l -methionine [SAM]) and shows a high structural similarity to the dengue virus (DENV) MTase. The second chain lacks SAM and displays conformational changes in the αX α-helix contributing to the SAM and RNA binding. These conformational modifications reveal a possible molecular mechanism of the enzymatic turnover involving a conserved Ser/Arg motif. In the second chain, the SAM binding site accommodates a sulfate close to a glycerol that could serve as a basis for structure-based drug design. In addition, compounds known to inhibit the DENV MTase show similar inhibition potency on the ZIKV MTase. Altogether these results contribute to a better understanding of the ZIKV MTase, a central player in viral replication and host innate immune response, and lay the basis for the development of potential antiviral drugs. IMPORTANCE The Zika virus (ZIKV) is associated with microcephaly in newborns, and other neurological disorders such as Guillain-Barre syndrome. It is urgent to develop antiviral strategies inhibiting the viral replication. The ZIKV NS5 embeds a methyltransferase involved in the viral mRNA capping process, which is essential for viral replication and control of virus detection by innate immune mechanisms. We demonstrate that the ZIKV methyltransferase methylates the mRNA cap and adenosines located in RNA sequences. The structure of ZIKV methyltransferase shows high structural similarities to the dengue virus methyltransferase, but conformational specificities highlight the role of a conserved Ser/Arg motif, which participates in RNA and SAM recognition during the reaction turnover. In addition, the SAM binding site accommodates a sulfate and a glycerol, offering structural information to initiate structure-based drug design. Altogether, these results contribute to a better understanding of the Flavivirus methyltransferases, which are central players in the virus replication.

ABSTRACT RNA viruses are important emerging pathogens that cause human and animal infectious diseases. Antiviral therapies have to deal with the high mutational capacity of RNA viruses, which quickly adapt to new environments. A primary target for antiviral drug development is the viral RNA-dependent RNA polymerase (RdRp), which is the central enzyme of the viral RNA replication/transcription machinery. Here, we review the current mechanistic and structural knowledge on RdRps of positive-strand RNA viruses gained through crystallography and biochemistry. In addition, we review the growing body of information on RdRp-mediated strategies, such as proofreading and genome end repair, used by positive-strand RNA viruses to maintain their genome integrity.

ABSTRACT: Upon the discovery of HCV, dengue virus (DENV) and other flaviviruses have served as models to unravel the biology and mechanisms at play during HCV replication. HCV research has rapidly become a well-established field. Recently, several specific anti-HCV antiviral drugs have been discovered and approved for use in the clinic. Now, the strong emergence of DENV in the world and the associated increasing burden is casting light back to dengue virology and anti-dengue drug discovery. HCV polymerase (NS5B) is a prime target in antiviral therapies, and the analogous DENV polymerase (NS5) is also becoming one. Although both enzymes share common fold and function to some extent, a significant amount of unique structural and functional features have to be clearly delineated to efficiently translate drug design potential between these two essential enzymes.

The dengue fever virus (DENV) and the yellow fever virus (YFV) are members of the genus flavivirus in the family Flaviviridae. An estimated 50-100 million cases of DENV infections occur each year and approximately half a million patients require hospitalization. There is no vaccine or effective antiviral treatment available. There is an urgent need for potent and safe inhibitors of DENV replication; ideally such compounds should have broad-spectrum activity against flaviviruses. We here report on the in vitro activity of 3',5'di-O-trityluridine on flavivirus replication. The compound results in a dose-dependent inhibition of (i) DENV- and YFV-induced cytopathic effect (CPE) (EC50 values in the low micromolar range for the 4 DENV serotypes), (ii) RNA replication (DENV-2 EC50=1.5μM; YFV-17D EC50=0.83μM) and (iii) viral antigen production. Antiviral activity was also demonstrated in DENV subgenomic replicons (which do not encode the structural viral proteins) (EC50=2.3μM), indicating that the compound inhibits intracellular events of the viral replication cycle. Preliminary data indicate that the molecule may inhibit the viral RNA-dependent RNA polymerase. publisher: Elsevier articletitle: 3′,5′Di-O-trityluridine inhibits in vitro flavivirus replication journaltitle: Antiviral Research articlelink: http://dx.doi.org/10.1016/j.antiviral.2013.01.011 content_type: article copyright: Copyright © 2013 Elsevier B.V. All rights reserved. ispartof: Antiviral Research vol:98 issue:2 pages:242-247 ispartof: location:Netherlands status: published

Dengue virus (DENV) is an important human pathogen, especially in the tropical and subtropical parts of the world, causing considerable morbidity and mortality. DENV replication occurs in the cytoplasm; however, a high proportion of nonstructural protein 5 (NS5), containing methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) activities, accumulates in the nuclei of infected cells. The present study investigates the impact of nuclear localization of NS5 on its known functions, including viral RNA replication and subversion of the type I interferon response. By using a mutation analysis approach, we identified the most critical residues within the αβ nuclear localization signal (αβNLS), which are essential for the nuclear accumulation of this protein. Although we observed an overall correlation between reduced nuclear accumulation of NS5 and impaired RNA replication, we identified one mutant with drastically reduced amounts of nuclear NS5 and virtually unaffected RNA replication, arguing that nuclear localization of NS5 does not correlate strictly with DENV replication, at least in cell culture. Because NS5 plays an important role in blocking interferon signaling via STAT-2 (signal transducer and activator of transcription 2) degradation, the abilities of the NLS mutants to block this pathway were investigated. All mutants were able to degrade STAT-2, with accordingly similar type I interferon resistance phenotypes. Since the NLS is contained within the RdRp domain, the MTase and RdRp activities of the mutants were determined by using recombinant full-length NS5. We found that the C-terminal region of the αβNLS is a critical functional element of the RdRp domain required for polymerase activity. These results indicate that efficient DENV RNA replication requires only minimal, if any, nuclear NS5, and they identify the αβNLS as a structural element required for proper RdRp activity.

Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.

Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential.

Highlights ► RNA viral capping mechanisms. ► Enzymology of viral RNA capping. ► Structures of viral RNA capping enzymes. ► Structures of viral RNA capping main «lead» inhibitors., Most viruses modify their genomic and mRNA 5′-ends with the addition of an RNA cap, allowing efficient mRNA translation, limiting degradation by cellular 5′–3′ exonucleases, and avoiding its recognition as foreign RNA by the host cell. Viral RNA caps can be synthesized or acquired through the use of a capping machinery which exhibits a significant diversity in organization, structure and mechanism relative to that of their cellular host. Therefore, viral RNA capping has emerged as an interesting field for antiviral drug design. Here, we review the different pathways and mechanisms used to produce viral mRNA 5′-caps, and present current structures, mechanisms, and inhibitors known to act on viral RNA capping.

The West Nile virus (WNV) NS5 protein contains a methyltransferase (MTase) domain involved in RNA capping and an RNA-dependent RNA polymerase (RdRp) domain essential for virus replication. Crystal structures of individual WNV MTase and RdRp domains have been solved; however, the structure of full-length NS5 has not been determined. To gain more insight into the structure of NS5 and interactions between the MTase and RdRp domains, we generated a panel of seven monoclonal antibodies (mAbs) to the NS5 protein of WNV (Kunjin strain) and mapped their binding sites using a series of truncated NS5 proteins and synthetic peptides. Binding sites of four mAbs (5D4, 4B6, 5C11 and 6A10) were mapped to residues 354–389 in the fingers subdomain of the RdRp. This is consistent with the ability of these mAbs to inhibit RdRp activity in vitro and suggests that this region represents a potential target for RdRp inhibitors. Using a series of synthetic peptides, we also identified a linear epitope (bound by mAb 5H1) that mapped to a 13 aa stretch surrounding residues 47 and 49 in the MTase domain, a region predicted to interact with the palm subdomain of the RdRp. The failure of one mAb (7G6) to bind both N- and C-terminally truncated NS5 recombinants indicates that the antibody recognizes a conformational epitope that requires the presence of residues in both the MTase and RdRp domains. These data support a structural model of the full-length NS5 molecule that predicts a physical interaction between the MTase and the RdRp domains.

The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3D pol ) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3D pol at this site excludes its uridylation by the carrier 3D pol . We suggest that VPg at this position is either uridylated by another 3D pol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3D pol /VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.

International audience; Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product formation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation.

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.

The murine monoclonal antibody BCF2 is able to neutralize the venom of the scorpion Centruroides noxius Hoffmann. A chimeric Fab of BCF2 (chFab-BCF2) comprising the variable regions of murine BCF2 and human constant regions was assembled. chFab-BCF2 was expressed as a soluble and functional protein in the periplasmic space of Escherichia coli. An expression yield of 1 mg/l was reached by combination of late-log-phase induction, rich culture medium, low expression temperature and addition of sucrose (0.3 M) to the culture medium. The addition of sucrose induced secretion of 60% of the protein into the medium. After expression for 23 h, a novel process was used to release the remaining periplasmic protein in situ consisting in the addition of lysozyme and sucrose up to 0.6 M (20%) directly to the culture medium. chFab-BCF2 was recovered by ammonium sulfate precipitation and purified in a single step by affinity chromatography using anti-human anti-F(ab′)2 IgG coupled to Sepharose-proteinG. Pure chFab-BCF2 maintained a similar nanomolar affinity as BCF2 to its cognate antigen, the Na+-channel-affecting toxin Cn2. Recombinant chFab-BCF2 was able to neutralize Cn2 in vivo even at a molar ratio of 1:1, as well as the whole venom of C. noxius. Thus, it is a promising candidate to be used as a specific and efficient recombinant antidote against scorpion stings.

Viruses represent an attractive system with which to study the molecular basis of mRNA capping and its relation to the RNA transcription machinery. The RNA-dependent RNA polymerase NS5 of flaviviruses presents a characteristic motif of S-adenosyl-L-methionine-dependent methyltransferases at its N-terminus, and polymerase motifs at its C-terminus. The crystal structure of an N-terminal fragment of Dengue virus type 2 NS5 is reported at 2.4 A resolution. We show that this NS5 domain includes a typical methyltransferase core and exhibits a (nucleoside-2'-O-)-methyltransferase activity on capped RNA. The structure of a ternary complex comprising S-adenosyl-L-homocysteine and a guanosine triphosphate (GTP) analogue shows that 54 amino acids N-terminal to the core provide a novel GTP-binding site that selects guanine using a previously unreported mechanism. Binding studies using GTP- and RNA cap-analogues, as well as the spatial arrangement of the methyltransferase active site relative to the GTP-binding site, suggest that the latter is a specific cap-binding site. As RNA capping is an essential viral function, these results provide a structural basis for the rational design of drugs against the emerging flaviviruses.

Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis.

Noxiustoxin (NTX) is a short-chain toxin from the venom of the scorpion Centruroides noxius Hoffmann, whose molecular structure and physiological effects have been characterized in detail, whereas the antigenic properties of this and other K(+) channel-blocking toxins are poorly studied. A monoclonal antibody against NTX, BNTX18, able to inhibit the binding of NTX to rat brain synaptosomes, was used in the present study for selecting immunoreactive peptides, mimotopes, from a 12mer and a 7mer phage library. The peptides were characterized immunologically and used for mapping the epitope on NTX. In total, 75 phage clones carrying 43 different peptides were analyzed of which 42 clones carrying 17 different peptides, twelve 12mer and five 7mer peptides, presented a single consensus motif: Leu(Ile, Val)-Tyr(Phe, Trp, Leu)-Gly-Met(Ala). All but three of the peptides containing this motif were reactive with selected mAb BNTX18 in a dot-blot assay of which eight were clearly positive in ELISA and exhibited in competition-inhibition assay the antibody binding specificity of the NTX epitope recognized by BNTX18. The two most reactive mimotopes injected into mice showed the ability to induce antibodies reacting with NTX, thus, to mimic the epitope of NTX antigenically. Sequence comparison and the analysis of the three-dimensional structure of NTX led to the proposal that residues Glu19-Leu20-Tyr21-Gly22 and the hydrophobic part of the side chain of Lys18 form the C-terminal part of the epitope. Due to the frequent presence of residues Pro, Leu, Thr, Arg, and Gln in the N-terminal part of the mimotopes, corresponding homologous residues in the N-terminal proximity of the partial epitope may be part of an additional more hydrophilic epitope element.

The antibody BCF2 generated against the mammal-specific toxin Cn2 of the scorpion Centruroides noxius Hoffmann neutralizes the effect of both the toxin and the venom. We cloned and sequenced the genes coding for the Fv fragment of BCF2. A three-dimensional (3D) model of the Fv fragment was generated using a knowledge-based approach. Furthermore, a 3D model of the complex Cn2–BCF2 was built using the nuclear magnetic resonance (NMR) structure of Cn2 and experimental results on a putative epitope region around the N and C termini. The initial complex conformations were submitted to a new refinement procedure of rigid-body energy minimization combined with flexible-side-chain molecular dynamics. The final complex, selected after an extensive evaluation, uses the loop 7–11 as the central part of the epitope. The generated complex allows the following conclusions: 1) the neutralizing capacity of BCF2 toward the venom of C. noxius might rather be caused by the high venom concentration and toxicity of Cn2 than by a broad specificity, 2) the region involved in the binding of Cn2 to the Na+ channel, should overlap with the employed epitope region, and 3) contact residues SerL91, AsnL92, LeuH50, AspH56, TyrH95, and TyrH98 of BCF2 are candidates for mutations to broaden its specificity. Proteins 1999;37:130–143. © 1999 Wiley-Liss, Inc.

This chapter reviews current literature dealing with peptides isolated from the venom of scorpions. Only peptides that recognize K+-channels are reported. They are called K+-channel-ligands or simply peptides, because the actual toxicity effects of all these peptides has not been demonstrated. The primary structures of 35 peptides are reviewed, and a general nomenclature has been proposed to define 9 distinct sub-families of related sequences. Partial sequences were not included in this classification. They are 29 to 39 amino acid residues long peptides with a common structural motif composed of an α-helix segment and three anti-parallel β-sheet strands stabilized by three or four disulfide bridges. Binding and/or electrophysiological experiments showed that the affinity of these peptides for the various sub-types of K+-channels varies from micromolar to picomolar concentrations. Some data on the three-dimensional structures and on specific functions of the various peptides on voltage-dependent K+-channels, high-conductance and small-conductance, Ca2+-dependent K+-channels are also briefly reviewed.

Scorpion toxins acting on sodium channels differ in their specificity. Toxic peptides specific towards mammals and arthropods (insects and/or crustaceans) have been described. Because of the similar three-dimensional fold of these peptides, the molecular base of their specificity is thought to reside in certain differences at the level of amino acid residues especially within or near the binding site of the toxin to the particular ion channel. The cDNA, amino acid sequence and biological activity of an insect-specific toxin, Cn10, from the scorpion Centruroides noxius Hoffmann is reported. The electrostatic potential surface around a three-dimensional model of Cn10 was calculated. It revealed that residues Tyr4, Lys13, Ile18, Leu19, Gly20, Lys43, Leu44, Thr57, Tyr58, Pro59, Thr64 and Cys65, situated at the side of the toxin proposed in the literature to bind to the sodium channel, constitute a positive surface region. Therefore, they may form the site that binds to the channel. Cn10 was included in a comparative analysis of two groups of natural variants, highly similar peptides of the genus Centruroides with specificities towards mammals or arthropods. A number of surface-accessible residues, consistently different between the two groups and situated near the putative binding site, may be of importance for the specificity of the analyzed toxins.

Binding of the Dengue virus S-adenosyl-L-methionine (AdoMet)-dependent mRNA cap methyltransferase (NS5MTaseDV ) with adamantane derivatives was explored using molecular modeling methods and (nucleoside-2'O)-methyltransferase bioassay. The studied compounds include urea derivatives of adamantane and the antiviral drugs amantadine and rimantadine. The urea derivatives of adamantanes had previously been identified as inhibitors of NS5MTaseDV . The docking simulations using GOLD, Glide, and Dock give consistent binding modes and binding affinities of adamantanes in the AdoMet-binding site of NS5MTaseDV and, in particular, yield similar positions for the previously found inhibitors. Combined, they perfectly correspond to the bioassay measurements of nucleoside-2'O-methyltransferase activity of NS5TaseDV , which confirmed inhibitory properties of the active urea adamantane but did not show inhibitory activity for amantadine and rimantadine. We also employed microscopic molecular dynamics (MD) simulations and a linear interaction energy (LIE) method to verify the docking results. The MD/LIE binding free energies of selected protein-inhibitor complexes agree overall with the binding affinities from docking and demonstrate that amantadine and rimantadine only weakly bind at the explored site. The MD simulations also demonstrated the flexible character of a protein loop that is located between the β2 and β3 strands and is part of the AdoMet-binding pocket of NS5MTaseDV .

Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential.

Picornaviridae is one of the largest viral families and is composed of 14 genera, six of which include human pathogens. The best known picornaviruses are enteroviruses (including polio, PV, and rhinoviruses), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Although infections often are mild, certain strains may cause pandemic outbreaks accompanied with meningitis and/or paralysis. Vaccines are available for PV, HAV and FMDV. When the oral vaccines are given to immunocompromised individuals, they may be chronically infected, and remain secretors of vaccine-derived variants of virus for years. There is no effective prophylaxis available for these or other picornaviruses. So far, only the 3C protease from viruses in three genera has been fully characterized as an anti-viral target, whereas the mode of action of compounds targeting other non-structural proteins have remained largely unaddressed. Within the EU-supported FP6 project-VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication), the non-structural proteins were studied to identify conserved binding sites for broadly reactive anti-virals. The putative 2C helicase from echovirus-30 was shown to form ring-shaped hexamers typical for DNA-encoded SF3 helicases, and to possess ATPase activity. Hexamer formation of 2C from enterovirus 76 was in vitro shown to be dependent on the 44 N-terminal residues. Crystal structures of three enterovirus 3C proteases were solved and shown to be similar to those of other picornaviruses. A new binding site of VPg to the bottom of the thumb domain of CV-B3 3D polymerase was identified as a potential target. Broad anti-enterovirus compounds against 2C and 3A proteins were also identified, including thiazolobenzimidazoles (active against 2C) and TTP-8307 (targeting 3A). There is a need for more potent inhibitors against PV and other picornaviruses, which are potential silent reservoirs for re-emerging PV-like disease., This work was supported the EU (Integrated Project VIZIER FP-6 contract No. 511960).

Vesicular packing material (VP) made of clusters of extracted higher plant cells with the intact framework of their cell wall was used so far for permeation chromatography (vesicle chromatography). The objective of this study was to devise a method to entrap dextran in the vesicles. This can provide a means to entrap biocatalysts and secondly, to create aqueous two-phase systems with a stationary dextran phase for liquid-liquid partition chromatography. Dextran of molecular sizes above the separation limit of the plant cell wall cannot permeate into the intracellular space in aqueous medium. However, in hydrophilic organic solvent/water mixtures, dextran molecules can diffuse into the capsules. The removal of the organic solvent leaves the dextran trapped inside. There was an inverse correlation between the percentage of dextran permeating through the cell wall (Pperm) and the concentration of solvent required for dextran precipitation. The increase of permeability is therefore considered to be caused, to a great extent, by the decrease of the effective size of dextran molecules due to decreased solvation. Pperm was inversely correlated to the dielectric constants and the polarities of the solvents and, in the case of protic solvents, the hydrogen-bond acidities. No correlation was found to the hydrogen-bond basicities.

The efficiency of enzymes in organic solvents is determined by the catalytic parameters, which mostly are changed in comparison with those in aqueous solutions, as well as by reversible and irreversible denaturation of the enzyme protein. The interplay of these processes was studied on soluble and immobilized glucoamylase and invertase in mixtures of buffer and several water-miscible solvents such as methanol, glycerol, dimethylformamide, and dioxane. A comparison of the catalytic activity in 50 or 30% (v/v) solvents with the extent of irreversible inactivation during the time of assay showed that the solvents act on the catalytic parameters of the enzymes and their conformational stability in different ways. Irreversible inactivation is especially strong in dimethylformamide, whereas glycerol does not induce denaturation but strongly reduces the catalytic efficiency. Striking effects were observed in dioxane, where high stability to irreversible denaturation but strong reduction of catalytic power were observed. In all solvents K m values were more affected than V max values, suggesting that reversible denaturation plays an inferior role in activity losses. Similar effects of the individual solvents were also observed after immobilization of the enzymes by covalent binding to a macroporous aminomethyl polystyrene-divinylbenzene copolymer or a macroporous aminopropyl silica. In all cases, however, immobilized enzymes showed better long-term stability than the soluble enzymes.

An error was introduced in the preparation of this article for publication. The incorrect figure file was provided for Figure 4. Please view the correct Figure 4 here

The flavivirus RNA genome contains a conserved cap-1 structure, (7Me)GpppA(2'OMe)G, at the 5' end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)- and the (nucleoside-2'O)-MTases (2'O-MTase), reside in a single domain of non-structural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 2'O-MTase activity of NS5MTase of dengue virus (NS5MTase(DV)) using purified, short, capped RNA substrates ((7Me)GpppAC(n) or GpppAC(n)). NS5MTase(DV) methylated both types of substrate exclusively at the 2'O position. The efficiency of 2'O-methylation did not depend on the methylation of the N7 position. Using (7Me)GpppAC(n) and GpppAC(n) substrates of increasing chain lengths, it was found that both NS5MTase(DV) 2'O activity and substrate binding increased before reaching a plateau at n=5. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K(m) values for (7Me)GpppAC(5) and the co-substrate S-adenosyl-L-methionine (AdoMet) were determined (0.39 and 3.26 microM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 2'O-MTase activity of NS5MTase(DV) showed a low turnover of 3.25x10(-4) s(-1). Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTase(DV), which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC(50) 0.34 microM) and sinefungin (IC(50) 0.63 microM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays.

Flaviviruses are the causative agents of severe diseases such as Dengue or Yellow fever. The replicative machinery used by the virus is based on few enzymes including a methyltransferase, located in the N-terminal domain of the NS5 protein. Flaviviral methyltransferases are involved in the last two steps of the mRNA capping process, transferring a methyl group from S-adenosyl-l-methionine onto the N7 position of the cap guanine (guanine-N7 methyltransferase) and the ribose 2?O position of the first nucleotide following the cap guanine (nucleoside-2?O methyltransferase). The RNA capping process is crucial for mRNA stability, protein synthesis and virus replication. Such an essential function makes methyltransferases attractive targets for the design of antiviral drugs. In this context, starting from the crystal structure of Wesselsbron flavivirus methyltransferase, we elaborated a mechanistic model describing protein/RNA interaction during N7 methyl transfer. Next we used an in silico docking procedure to identify commercially available compounds that would display high affinity for the methyltransferase active site. The best candidates selected were tested in vitro to assay their effective inhibition on 2?O and N7 methyltransferase activities on Wesselsbron and Dengue virus (Dv) methyltransferases. The results of such combined computational and experimental screening approach led to the identification of a high-potency inhibitor.