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... Show moreEnzymes 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 designand synthesis of nucleoside-derived inhibitors against the SARS-CoV-2 nsp14 (N7-guanine)-methyltransferase (N7-MTase) thatcatalyzes the transfer of the methyl group from theS-adenosyl-L-methionine (SAM) cofactor to theN7-guanosine cap. Sevencompounds out of 39 SAM analogues showed remarkable double-digit nanomolar inhibitory activity against theN7-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 (Delta Tm approximate to 11 degrees C), and the best inhibitor demonstrated high selectivityfor nsp14 over human RNAN7-MTase. Show less
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... Show moreDespite 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. Show less
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... Show morePicornaviridae 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-vitals. 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 picornavinises, which are potential silent reservoirs for re-emerging PV-like disease. (C) 2011 Elsevier B.V. All rights reserved. Show less
The 2C protein, which is an essential ATPase and one of the most conserved proteins across the Picornaviridae family, is an emerging antiviral target for which structural and functional... Show moreThe 2C protein, which is an essential ATPase and one of the most conserved proteins across the Picornaviridae family, is an emerging antiviral target for which structural and functional characterization remain elusive. Based on a distant relationship to helicases of small DNA viruses, piconavirus 2C proteins have been predicted to unwind double-stranded RNAs. Here, a terminally extended variant of the 2C protein from echovirus 30 has been studied by means of enzymatic activity assays, transmission electron microscopy, atomic force microscopy and dynamic light scattering. The transmission electron-microscopy technique showed the existence of ring-shaped particles with similar to 12 nm external diameter. Image analysis revealed that these particles were hexameric and resembled those formed by superfamily 3 DNA virus helicases. Show less
Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an... Show moreSome mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coil or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication. (C) 2010 Elsevier B.V. All rights reserved. Show less
This review focuses on bioinformatics technologies employed by the EU-sponsored multidisciplinary VIZIER consortium (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6... Show moreThis review focuses on bioinformatics technologies employed by the EU-sponsored multidisciplinary VIZIER consortium (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960, active from 1 November 2004 to 30 April 2009), to achieve its goals. From the management of the information flow of the project, to bioinformatics-mediated selection of RNA viruses and prediction of protein targets, to the analysis of 3D protein structures and antiviral compounds, these technologies provided a communication framework and integrated solutions for steady and timely advancement of the project. RNA viruses form a large class of major pathogens that affect humans and domestic animals. Such RNA viruses as HIV, Influenza virus and Hepatitis C virus are of prime medical concern today, but the identities of viruses that will threaten human population tomorrow are far from certain. To contain outbreaks of common or newly emerging infections, prototype drugs against viruses representing the Virus Universe must be developed. This concept was championed by the VIZIER project which brought together experts in diverse fields to produce a concerted and sustained effort for identifying and validating targets for antivirus therapy in dozens of RNA virus lineages. (C) 2010 Elsevier B.V. All rights reserved. Show less
The alphaviruses were amongst the first arboviruses to be isolated, characterized and assigned a taxonomic status. They are globally very widespread, infecting a large variety of terrestrial... Show moreThe alphaviruses were amongst the first arboviruses to be isolated, characterized and assigned a taxonomic status. They are globally very widespread, infecting a large variety of terrestrial animals, insects and even fish, and circulate both in the sylvatic and urban/peri-urban environment, causing considerable human morbidity and mortality. Nevertheless, despite their obvious importance as pathogens, there are currently no effective antiviral drugs with which to treat humans or animals infected by any of these viruses. The EU-supported project VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the alphaviruses [Coutard, B., Gorbalenya, A.E., Snijder, E.J., Leontovich, A.M., Poupon, A., De Lamballerie, X., Charrel, R., Gould, E.A., Gunther, S., Norder, H., Klempa, B., Bourhy, H., Rohayemj, J., L'hermite, E., Nordlund, P., Stuart, D.I., Owens, R.J., Grimes, J.M., Tuckerm, PA., Bolognesi, M., Mattevi, A., Coll, M., Jones, TA., Aqvist, J., Unger, T., Hilgenfeld, R., Bricogne, G., Neyts, J., La Colla, P., Puerstinger, G., Gonzalez, J.P., Leroy, E., Cambillau, C., Romette, J.L., Canard, B., 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res. 78, 37-46]. This review highlights some of the major features of alphaviruses that have been investigated during recent years. After describing their classification, epidemiology and evolutionary history and the expanding geographic distribution of Chikungunya virus, we review progress in understanding the structure and function of alphavirus replicative enzymes achieved under the VIZIER programme and the development of new disease control strategies. (C) 2009 Elsevier B.V. All rights reserved. Show less
This review focuses on bioinformatics technologies employed by the EU-sponsored multidisciplinary VIZIER consortium (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6... Show moreThis review focuses on bioinformatics technologies employed by the EU-sponsored multidisciplinary VIZIER consortium (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960, active from 1 November 2004 to 30 April 2009), to achieve its goals. From the management of the information flow of the project, to bioinformatics-mediated selection of RNA viruses and prediction of protein targets, to the analysis of 3D protein structures and antiviral compounds, these technologies provided a communication framework and integrated solutions for steady and timely advancement of the project. RNA viruses form a large class of major pathogens that affect humans and domestic animals. Such RNA viruses as HIV, Influenza virus and Hepatitis C virus are of prime medical concern today, but the identities of viruses that will threaten human population tomorrow are far from certain. To contain outbreaks of common or newly emerging infections, prototype drugs against viruses representing the Virus Universe must be developed. This concept was championed by the VIZIER project which brought together experts in diverse fields to produce a concerted and sustained effort for identifying and validating targets for antivirus therapy in dozens of RNA virus lineages. Show less
