Monkeypox is a disease with pandemic potential. It is caused by the monkeypox virus (MPXV), a double-stranded DNA virus from the Poxviridae family, that replicates in the cytoplasm and must encode for its own RNA processing machinery including the capping machinery. Here, we present crystal structures of its 2'-O-RNA methyltransferase (MTase) VP39 in complex with the pan-MTase inhibitor sinefungin and a series of inhibitors that were discovered based on it. A comparison of this 2'-O-RNA MTase with enzymes from unrelated single-stranded RNA viruses (SARS-CoV-2 and Zika) reveals a conserved sinefungin binding mode, implicating that a single inhibitor could be used against unrelated viral families. Indeed, several of our inhibitors such as TO507 also inhibit the coronaviral nsp14 MTase.

Faculty of Food and Biochemical Technology University of Chemistry and Technology Prague 6 Czech Republic

Institute of Organic Chemistry and Biochemistry AS CR Prague 6 Czech Republic

Zobrazit více v PubMed

Rizk JG, Lippi G, Henry BM, Forthal DN, Rizk Y. Prevention and treatment of monkeypox. Drugs. 2022;82:957–963. doi: 10.1007/s40265-022-01742-y. PubMed DOI PMC

Beer EM, Rao VB. A systematic review of the epidemiology of human monkeypox outbreaks and implications for outbreak strategy. PLoS Negl. Trop. Dis. 2019;13:e0007791. doi: 10.1371/journal.pntd.0007791. PubMed DOI PMC

https://www.cdc.gov/poxvirus/monkeypox/index.html (2022).

Grimm C, Bartuli J, Fischer U. Cytoplasmic gene expression: lessons from poxviruses. Trends Biochem. Sci. 2022;47:892–902. doi: 10.1016/j.tibs.2022.04.010. PubMed DOI

Paterson BM, Rosenberg M. Efficient translation of prokaryotic mRNAs in a eukaryotic cell-free system requires addition of a cap structure. Nature. 1979;279:692–696. doi: 10.1038/279692a0. PubMed DOI

Both GW, Furuichi Y, Muthukrishnan S, Shatkin AJ. Ribosome binding to reovirus mRNA in protein synthesis requires 5’ terminal 7-methylguanosine. Cell. 1975;6:185–195. doi: 10.1016/0092-8674(75)90009-4. PubMed DOI

Mears HV, Sweeney TR. Better together: the role of IFIT protein-protein interactions in the antiviral response. J. Gen. Virol. 2018;99:1463–1477. doi: 10.1099/jgv.0.001149. PubMed DOI

Thoresen D, et al. The molecular mechanism of RIG-I activation and signaling. Immunol. Rev. 2021;304:154–168. doi: 10.1111/imr.13022. PubMed DOI PMC

Liu SW, Katsafanas GC, Liu R, Wyatt LS, Moss B. Poxvirus decapping enzymes enhance virulence by preventing the accumulation of dsRNA and the induction of innate antiviral responses. Cell Host Microbe. 2015;17:320–331. doi: 10.1016/j.chom.2015.02.002. PubMed DOI PMC

Georgana I, Sumner RP, Towers GJ, Maluquer de Motes C. Virulent poxviruses inhibit DNA sensing by preventing STING activation. J. Virol. 2018;92:e02145–17. doi: 10.1128/JVI.02145-17. PubMed DOI PMC

Shuman S. RNA capping: progress and prospects. RNA. 2015;21:735–737. doi: 10.1261/rna.049973.115. PubMed DOI PMC

Hyde JL, Diamond MS. Innate immune restriction and antagonism of viral RNA lacking 2-O methylation. Virology. 2015;479–480:66–74. doi: 10.1016/j.virol.2015.01.019. PubMed DOI PMC

Hodel AE, Gershon PD, Shi XN, Quiocho FA. The 1.85 angstrom structure of vaccinia protein VP39: a bifunctional enzyme that participates in the modification of both mRNA ends. Cell. 1996;85:247–256. doi: 10.1016/S0092-8674(00)81101-0. PubMed DOI

Krafcikova P, Silhan J, Nencka R, Boura E. Structural analysis of the SARS-CoV-2 methyltransferase complex involved in RNA cap creation bound to sinefungin. Nat. Commun. 2020;11:3717. doi: 10.1038/s41467-020-17495-9. PubMed DOI PMC

Benoni R, et al. Substrate specificity of SARS-CoV-2 Nsp10-Nsp16 methyltransferase. Viruses. 2021;13:1722. doi: 10.3390/v13091722. PubMed DOI PMC

Hodel AE, Gershon PD, Quiocho FA. Structural basis for sequence-nonspecific recognition of 5’-capped mRNA by a cap-modifying enzyme. Mol. Cell. 1998;1:443–447. doi: 10.1016/S1097-2765(00)80044-1. PubMed DOI

Li Z, Lazaridis T. Water at biomolecular binding interfaces. Phys. Chem. Chem. Phys. 2007;9:573–581. doi: 10.1039/B612449F. PubMed DOI

Samways ML, Taylor RD, Macdonald HEB, Essex JW. Water molecules at protein-drug interfaces: computational prediction and analysis methods. Chem. Soc. Rev. 2021;50:9104–9120. doi: 10.1039/D0CS00151A. PubMed DOI

Otava T, et al. The structure-based design of SARS-CoV-2 nsp14 methyltransferase ligands yields nanomolar inhibitors. ACS Infect. Dis. 2021;7:2214–2220. doi: 10.1021/acsinfecdis.1c00131. PubMed DOI

Czarna A, et al. Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity. Structure. 2022;30:1050–1054 e1052. doi: 10.1016/j.str.2022.04.014. PubMed DOI PMC

Kmiec D, Kirchhoff F. Monkeypox: a new threat? Int J. Mol. Sci. 2022;23:7866. doi: 10.3390/ijms23147866. PubMed DOI PMC

Bunge EM, et al. The changing epidemiology of human monkeypox-A potential threat? A systematic review. PLoS Negl. Trop. Dis. 2022;16:e0010141. doi: 10.1371/journal.pntd.0010141. PubMed DOI PMC

Rao, A. K. et al. Use of JYNNEOS Smallpox and Monkeypox Vaccine, Live, Nonreplicating for Preexposure Vaccination of Persons at Risk for Occupational Exposure to Orthopoxviruses: Recommendations of the Advisory Committee on Immunization Practices - United States, 2022. Mmwr-Morbid Mortal W.71, 734–742 (2022). PubMed PMC

Russo AT, et al. An overview of tecovirimat for smallpox treatment and expanded anti-orthopoxvirus applications. Expert Rev. Anti Infect. Ther. 2021;19:331–344. doi: 10.1080/14787210.2020.1819791. PubMed DOI PMC

Decroly E, Ferron F, Lescar J, Canard B. Conventional and unconventional mechanisms for capping viral mRNA. Nat. Rev. Microbiol. 2011;10:51–65. doi: 10.1038/nrmicro2675. PubMed DOI PMC

Brecher M, et al. Identification and characterization of novel broad-spectrum inhibitors of the flavivirus methyltransferase. ACS Infect. Dis. 2015;1:340–349. doi: 10.1021/acsinfecdis.5b00070. PubMed DOI PMC

Lim SP, et al. Small molecule inhibitors that selectively block dengue virus methyltransferase. J. Biol. Chem. 2011;286:6233–6240. doi: 10.1074/jbc.M110.179184. PubMed DOI PMC

Devkota K, et al. Probing the SAM binding site of SARS-CoV-2 Nsp14 in vitro using SAM competitive inhibitors guides developing selective bisubstrate inhibitors. SLAS Disco. 2021;26:1200–1211. doi: 10.1177/24725552211026261. PubMed DOI PMC

Khalili Yazdi A, et al. A high-throughput radioactivity-based assay for screening SARS-CoV-2 nsp10-nsp16 complex. SLAS Disco. 2021;26:757–765. doi: 10.1177/24725552211008863. PubMed DOI PMC

Klima M, et al. Crystal structure of SARS-CoV-2 nsp10-nsp16 in complex with small molecule inhibitors, SS148 and WZ16. Protein Sci. 2022;31:e4395. doi: 10.1002/pro.4395. PubMed DOI PMC

Ahmed-Belkacem R, et al. Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues. J. Med Chem. 2022;65:6231–6249. doi: 10.1021/acs.jmedchem.2c00120. PubMed DOI

Dostalik P, et al. Structural analysis of the OC43 coronavirus 2’-O-RNA methyltransferase. J. Virol. 2021;95:e0046321. doi: 10.1128/JVI.00463-21. PubMed DOI PMC

Li F, et al. SS148 and WZ16 inhibit the activities of nsp10-nsp16 complexes from all seven human pathogenic coronaviruses. Biochim. Biophys. Acta Gen. Subj. 2023;1867:130319. doi: 10.1016/j.bbagen.2023.130319. PubMed DOI PMC

Nencka R, et al. Coronaviral RNA-methyltransferases: function, structure and inhibition. Nucleic Acids Res. 2022;50:635–650. doi: 10.1093/nar/gkab1279. PubMed DOI PMC

Hercik K, Brynda J, Nencka R, Boura E. Structural basis of Zika virus methyltransferase inhibition by sinefungin. Arch. Virol. 2017;162:2091–2096. doi: 10.1007/s00705-017-3345-x. PubMed DOI

Mueller, U. et al. The macromolecular crystallography beamlines at BESSY II of the Helmholtz-Zentrum Berlin: Current status and perspectives. Eur. Phys. J. Plus130. 10.1140/epjp/i2015-15141-2, (2015).

Kabsch W. Xds. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2010;66:125–132. doi: 10.1107/S0907444909047337. PubMed DOI PMC

Hodel AE, Gershon PD, Shi XN, Wang SM, Quiocho FA. Specific protein recognition of an mRNA cap through its alkylated base. Nat. Struct. Biol. 1997;4:350–354. doi: 10.1038/nsb0597-350. PubMed DOI

McCoy AJ, et al. Phaser crystallographic software. J. Appl. Crystallogr. 2007;40:658–674. doi: 10.1107/S0021889807021206. PubMed DOI PMC

Liebschner D, et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D. 2019;75:861–877. doi: 10.1107/S2059798319011471. PubMed DOI PMC

Afonine PV, et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D. 2018;74:531–544. doi: 10.1107/S2059798318006551. PubMed DOI PMC

Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2010;66:486–501. doi: 10.1107/S0907444910007493. PubMed DOI PMC

Baranowski MR, et al. Synthesis of fluorophosphate nucleotide analogues and their characterization as tools for (1)(9)F NMR studies. J. Org. Chem. 2015;80:3982–3997. doi: 10.1021/acs.joc.5b00337. PubMed DOI

Structure of SARS-CoV-2 MTase nsp14 with the inhibitor STM957 reveals inhibition mechanism that is shared with a poxviral MTase VP39

Meeting report of the 37th International Conference on Antiviral Research in Gold Coast, Australia, May 20-24, 2024, organized by the International Society for Antiviral Research

Discovery of highly potent SARS-CoV-2 nsp14 methyltransferase inhibitors based on adenosine 5'-carboxamides

Structure of monkeypox virus poxin: implications for drug design