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.

• MeSH
• COVID-19 * MeSH
• infekce virem zika * MeSH
• lidé MeSH
• methyltransferasy metabolismus   MeSH
• RNA virová genetika   MeSH
• RNA MeSH
• SARS-CoV-2 genetika   MeSH
• virové nestrukturální proteiny chemie   MeSH
• virus opičích neštovic genetika   metabolismus   MeSH
• virus zika * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methyltransferasy MeSH
• RNA virová MeSH
• RNA MeSH
• virové nestrukturální proteiny MeSH

The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2'-O-MTase.

• Klíčová slova
• SARS-CoV-2, inhibitor, methylation, virus,
• MeSH
• chromatografie kapalinová MeSH
• COVID-19 virologie   MeSH
• hmotnostní spektrometrie MeSH
• lidé MeSH
• methyltransferasy genetika   metabolismus   MeSH
• metylace MeSH
• regulace exprese virových genů MeSH
• RNA čepičky MeSH
• RNA virová genetika   MeSH
• SARS-CoV-2 enzymologie   genetika   MeSH
• substrátová specifita MeSH
• virové nestrukturální proteiny genetika   metabolismus   MeSH
• virové regulační a přídatné proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methyltransferasy MeSH
• NSP10 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP16 protein, SARS-CoV-2 MeSH Prohlížeč
• RNA čepičky MeSH
• RNA virová MeSH
• virové nestrukturální proteiny MeSH
• virové regulační a přídatné proteiny MeSH

The OC43 coronavirus is a human pathogen that usually causes only the common cold. One of its key enzymes, similar to other coronaviruses, is the 2'-O-RNA methyltransferase (MTase), which is essential for viral RNA stability and expression. Here, we report the crystal structure of the 2'-O-RNA MTase in a complex with the pan-methyltransferase inhibitor sinefungin solved at 2.2-Å resolution. The structure reveals an overall fold consistent with the fold observed in other coronaviral MTases. The major differences are in the conformation of the C terminus of the nsp16 subunit and an additional helix in the N terminus of the nsp10 subunits. The structural analysis also revealed very high conservation of the S-adenosyl methionine (SAM) binding pocket, suggesting that the SAM pocket is a suitable spot for the design of antivirals effective against all human coronaviruses. IMPORTANCE Some coronaviruses are dangerous pathogens, while some cause only common colds. The reasons are not understood, although the spike proteins probably play an important role. However, to understand the coronaviral biology in sufficient detail, we need to compare the key enzymes from different coronaviruses. We solved the crystal structure of 2'-O-RNA methyltransferase of the OC43 coronavirus, a virus that usually causes mild colds. The structure revealed some differences in the overall fold but also revealed that the SAM binding site is conserved, suggesting that development of antivirals against multiple coronaviruses is feasible.

• Klíčová slova
• OC43, coronavirus, crystal structure, methyltransferase,
• MeSH
• Betacoronavirus enzymologie   genetika   MeSH
• konformace proteinů, alfa-helix MeSH
• krystalografie rentgenová MeSH
• methyltransferasy chemie   genetika   MeSH
• vazebná místa MeSH
• virové proteiny chemie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methyltransferasy MeSH
• RNA 2'-O-methyltransferase MeSH Prohlížeč
• virové proteiny MeSH