The Czech Republic, a part of the former Czechoslovakia, has been at the forefront of several research directions in virology, genetics and physiology [...].

• MeSH
• virologie * MeSH
• Publikační typ
• práce podpořená grantem MeSH
• úvodníky MeSH
• Geografické názvy
• Česká republika MeSH

Positive-sense single-stranded RNA (+RNA) viruses have proven to be important pathogens that are able to threaten and deeply damage modern societies, as illustrated by the ongoing COVID-19 pandemic. Therefore, compounds active against most or many +RNA viruses are urgently needed. Here, we present PR673, a helquat-like compound that is able to inhibit the replication of SARS-CoV-2 and tick-borne encephalitis virus in cell culture. Using in vitro polymerase assays, we demonstrate that PR673 inhibits RNA synthesis by viral RNA-dependent RNA polymerases (RdRps). Our results illustrate that the development of broad-spectrum non-nucleoside inhibitors of RdRps is feasible.

• Klíčová slova
• Flaviruses, RNA-dependent RNA-polymerase, SARS-CoV-2, antiviral agents, helquat-like compound,
• MeSH
• COVID-19 * MeSH
• lidé MeSH
• pandemie MeSH
• RNA-dependentní RNA-polymerasa MeSH
• SARS-CoV-2 MeSH
• viry klíšťové encefalitidy * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA-dependentní RNA-polymerasa MeSH

Coronaviral methyltransferases (MTases), nsp10/16 and nsp14, catalyze the last two steps of viral RNA-cap creation that takes place in cytoplasm. This cap is essential for the stability of viral RNA and, most importantly, for the evasion of innate immune system. Non-capped RNA is recognized by innate immunity which leads to its degradation and the activation of antiviral immunity. As a result, both coronaviral MTases are in the center of scientific scrutiny. Recently, X-ray and cryo-EM structures of both enzymes were solved even in complex with other parts of the viral replication complex. High-throughput screening as well as structure-guided inhibitor design have led to the discovery of their potent inhibitors. Here, we critically summarize the tremendous advancement of the coronaviral MTase field since the beginning of COVID pandemic.

• MeSH
• aminokyseliny chemie   MeSH
• Coronavirus účinky léků   enzymologie   genetika   MeSH
• lidé MeSH
• methyltransferasy antagonisté a inhibitory   chemie   metabolismus   MeSH
• metylace MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• objevování léků MeSH
• RNA virová chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• methyltransferasy MeSH
• RNA virová 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

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.

• Klíčová slova
• SARS-CoV-2, capping enzyme, coronavirus, methyltransferase, nsp10, nsp14, nsp16,
• MeSH
• COVID-19 virologie   MeSH
• lidé MeSH
• methyltransferasy analýza   genetika   metabolismus   MeSH
• monoklonální protilátky analýza   MeSH
• RNA čepičky genetika   metabolismus   MeSH
• RNA virová genetika   metabolismus   MeSH
• SARS-CoV-2 chemie   enzymologie   genetika   MeSH
• transport proteinů MeSH
• virové nestrukturální proteiny analýza   genetika   metabolismus   MeSH
• virové regulační a přídatné proteiny analýza   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methyltransferasy MeSH
• monoklonální protilátky 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