The nucleocapsid protein of the SARS-CoV-2 virus comprises two RNA-binding domains and three regions that are intrinsically disordered. While the structures of the RNA-binding domains have been solved using protein crystallography and NMR, current knowledge of the conformations of the full-length nucleocapsid protein is rather limited. To fill in this knowledge gap, we combined coarse-grained molecular simulations with data from small-angle X-ray scattering (SAXS) experiments using the ensemble refinement of SAXS (EROS) method. Our results show that the dimer of the full-length nucleocapsid protein exhibits large conformational fluctuations with its radius of gyration ranging from about 4 to 8 nm. The RNA-binding domains do not make direct contacts. The disordered region that links these two domains comprises a hydrophobic α-helix which makes frequent and nonspecific contacts with the RNA-binding domains. Each of the intrinsically disordered regions adopts conformations that are locally compact, yet on average, much more extended than Gaussian chains of equivalent lengths. We offer a detailed picture of the conformational ensemble of the nucleocapsid protein dimer under near-physiological conditions, which will be important for understanding the nucleocapsid assembly process.

• Keywords
• EROS, Nucleocapsid, SARS-CoV-2, SAXS,
• MeSH
• COVID-19 * MeSH
• X-Ray Diffraction MeSH
• Protein Conformation MeSH
• Humans MeSH
• Scattering, Small Angle MeSH
• Nucleocapsid Proteins chemistry   MeSH
• Nucleocapsid MeSH
• SARS-CoV-2 * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Nucleocapsid Proteins 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
• Amino Acids chemistry   MeSH
• Coronavirus drug effects   enzymology   genetics   MeSH
• Humans MeSH
• Methyltransferases antagonists & inhibitors   chemistry   metabolism   MeSH
• Methylation MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Drug Discovery MeSH
• RNA, Viral chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids MeSH
• Methyltransferases MeSH
• RNA, Viral 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.

• Keywords
• OC43, coronavirus, crystal structure, methyltransferase,
• MeSH
• Betacoronavirus enzymology   genetics   MeSH
• Protein Conformation, alpha-Helical MeSH
• Crystallography, X-Ray MeSH
• Methyltransferases chemistry   genetics   MeSH
• Binding Sites MeSH
• Viral Proteins chemistry   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Methyltransferases MeSH
• RNA 2'-O-methyltransferase MeSH Browser
• Viral Proteins MeSH

There is an urgent need for specific antiviral treatments directed against SARS-CoV-2 to prevent the most severe forms of COVID-19. By drug repurposing, affordable therapeutics could be supplied worldwide in the present pandemic context. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus could be a strategy to impede viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, a non-steroidal anti-inflammatory drug (NSAID) that was previously demonstrated to be active against Influenza A virus, were evaluated against SARS-CoV-2. Intrinsic fluorescence spectroscopy, fluorescence anisotropy, and dynamic light scattering assays demonstrated naproxen binding to the nucleoprotein of SARS-Cov-2 as predicted by molecular modeling. Naproxen impeded recombinant N oligomerization and inhibited viral replication in infected cells. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen specifically inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2-induced damage. No inhibition of viral replication was observed with paracetamol or the COX-2 inhibitor celecoxib. Thus, among the NSAID tested, only naproxen combined antiviral and anti-inflammatory properties. Naproxen addition to the standard of care could be beneficial in a clinical setting, as tested in an ongoing clinical study.

• Keywords
• SARS-CoV-2, antiviral, drug repurposing, inflammation, influenza, nucleoprotein, oligomerization, structure-based drug design,
• MeSH
• Anti-Inflammatory Agents, Non-Steroidal pharmacology   MeSH
• Antiviral Agents pharmacology   MeSH
• Cell Line MeSH
• Chlorocebus aethiops MeSH
• COVID-19 Drug Treatment * MeSH
• Humans MeSH
• Naproxen pharmacology   MeSH
• Nucleoproteins antagonists & inhibitors   metabolism   MeSH
• Drug Repositioning MeSH
• Virus Replication drug effects   MeSH
• SARS-CoV-2 drug effects   physiology   MeSH
• Molecular Docking Simulation MeSH
• Vero Cells MeSH
• Viral Proteins antagonists & inhibitors   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Inflammatory Agents, Non-Steroidal MeSH
• Antiviral Agents MeSH
• Naproxen MeSH
• Nucleoproteins MeSH
• Viral Proteins MeSH