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1.
Nucleic Acids Res ; 50(2): 635-650, 2022 01 25.
Article in English | MEDLINE | ID: covidwho-1621653

ABSTRACT

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.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus/drug effects , Coronavirus/enzymology , Methyltransferases/antagonists & inhibitors , Methyltransferases/chemistry , Methyltransferases/metabolism , Amino Acid Sequence , Amino Acids/chemistry , Binding Sites , Coronavirus/genetics , Drug Discovery , Humans , Methylation , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Binding , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , Structure-Activity Relationship
2.
2021.
Preprint in English | Other preprints | ID: ppcovidwho-294618

ABSTRACT

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) that 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 revealed 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 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.

3.
J Virol ; 95(15): e0046321, 2021 07 12.
Article in English | MEDLINE | ID: covidwho-1486505

ABSTRACT

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.


Subject(s)
Betacoronavirus/enzymology , Methyltransferases/chemistry , Viral Proteins/chemistry , Betacoronavirus/genetics , Binding Sites , Crystallography, X-Ray , Methyltransferases/genetics , Protein Conformation, alpha-Helical , Viral Proteins/genetics
4.
Molecules ; 26(13)2021 Jun 22.
Article in English | MEDLINE | ID: covidwho-1288958

ABSTRACT

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.


Subject(s)
Antiviral Agents/pharmacology , Fluorescence Resonance Energy Transfer/methods , High-Throughput Screening Assays/methods , Protease Inhibitors/pharmacology , RNA Viruses/enzymology , COVID-19/drug therapy , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/genetics , Coronavirus Papain-Like Proteases/metabolism , Drug Evaluation, Preclinical , Encephalitis Viruses, Tick-Borne/enzymology , Fluorescent Dyes/chemistry , Humans , RNA Helicases/antagonists & inhibitors , RNA Helicases/chemistry , RNA Helicases/genetics , RNA Helicases/metabolism , SARS-CoV-2/enzymology , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
5.
J Virol ; 95(15): e0046321, 2021 07 12.
Article in English | MEDLINE | ID: covidwho-1236419

ABSTRACT

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.


Subject(s)
Betacoronavirus/enzymology , Methyltransferases/chemistry , Viral Proteins/chemistry , Betacoronavirus/genetics , Binding Sites , Crystallography, X-Ray , Methyltransferases/genetics , Protein Conformation, alpha-Helical , Viral Proteins/genetics
6.
PLoS Pathog ; 16(12): e1009100, 2020 12.
Article in English | MEDLINE | ID: covidwho-954543

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition.


Subject(s)
COVID-19 , Molecular Docking Simulation , Nucleocapsid Proteins/chemistry , Phosphoproteins/chemistry , RNA, Viral/chemistry , SARS-CoV-2/chemistry , Humans , Magnetic Resonance Spectroscopy , Nucleocapsid Proteins/genetics , Phosphoproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics
7.
Nat Commun ; 11(1): 3717, 2020 07 24.
Article in English | MEDLINE | ID: covidwho-680539

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.


Subject(s)
Betacoronavirus/enzymology , Methyltransferases/chemistry , Viral Nonstructural Proteins/chemistry , Viral Regulatory and Accessory Proteins/chemistry , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine/pharmacology , COVID-19 , Catalytic Domain , Coronavirus Infections/virology , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Methyltransferases/metabolism , Models, Chemical , Models, Molecular , Pandemics , Pneumonia, Viral/virology , RNA Caps , RNA Stability , RNA, Viral/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism
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