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Article: Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants.

Wilson, Isha M / Frazier, Meredith N / Li, Jian-Liang / Randall, Thomas A / Stanley, Robin E

bioRxiv : the preprint server for biology

2022

Abstract: Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of ... ...

Abstract	Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9+ million SARS-CoV-2 sequences revealed mutations across Nsp15â€™s three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants
Language	English
Publishing date	2022-05-12
Publishing country	United States
Document type	Preprint
DOI	10.1101/2022.05.10.491349
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Recent insights into the structure and function of coronavirus ribonucleases.

Frazier, Meredith N / Riccio, Amanda A / Wilson, Isha M / Copeland, William C / Stanley, Robin E

FEBS open bio

2022 Volume 12, Issue 9, Page(s) 1567–1583

Abstract: Coronaviruses use approximately two-thirds of their 30-kb genomes to encode nonstructural proteins (nsps) with diverse functions that assist in viral replication and transcription, and evasion of the host immune response. The SARS-CoV-2 pandemic has led ... ...

Abstract	Coronaviruses use approximately two-thirds of their 30-kb genomes to encode nonstructural proteins (nsps) with diverse functions that assist in viral replication and transcription, and evasion of the host immune response. The SARS-CoV-2 pandemic has led to renewed interest in the molecular mechanisms used by coronaviruses to infect cells and replicate. Among the 16 Nsps involved in replication and transcription, coronaviruses encode two ribonucleases that process the viral RNA-an exonuclease (Nsp14) and an endonuclease (Nsp15). In this review, we discuss recent structural and biochemical studies of these nucleases and the implications for drug discovery.
MeSH term(s)	COVID-19 ; Humans ; Mutation ; Ribonucleases ; SARS-CoV-2 ; Viral Nonstructural Proteins/chemistry ; Viral Nonstructural Proteins/genetics ; Viral Nonstructural Proteins/metabolism
Chemical Substances	Viral Nonstructural Proteins ; Ribonucleases (EC 3.1.-)
Language	English
Publishing date	2022-04-29
Publishing country	England
Document type	Journal Article ; Review ; Research Support, N.I.H., Extramural ; Research Support, N.I.H., Intramural
ZDB-ID	2651702-4
ISSN	2211-5463 ; 2211-5463
ISSN (online)	2211-5463
ISSN	2211-5463
DOI	10.1002/2211-5463.13414
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants.

Wilson, Isha M / Frazier, Meredith N / Li, Jian-Liang / Randall, Thomas A / Stanley, Robin E

Journal of molecular biology

2022 Volume 434, Issue 20, Page(s) 167796

Abstract	Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9 + million SARS-CoV-2 sequences revealed mutations across Nsp15's three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo.
MeSH term(s)	COVID-19/virology ; Endoribonucleases/chemistry ; Endoribonucleases/genetics ; Humans ; Recombinant Proteins/chemistry ; SARS-CoV-2/enzymology ; Uridylate-Specific Endoribonucleases/chemistry ; Uridylate-Specific Endoribonucleases/genetics ; Viral Nonstructural Proteins/chemistry ; Viral Nonstructural Proteins/genetics
Chemical Substances	Recombinant Proteins ; Viral Nonstructural Proteins ; Endoribonucleases (EC 3.1.-) ; Uridylate-Specific Endoribonucleases (EC 3.1.-) ; nidoviral uridylate-specific endoribonuclease (EC 3.1.-)
Language	English
Publishing date	2022-08-19
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Intramural
ZDB-ID	80229-3
ISSN	1089-8638 ; 0022-2836
ISSN (online)	1089-8638
ISSN	0022-2836
DOI	10.1016/j.jmb.2022.167796
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Recent insights into the structure and function of coronavirus ribonucleases

Frazier, Meredith N. / Riccio, Amanda A. / Wilson, Isha M. / Copeland, William C. / Stanley, Robin E.

FEBS Open Bio. 2022 Sept., v. 12, no. 9

2022

Abstract: Coronaviruses use approximately two‐thirds of their 30‐kb genomes to encode nonstructural proteins (nsps) with diverse functions that assist in viral replication and transcription, and evasion of the host immune response. The SARS‐CoV‐2 pandemic has led ... ...

Abstract	Coronaviruses use approximately two‐thirds of their 30‐kb genomes to encode nonstructural proteins (nsps) with diverse functions that assist in viral replication and transcription, and evasion of the host immune response. The SARS‐CoV‐2 pandemic has led to renewed interest in the molecular mechanisms used by coronaviruses to infect cells and replicate. Among the 16 Nsps involved in replication and transcription, coronaviruses encode two ribonucleases that process the viral RNA—an exonuclease (Nsp14) and an endonuclease (Nsp15). In this review, we discuss recent structural and biochemical studies of these nucleases and the implications for drug discovery.
Keywords	Severe acute respiratory syndrome coronavirus 2 ; drugs ; genome ; immune response ; pandemic ; ribonucleases ; virus replication
Language	English
Dates of publication	2022-09
Size	p. 1567-1583.
Publishing place	John Wiley & Sons, Ltd
Document type	Article
Note	REVIEW
ZDB-ID	2651702-4
ISSN	2211-5463
ISSN	2211-5463
DOI	10.1002/2211-5463.13414
Database	NAL-Catalogue (AGRICOLA)

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Article: Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants

Wilson, Isha M. / Frazier, Meredith N. / Li, Jian-Liang / Randall, Thomas A. / Stanley, Robin E.

Journal of molecular biology. 2022 Aug. 15,

2022

Abstract	Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9 + million SARS-CoV-2 sequences revealed mutations across Nsp15′s three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo.
Keywords	COVID-19 infection ; RNA ; Severe acute respiratory syndrome coronavirus 2 ; bioinformatics ; drug design ; evolution ; immune response ; innate immunity ; molecular biology ; oligomerization ; pandemic ; patients ; recombinant proteins ; viral genome ; virus replication ; viruses
Language	English
Dates of publication	2022-0815
Publishing place	Elsevier Ltd
Document type	Article
Note	Pre-press version
ZDB-ID	80229-3
ISSN	1089-8638 ; 0022-2836
ISSN (online)	1089-8638
ISSN	0022-2836
DOI	10.1016/j.jmb.2022.167796
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Flipped over U: structural basis for dsRNA cleavage by the SARS-CoV-2 endoribonuclease.

Frazier, Meredith N / Wilson, Isha M / Krahn, Juno M / Butay, Kevin John / Dillard, Lucas B / Borgnia, Mario J / Stanley, Robin E

Nucleic acids research

2022 Volume 50, Issue 14, Page(s) 8290–8301

Abstract: Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is a uridine-specific ... ...

Abstract	Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is a uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.
MeSH term(s)	COVID-19 ; Endoribonucleases/metabolism ; Humans ; RNA, Double-Stranded/genetics ; SARS-CoV-2/genetics ; Uridine ; Viral Nonstructural Proteins/metabolism
Chemical Substances	RNA, Double-Stranded ; Viral Nonstructural Proteins ; Endoribonucleases (EC 3.1.-) ; Uridine (WHI7HQ7H85)
Language	English
Publishing date	2022-07-08
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Intramural
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkac589
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Zs.A 1067: Show issues

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Article: Flipped Over U: Structural Basis for dsRNA Cleavage by the SARS-CoV-2 Endoribonuclease.

Frazier, Meredith N / Wilson, Isha M / Krahn, Juno M / Butay, Kevin John / Dillard, Lucas B / Borgnia, Mario J / Stanley, Robin E

bioRxiv : the preprint server for biology

2022

Abstract	Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.
Language	English
Publishing date	2022-03-02
Publishing country	United States
Document type	Preprint
DOI	10.1101/2022.03.02.480688
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants

Wilson, Isha M. / Frazier, Meredith N. / Li, Jian-Liang / Randall, Thomas A. / Stanley, Robin E.

bioRxiv

Abstract	Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9+ million SARS-CoV-2 sequences revealed mutations across Nsp159s three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo.
Keywords	covid19
Language	English
Publishing date	2022-05-12
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2022.05.10.491349
Database	COVID19

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Article ; Online: Flipped Over U: Structural Basis for dsRNA Cleavage by the SARS-CoV-2 Endoribonuclease

Frazier, Meredith N / Wilson, Isha M / Krahn, Juno M / Butay, Kevin John / Dillard, Lucas B / Borgnia, Mario J. N / Stanley, Robin E

bioRxiv

Abstract	Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.
Keywords	covid19
Language	English
Publishing date	2022-03-02
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2022.03.02.480688
Database	COVID19

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Article ; Online: Characterization of SARS2 Nsp15 nuclease activity reveals it's mad about U.

Frazier, Meredith N / Dillard, Lucas B / Krahn, Juno M / Perera, Lalith / Williams, Jason G / Wilson, Isha M / Stewart, Zachary D / Pillon, Monica C / Deterding, Leesa J / Borgnia, Mario J / Stanley, Robin E

Nucleic acids research

2021 Volume 49, Issue 17, Page(s) 10136–10149

Abstract: Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved ...

Abstract	Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3' of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here, we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3' of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA, and will aid in the development of new anti-viral therapeutics.
MeSH term(s)	COVID-19/virology ; Catalytic Domain/genetics ; Cryoelectron Microscopy ; Crystallography, X-Ray ; Endoribonucleases/metabolism ; Humans ; Molecular Dynamics Simulation ; Protein Multimerization/physiology ; RNA, Viral/genetics ; RNA, Viral/metabolism ; SARS-CoV-2/genetics ; Substrate Specificity ; Uridine/chemistry ; Viral Nonstructural Proteins/metabolism
Chemical Substances	RNA, Viral ; Viral Nonstructural Proteins ; Endoribonucleases (EC 3.1.-) ; nidoviral uridylate-specific endoribonuclease (EC 3.1.-) ; Uridine (WHI7HQ7H85)
Language	English
Publishing date	2021-08-13
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Intramural
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkab719
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