Mutagenesis of S-Adenosyl-l-Methionine-Binding Residues in Coronavirus nsp14 N7-Methyltransferase Demonstrates Differing Requirements for Genome Translation and Resistance to Innate Immunity.
Identifieur interne : 001099 ( Main/Exploration ); précédent : 001098; suivant : 001100Mutagenesis of S-Adenosyl-l-Methionine-Binding Residues in Coronavirus nsp14 N7-Methyltransferase Demonstrates Differing Requirements for Genome Translation and Resistance to Innate Immunity.
Auteurs : James Brett Case [États-Unis] ; Alison W. Ashbrook [États-Unis] ; Terence S. Dermody [États-Unis] ; Mark R. Denison [États-Unis]Source :
- Journal of virology [ 1098-5514 ] ; 2016.
Descripteurs français
- KwdFr :
- ARN viral (génétique), ARN viral (métabolisme), Adémétionine (), Adémétionine (métabolisme), Analyse de mutations d'ADN, Animaux, Antiviraux (pharmacologie), Biosynthèse des protéines, Cellules cultivées, Coronavirus (enzymologie), Génome viral (physiologie), Humains, Immunité innée (immunologie), Immunomodulation, Interféron bêta (pharmacologie), Mutagenèse, Mutation (génétique), Protéines virales non structurales (), Protéines virales non structurales (génétique), Protéines virales non structurales (métabolisme), Réaction de polymérisation en chaine en temps réel, Réplication virale, Similitude de séquences d'acides aminés, Souris, Souris de lignée C57BL, Souris knockout, Séquence d'acides aminés, Tumeurs du cerveau (enzymologie), Tumeurs du cerveau (génétique), Tumeurs du cerveau (immunologie), Tumeurs du cerveau (virologie).
- MESH :
- enzymologie : Coronavirus, Tumeurs du cerveau.
- génétique : ARN viral, Mutation, Protéines virales non structurales, Tumeurs du cerveau.
- immunologie : Immunité innée, Tumeurs du cerveau.
- métabolisme : ARN viral, Adémétionine, Protéines virales non structurales.
- pharmacologie : Antiviraux, Interféron bêta.
- physiologie : Génome viral.
- virologie : Tumeurs du cerveau.
- Adémétionine, Analyse de mutations d'ADN, Animaux, Biosynthèse des protéines, Cellules cultivées, Humains, Immunomodulation, Mutagenèse, Protéines virales non structurales, Réaction de polymérisation en chaine en temps réel, Réplication virale, Similitude de séquences d'acides aminés, Souris, Souris de lignée C57BL, Souris knockout, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Amino Acid Sequence, Animals, Antiviral Agents (pharmacology), Brain Neoplasms (enzymology), Brain Neoplasms (genetics), Brain Neoplasms (immunology), Brain Neoplasms (virology), Cells, Cultured, Coronavirus (enzymology), DNA Mutational Analysis, Genome, Viral (physiology), Humans, Immunity, Innate (immunology), Immunomodulation, Interferon-beta (pharmacology), Mice, Mice, Inbred C57BL, Mice, Knockout, Mutagenesis, Mutation (genetics), Protein Biosynthesis, RNA, Viral (genetics), RNA, Viral (metabolism), Real-Time Polymerase Chain Reaction, S-Adenosylmethionine (chemistry), S-Adenosylmethionine (metabolism), Sequence Homology, Amino Acid, Viral Nonstructural Proteins (chemistry), Viral Nonstructural Proteins (genetics), Viral Nonstructural Proteins (metabolism), Virus Replication.
- MESH :
- chemical , chemistry : S-Adenosylmethionine, Viral Nonstructural Proteins.
- chemical , genetics : RNA, Viral, Viral Nonstructural Proteins.
- chemical , metabolism : RNA, Viral, S-Adenosylmethionine, Viral Nonstructural Proteins.
- chemical , pharmacology : Antiviral Agents, Interferon-beta.
- enzymology : Brain Neoplasms, Coronavirus.
- genetics : Brain Neoplasms, Mutation.
- immunology : Brain Neoplasms, Immunity, Innate.
- physiology : Genome, Viral.
- virology : Brain Neoplasms.
- Amino Acid Sequence, Animals, Cells, Cultured, DNA Mutational Analysis, Humans, Immunomodulation, Mice, Mice, Inbred C57BL, Mice, Knockout, Mutagenesis, Protein Biosynthesis, Real-Time Polymerase Chain Reaction, Sequence Homology, Amino Acid, Virus Replication.
Abstract
Eukaryotic mRNAs possess a methylated 5'-guanosine cap that is required for RNA stability, efficient translation, and protection from cell-intrinsic defenses. Many viruses use 5' caps or other mechanisms to mimic a cap structure to limit detection of viral RNAs by intracellular innate sensors and to direct efficient translation of viral proteins. The coronavirus (CoV) nonstructural protein 14 (nsp14) is a multifunctional protein with N7-methyltransferase (N7-MTase) activity. The highly conserved S-adenosyl-l-methionine (SAM)-binding residues of the DxG motif are required for nsp14 N7-MTase activity in vitro However, the requirement for CoV N7-MTase activity and the importance of the SAM-binding residues during viral replication have not been determined. Here, we engineered mutations in murine hepatitis virus (MHV) nsp14 N7-MTase at residues D330 and G332 and determined the effects of these mutations on viral replication, sensitivity to mutagen, inhibition by type I interferon (IFN), and translation efficiency. Virus encoding a G332A substitution in nsp14 displayed delayed replication kinetics and decreased peak titers relative to wild-type (WT) MHV. In addition, replication of nsp14 G332A virus was diminished following treatment of cells with IFN-β, and nsp14 G332A genomes were translated less efficiently both in vitro and during viral infection. In contrast, substitution of alanine at MHV nsp14 D330 did not affect viral replication, sensitivity to mutagen, or inhibition by IFN-β compared to WT MHV. Our results demonstrate that the conserved MHV N7-MTase SAM-binding-site residues are not required for MHV viability and suggest that the determinants of CoV N7-MTase activity differ in vitro and during virus infection.
DOI: 10.1128/JVI.00542-16
PubMed: 27252528
Affiliations:
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protéines</term><term>Cellules cultivées</term><term>Humains</term><term>Immunomodulation</term><term>Mutagenèse</term><term>Protéines virales non structurales</term><term>Réaction de polymérisation en chaine en temps réel</term><term>Réplication virale</term><term>Similitude de séquences d'acides aminés</term><term>Souris</term><term>Souris de lignée C57BL</term><term>Souris knockout</term><term>Séquence d'acides aminés</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Eukaryotic mRNAs possess a methylated 5'-guanosine cap that is required for RNA stability, efficient translation, and protection from cell-intrinsic defenses. Many viruses use 5' caps or other mechanisms to mimic a cap structure to limit detection of viral RNAs by intracellular innate sensors and to direct efficient translation of viral proteins. The coronavirus (CoV) nonstructural protein 14 (nsp14) is a multifunctional protein with N7-methyltransferase (N7-MTase) activity. The highly conserved S-adenosyl-l-methionine (SAM)-binding residues of the DxG motif are required for nsp14 N7-MTase activity in vitro However, the requirement for CoV N7-MTase activity and the importance of the SAM-binding residues during viral replication have not been determined. Here, we engineered mutations in murine hepatitis virus (MHV) nsp14 N7-MTase at residues D330 and G332 and determined the effects of these mutations on viral replication, sensitivity to mutagen, inhibition by type I interferon (IFN), and translation efficiency. Virus encoding a G332A substitution in nsp14 displayed delayed replication kinetics and decreased peak titers relative to wild-type (WT) MHV. In addition, replication of nsp14 G332A virus was diminished following treatment of cells with IFN-β, and nsp14 G332A genomes were translated less efficiently both in vitro and during viral infection. In contrast, substitution of alanine at MHV nsp14 D330 did not affect viral replication, sensitivity to mutagen, or inhibition by IFN-β compared to WT MHV. Our results demonstrate that the conserved MHV N7-MTase SAM-binding-site residues are not required for MHV viability and suggest that the determinants of CoV N7-MTase activity differ in vitro and during virus infection.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Tennessee</li></region></list><tree><country name="États-Unis"><region name="Tennessee"><name sortKey="Case, James Brett" sort="Case, James Brett" uniqKey="Case J" first="James Brett" last="Case">James Brett Case</name></region><name sortKey="Ashbrook, Alison W" sort="Ashbrook, Alison W" uniqKey="Ashbrook A" first="Alison W" last="Ashbrook">Alison W. Ashbrook</name><name sortKey="Denison, Mark R" sort="Denison, Mark R" uniqKey="Denison M" first="Mark R" last="Denison">Mark R. Denison</name><name sortKey="Dermody, Terence S" sort="Dermody, Terence S" uniqKey="Dermody T" first="Terence S" last="Dermody">Terence S. 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