The 29-Nucleotide Deletion Present in Human but Not in Animal Severe Acute Respiratory Syndrome Coronaviruses Disrupts the Functional Expression of Open Reading Frame 8▿
Identifieur interne : 001A83 ( Ncbi/Merge ); précédent : 001A82; suivant : 001A84The 29-Nucleotide Deletion Present in Human but Not in Animal Severe Acute Respiratory Syndrome Coronaviruses Disrupts the Functional Expression of Open Reading Frame 8▿
Auteurs : Monique Oostra [Pays-Bas] ; Cornelis A. M. De Haan [Pays-Bas] ; Peter J. M. Rottier [Pays-Bas]Source :
- Journal of Virology [ 0022-538X ] ; 2007.
Descripteurs français
- KwdFr :
- Animaux, Cellules Vero, Données de séquences moléculaires, Délétion de séquence, Humains, Lignée cellulaire, Protéines de la matrice virale (), Protéines de la matrice virale (génétique), Protéines de la matrice virale (métabolisme), Protéines virales (), Protéines virales (génétique), Protéines virales (métabolisme), Régulation de l'expression des gènes viraux, Spécificité d'espèce, Syndrome respiratoire aigu sévère (virologie), Séquence d'acides aminés, Virus du SRAS (génétique), Virus du SRAS (isolement et purification), Virus du SRAS (physiologie).
- MESH :
- génétique : Protéines de la matrice virale, Protéines virales, Virus du SRAS.
- isolement et purification : Virus du SRAS.
- métabolisme : Protéines de la matrice virale, Protéines virales.
- physiologie : Virus du SRAS.
- virologie : Syndrome respiratoire aigu sévère.
- Animaux, Cellules Vero, Données de séquences moléculaires, Délétion de séquence, Humains, Lignée cellulaire, Protéines de la matrice virale, Protéines virales, Régulation de l'expression des gènes viraux, Spécificité d'espèce, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Amino Acid Sequence, Animals, Cell Line, Chlorocebus aethiops, Gene Expression Regulation, Viral, Humans, Molecular Sequence Data, SARS Virus (genetics), SARS Virus (isolation & purification), SARS Virus (physiology), Sequence Deletion, Severe Acute Respiratory Syndrome (virology), Species Specificity, Vero Cells, Viral Matrix Proteins (chemistry), Viral Matrix Proteins (genetics), Viral Matrix Proteins (metabolism), Viral Proteins (chemistry), Viral Proteins (genetics), Viral Proteins (metabolism).
- MESH :
- chemical , chemistry : Viral Matrix Proteins, Viral Proteins.
- genetics : SARS Virus, Viral Matrix Proteins, Viral Proteins.
- isolation & purification : SARS Virus.
- chemical , metabolism : Viral Matrix Proteins, Viral Proteins.
- physiology : SARS Virus.
- virology : Severe Acute Respiratory Syndrome.
- Amino Acid Sequence, Animals, Cell Line, Chlorocebus aethiops, Gene Expression Regulation, Viral, Humans, Molecular Sequence Data, Sequence Deletion, Species Specificity, Vero Cells.
Abstract
One of the most striking and dramatic genomic changes observed in the severe acute respiratory syndrome coronavirus (SARS-CoV) isolated from humans soon after its zoonotic transmission from palm civets was the acquisition of a characteristic 29-nucleotide deletion. This occurred in open reading frame 8 (ORF8), one of the accessory genes unique to the SARS-CoV. The function of ORF8 and the significance of the deletion are unknown. The intact ORF8 present in animal and some early human isolates encodes a 122-amino-acid polypeptide (8ab+), which we expressed in cells using the vaccinia virus T7 expression system. It was found to contain a cleavable signal sequence, which directs the precursor to the endoplasmic reticulum (ER) and mediates its translocation into the lumen. The cleaved protein became N-glycosylated, assembled into disulfide-linked homomultimeric complexes, and remained stably in the ER. The 29-nucleotide deletion splits ORF8 into two ORFs, 8a and 8b, encoding 39- and 84-residue polypeptides. The 8a polypeptide is likely to remain in the cytoplasm, as it is too small for its signal sequence to function and will therefore be directly released from the ribosome. However, we could not confirm this experimentally due to the lack of proper antibodies. ORF8b appeared not to be expressed in SARS-CoV-infected cells or when expressed from mRNA's mimicking mRNA8. This was due to the context of the internal AUG initiation codon, as we demonstrated after placing the ORF8b immediately behind the T7 promoter. A soluble, unmodified and monomeric 8b protein was now expressed in the cytoplasm, which was highly unstable and rapidly degraded. Clearly, the 29-nucleotide deletion disrupts the proper expression of the SARS-CoV ORF8, the implications of which are discussed.
Url:
DOI: 10.1128/JVI.01631-07
PubMed: 17928347
PubMed Central: 2168875
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PMC:2168875Le document en format XML
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<front><div type="abstract" xml:lang="en"><p>One of the most striking and dramatic genomic changes observed in the severe acute respiratory syndrome coronavirus (SARS-CoV) isolated from humans soon after its zoonotic transmission from palm civets was the acquisition of a characteristic 29-nucleotide deletion. This occurred in open reading frame 8 (ORF8), one of the accessory genes unique to the SARS-CoV. The function of ORF8 and the significance of the deletion are unknown. The intact ORF8 present in animal and some early human isolates encodes a 122-amino-acid polypeptide (8ab<sup>+</sup>
), which we expressed in cells using the vaccinia virus T7 expression system. It was found to contain a cleavable signal sequence, which directs the precursor to the endoplasmic reticulum (ER) and mediates its translocation into the lumen. The cleaved protein became N-glycosylated, assembled into disulfide-linked homomultimeric complexes, and remained stably in the ER. The 29-nucleotide deletion splits ORF8 into two ORFs, 8a and 8b, encoding 39- and 84-residue polypeptides. The 8a polypeptide is likely to remain in the cytoplasm, as it is too small for its signal sequence to function and will therefore be directly released from the ribosome. However, we could not confirm this experimentally due to the lack of proper antibodies. ORF8b appeared not to be expressed in SARS-CoV-infected cells or when expressed from mRNA's mimicking mRNA8. This was due to the context of the internal AUG initiation codon, as we demonstrated after placing the ORF8b immediately behind the T7 promoter. A soluble, unmodified and monomeric 8b protein was now expressed in the cytoplasm, which was highly unstable and rapidly degraded. Clearly, the 29-nucleotide deletion disrupts the proper expression of the SARS-CoV ORF8, the implications of which are discussed.</p>
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<front><div type="abstract" xml:lang="en"><p>One of the most striking and dramatic genomic changes observed in the severe acute respiratory syndrome coronavirus (SARS-CoV) isolated from humans soon after its zoonotic transmission from palm civets was the acquisition of a characteristic 29-nucleotide deletion. This occurred in open reading frame 8 (ORF8), one of the accessory genes unique to the SARS-CoV. The function of ORF8 and the significance of the deletion are unknown. The intact ORF8 present in animal and some early human isolates encodes a 122-amino-acid polypeptide (8ab<sup>+</sup>
), which we expressed in cells using the vaccinia virus T7 expression system. It was found to contain a cleavable signal sequence, which directs the precursor to the endoplasmic reticulum (ER) and mediates its translocation into the lumen. The cleaved protein became N-glycosylated, assembled into disulfide-linked homomultimeric complexes, and remained stably in the ER. The 29-nucleotide deletion splits ORF8 into two ORFs, 8a and 8b, encoding 39- and 84-residue polypeptides. The 8a polypeptide is likely to remain in the cytoplasm, as it is too small for its signal sequence to function and will therefore be directly released from the ribosome. However, we could not confirm this experimentally due to the lack of proper antibodies. ORF8b appeared not to be expressed in SARS-CoV-infected cells or when expressed from mRNA's mimicking mRNA8. This was due to the context of the internal AUG initiation codon, as we demonstrated after placing the ORF8b immediately behind the T7 promoter. A soluble, unmodified and monomeric 8b protein was now expressed in the cytoplasm, which was highly unstable and rapidly degraded. Clearly, the 29-nucleotide deletion disrupts the proper expression of the SARS-CoV ORF8, the implications of which are discussed.</p>
</div>
</front>
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<pubmed><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">The 29-nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame 8.</title>
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<author><name sortKey="De Haan, Cornelis A M" sort="De Haan, Cornelis A M" uniqKey="De Haan C" first="Cornelis A M" last="De Haan">Cornelis A M. De Haan</name>
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<term>Chlorocebus aethiops</term>
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<term>Humans</term>
<term>Molecular Sequence Data</term>
<term>SARS Virus (genetics)</term>
<term>SARS Virus (isolation & purification)</term>
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<term>Données de séquences moléculaires</term>
<term>Délétion de séquence</term>
<term>Humains</term>
<term>Lignée cellulaire</term>
<term>Protéines de la matrice virale ()</term>
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<term>Protéines de la matrice virale (métabolisme)</term>
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<front><div type="abstract" xml:lang="en">One of the most striking and dramatic genomic changes observed in the severe acute respiratory syndrome coronavirus (SARS-CoV) isolated from humans soon after its zoonotic transmission from palm civets was the acquisition of a characteristic 29-nucleotide deletion. This occurred in open reading frame 8 (ORF8), one of the accessory genes unique to the SARS-CoV. The function of ORF8 and the significance of the deletion are unknown. The intact ORF8 present in animal and some early human isolates encodes a 122-amino-acid polypeptide (8ab(+)), which we expressed in cells using the vaccinia virus T7 expression system. It was found to contain a cleavable signal sequence, which directs the precursor to the endoplasmic reticulum (ER) and mediates its translocation into the lumen. The cleaved protein became N-glycosylated, assembled into disulfide-linked homomultimeric complexes, and remained stably in the ER. The 29-nucleotide deletion splits ORF8 into two ORFs, 8a and 8b, encoding 39- and 84-residue polypeptides. The 8a polypeptide is likely to remain in the cytoplasm, as it is too small for its signal sequence to function and will therefore be directly released from the ribosome. However, we could not confirm this experimentally due to the lack of proper antibodies. ORF8b appeared not to be expressed in SARS-CoV-infected cells or when expressed from mRNA's mimicking mRNA8. This was due to the context of the internal AUG initiation codon, as we demonstrated after placing the ORF8b immediately behind the T7 promoter. A soluble, unmodified and monomeric 8b protein was now expressed in the cytoplasm, which was highly unstable and rapidly degraded. Clearly, the 29-nucleotide deletion disrupts the proper expression of the SARS-CoV ORF8, the implications of which are discussed.</div>
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