title: 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.
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.
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Monique
authors.initials: M
authors.lastname: Oostra
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: de Haan
authors.forename: Peter J M
authors.initials: PJ
authors.lastname: Rottier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008969:Molecular Sequence Data; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D017384:Sequence Deletion; D045169:Severe Acute Respiratory Syndrome / Q000821:virology; D013045:Species Specificity; D014709:Vero Cells; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D014763:Viral Matrix Proteins; D014764:Viral Proteins; C488151:sars7a protein, SARS virus