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.

Translational control of the subgenomic RNAs of severe acute respiratory syndrome coronavirus

“…As shown above, we were unable to detect the protein expression of sgRNA 8 with the 5 0 viral leader sequence, which corroborated with a recent report on ORF8 expression [13]. Cells transfected with p8 displayed significant fluorescence (Figs.…”

“…Although SARS-CoV 8b gene product could be expressed in vivo when cloned directly behind a promoter [11][12][13]; the expression of 8a and 8b in SARS-CoVinfected cells is still controversial [9,10,13]. As shown above, we were unable to detect the protein expression of sgRNA 8 with the 5 0 viral leader sequence, which corroborated with a recent report on ORF8 expression [13].…”

“…Ten sgRNAs have been identified [5] and the SARS-CoV accessory proteins 3a, 3b, 6,7a, and 7b can be detected in infected cells or SARS patients besides structural proteins [7,8], while the expression of 8a and 8b is controversial [9][10][11][12][13]. Elucidation of the regulatory mechanism in the translation is important for understanding the pathogenesis of SARS-CoV; however, it is hard to compare the differential translation of sgRNAs because the steady-state level of viral proteins in infected cells reflects the sum of transcription, translation, and the relative stabilities of these transcriptional and translational products.…”

The Role of Severe Acute Respiratory Syndrome (SARS)-Coronavirus Accessory Proteins in Virus Pathogenesis

“…Interestingly, this difference is due to a 29 nucleotide deletion in the mRNA8 of SARS-CoV from some animal species [110,111,112,113,114], that splits ORF 8 into ORF8a and ORF8b, encoding 39- and 84-residue polypeptides, respectively [110]. In fact, larger deletions were reported in sequenced ORF8 genes from a cluster of viruses that were isolated from humans later during the SARS epidemic [111,115].…”

“…Vaccinia virus expressed SARS-CoV ORF8ab, contains a cleavable signal sequence, which directs the movement of the precursor protein to the ER and then mediates its translocation into the ER lumen, where it is N-glycosylated on the N81 residue [110,116] and subsequently assembled into disulfide-linked homo-multimeric complexes, which remains stably in the ER [110]. This retention and accumulation of ORF8ab in the ER occurs in the absence of a recognized ER retention signal [110,117].…”