Expression, post‐translational modification and biochemical characterization of proteins encoded by subgenomic mRNA8 of the severe acute respiratory syndrome coronavirus

Abstract

The most striking difference between the subgenomic mRNA8 of severe acute respiratory syndrome coronavirus isolated from human and some animal species is the deletion of 29 nucleotides, resulting in splitting of a single ORF (ORF8) into two ORFs (ORF8a and ORF8b). ORF8a and ORF8b are predicted to encode two small proteins, 8a and 8b, and ORF8 a single protein, 8ab (a fusion form of 8a and 8b). To understand the functions of these proteins, we cloned cDNA fragments covering these ORFs into expression plasmids, and expressed the constructs in both in vitro and in vivo systems. Expression of a construct containing ORF8a and ORF8b generated only a single protein, 8a; no 8b protein expression was obtained. Expression of a construct containing ORF8 generated the 8ab fusion protein. Site‐directed mutagenesis and enzymatic treatment revealed that protein 8ab is modified by N‐linked glycosylation on the N81 residue and by ubiquitination. In the absence of the 8a region, protein 8b undergoes rapid degradation by proteasomes, and addition of proteasome inhibitors inhibits the degradation of protein 8b as well as the protein 8b‐induced rapid degradation of the severe acute respiratory syndrome coronavirus E protein. Glycosylation could also stabilize protein 8ab. More interestingly, the two proteins could bind to monoubiquitin and polyubiquitin, suggesting the potential involvement of these proteins in the pathogenesis of severe acute respiratory syndrome coronavirus.

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

“…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

“…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].…”

“…This retention and accumulation of ORF8ab in the ER occurs in the absence of a recognized ER retention signal [110,117]. Not only do in vitro and in vivo studies show that ORF8ab is ubiquitinated, but both ORF8a and ORF8b also binds to monoubiquitin and polyubiquitin, suggesting the potential involvement of these proteins in the pathogenesis of SARS-CoV [116]. ORF8ab interacts with SARS-CoV proteins S, M, ORF3a and ORF7a [118], all of which have been shown to be structural proteins.…”

“…However, after it was cloned immediately downstream of a T7 promoter, a soluble, unmodified and monomeric ORF8b protein was expressed in the cytoplasm, which was extremely unstable and rapidly degraded [120]. Similarly, in the absence of the ORF8a region, ORF8b undergoes rapid degradation by proteasomes [116]. ORF8b down-regulates the expression of SARS-CoV E, a process that could be inhibited by the addition of proteasome inhibitors [116].…”