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. 2007 Aug;274(16):4211-22.
doi: 10.1111/j.1742-4658.2007.05947.x. Epub 2007 Jul 20.

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

Tra M Le  1 , Hui H Wong, Felicia P L Tay, Shouguo Fang, Choong-Tat Keng, Yee J Tan, Ding X Liu
Affiliations

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

Tra M Le et al. FEBS J. 2007 Aug.

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.

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Figures

Figure 1
Figure 1
Diagram showing the six constructs, pF‐8a, pF‐8b, pF‐8a/8b, p8a/b, pF‐8ab and pF‐8abM, used in this study. The nucleotide positions of ORF8a are shown in bold, and the positions of ORF8b are underlined. Also shown are the nucleotide sequence of the 29 nucleotide insertion, the position of the insertion, the numbers of amino acids for the putative proteins 8a and 8b, and the Flag tag at the N‐terminus of each construct.
Figure 2
Figure 2
Expression of constructs covering the 5′‐unique ORFs of the subgenomic mRNA8 of SARS‐CoV. (A) Expression of p8a/b (lane 1), pF‐8a/b (lane 2), pF‐8b (lane 3) and pF‐8ab (lane 4) in wheat germ extracts in the presence of [35S]methionine. The in vitro‐synthesized products were resolved on SDS/20% polyacrylamide gel and detected by autoradiography. The numbers on the left indicate molecular masses in kilodaltons. (B) Expression of p8a/b (lanes 1 and 5), pF‐8a/b (lanes 2 and 6), pF‐8b (lanes 3 and 7) and pF‐8ab (lanes 4 and 8) in rabbit reticulocyte lysates in the presence of [35S]methionine. The in vitro‐synthesized products were resolved on SDS/20% polyacrylamide gel either directly (lanes 1–4) or after immunoprecipitation with polyclonal antibodies to protein 8b (lanes 5–8). Polypeptides were detected by autoradiography. The numbers on the left indicate molecular masses in kilodaltons. (C) Expression of p8a/b (lane 1), pF‐8a/b (lane 2), pF‐8b (lane 3) and pF‐8ab (lane 4) in Cos‐7 cells. Cells were infected with the recombinant vaccinia/T7 virus, transfected with each of the four constructs, and harvested at 18 h post‐transfection. Polypeptides were resolved on SDS/20% polyacrylamide gel, and analyzed by western blot with antibody to Flag. The numbers on the left indicate molecular masses in kilodaltons.
Figure 3
Figure 3
Binding of proteins 8b and 8ab to monoubiquitin and polyubiquitin. (A) Cos‐7 cells were transfected with the Myc‐tagged ubiquitin (lanes 3, 6 and 9), the Myc‐tagged ubiquitin together with the Flag‐tagged protein 8b (lanes 1, 4 and 7), and the Myc‐tagged ubiquitin together with the Flag‐tagged protein 8ab (lanes 2, 5 and 8). Total cell lysates were prepared, separated on SDS/20% polyacrylamide gel, and analyzed either directly by western blot (lanes 1–3) or after coimmunoprecipitation with antibody to Flag (lanes 4–9). The numbers on the left indicate molecular masses in kilodaltons. (B) Cos‐7 cells were transfected with the Myc‐tagged ubiquitin (lanes 1 and 5), the Flag‐tagged protein 8ab (lanes 4 and 8), the Myc‐tagged ubiquitin together with the Flag‐tagged protein 8b (lanes 2 and 6), and the Myc‐tagged ubiquitin together with the Flag‐tagged protein 8ab (lanes 3 and 7). Total cell lysates were prepared, separated on SDS/20% polyacrylamide gel, and analyzed either directly by western blot (lanes 1–4) or after coimmunoprecipitation with antibody to Myc (lanes 5–8). The numbers on the left indicate molecular masses in kilodaltons. (C) Pull‐down of monoubiquitin and polyubiquitin by GST‐8b and GST‐8ab fusion proteins. GST, GST‐8b and GST‐8ab were purified from bacteria, separated on SDS/12% polyacrylamide gel, and visualized by staining with Comassie blue (lanes 1–3). Cos‐7 cells expressing the Myc‐tagged ubiquitin were labeled with [35S]methionine. Total cell lysates were prepared and precleared by incubation with GST beads at 4 °C for 2 h, and then incubated with beads prebound with GST, GST‐8b, and GST‐8ab, respectively, at 4 °C for 2 h. After washing three times with RIPA buffer, polypeptides were eluted from the beads by adding 30 µL of the loading dye and boiling for 5 min. The total cell lysates (lane 4) and the eluted polypeptides (lanes 5–7) were separated on 20% SDS/20% polyacrylamide gel and visualized by autoradiography. The numbers on the left of each panel indicate molecular masses in kilodaltons.
Figure 4
Figure 4
Expression of proteins 8b and 8ab using an infectious clone system based on coronavirus IBV, and inhibition of the rapid degradation of protein 8b by the proteasome inhibitors lactacystin and NLVS. (A) Diagram showing the genome organization of the IBV genome and the position of the inserted 8b or 8ab gene. (B) Northern blot analysis of the genomic and subgenomic RNAs in cells infected with rIBV (wild type), rIBV/8b, and rIBV/8ab. Ten micrograms of total RNA extracted from Vero cells infected with rIBV and the fourth passage (p4) of rIBV/8b and rIBV/8ab, respectively, were separated on 1% agarose gel and transferred to a Hybond N+ membrane. Viral RNAs were probed with a Dig‐labeled DNA probe corresponding to the 3′‐UTR of the IBV genome. The numbers on the right indicate the genomic and subgenomic RNA species of IBV, and the additional mRNA species derived from the inserted 8b and 8ab genes are indicated by asterisks. (C) Western blot analysis of proteins 8b and 8ab in cells infected with rIBV, rIBV/8b, and rIBV/8ab. Vero cells infected with rIBV, rIBV/8b and rIBV/8ab were harvested at 24 h postinfection, and lysates prepared and separated on SDS/20% polyacrylamide gel. Expression of proteins 8b and 8ab was analyzed by western blot with polyclonal antibodies to protein 8b. The numbers on the left indicate molecular masses in kilodaltons. (D) Inhibition of the rapid degradation of protein 8b by lactacystin and NLVS. Vero cells infected with rIBV/8b at a multiplicity of infection of approximately 1 were treated with dimethylsulfoxide alone (lane 1), 8 µm lactacystin (lane 2) and 32 µm NLVS (lane 3), respectively, and harvested at 24 h postinfection. Total cell lysates were separated on SDS/17.5% polyacrylamide gel and analyzed by western blot with antibodies to IBV N (upper panel) and protein 8b (lower panel).
Figure 5
Figure 5
Inhibition of protein 8b‐mediated rapid degradation of the SARS‐CoV E protein by the proteasome inhibitors lactacystin, NLVS, and MG132. Cos‐7 (top panel) and 293T (other panels) cells expressing the SARS‐CoV E protein Alone (lane 1) or coexpressing with protein 8b (lanes 2 and 3) were incubated either with dimethylsulfoxide (lanes 1 and 2), with 8 µm lactacystin plus 32 µm NLVS (top panel, lane 3), or with 50 µm MG132 (other panels, lane 3), and harvested at 18 h post‐transfection. Total cell lysates were separated on SDS/17.5% polyacrylamide gel and analyzed by western blot with appropriate antibodies.
Figure 6
Figure 6
N‐linked glycosylation of protein 8ab. (A) Western blot analysis of proteins 8b and 8ab treated with PNGaseF (lanes 1–4), and detection of variable amounts of the N‐linked glycosylated protein 8ab in Cos‐7 and HeLa cells. Cos‐7 (lanes 1–6) and HeLa (lanes 7–8) cells were transfected with pF‐8b (lanes 1, 3, 5 and 7) and pF‐8ab (lanes 2, 4, 6 and 8), and harvested at 18 h post‐transfection. Total cell lysates were prepared and treated with (lanes 3 and 4) or without (lanes 1, 2, 5–8) 4 µL of PNGase. Polypeptides were resolved on SDS/20% polyacrylamide gel and analyzed by western blot with antibody to Flag. The numbers on the left indicate molecular masses in kilodaltons. (B) Mutational analysis of the N‐linked glycosylation site and rapid degradation of the N81D mutant protein 8ab. Total cell lysates prepared from Cos‐7 cells expressing the wild‐type (lanes 2 and 4) and the mutant (lanes 1 and 2) protein 8ab were analyzed by western blot with either antibodies to Flag (lanes 1 and 2) or antibodies to protein 8b (lanes 3 and 4). In cells expressing the mutant protein 8ab, smaller bands representing degraded products of the mutant protein were detected (lanes 1 and 3). The upper diagram illustrates the regions that these fragments may be derived from. The numbers on the left indicate molecular masses in kilodaltons. (C) Inhibition of the degradation of the unglycosylated protein 8ab by lactacystin and NLVS. Cos‐7 cells expressing protein 8ab were incubated either with dimethylsulfoxide (lane 1) or with 8 µm lactacystin plus 32 µm NLVS (lane 2), and harvested at 18 h post‐transfection. Total cell lysates were separated on SDS/20% polyacrylamide gel and analyzed by western blot with antibodies to Flag. The same membrane was also probed with antibodies to actin as a control.
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