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. 2007 Jan;81(2):718-31.
doi: 10.1128/JVI.01691-06. Epub 2006 Nov 1.

The ORF7b protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is expressed in virus-infected cells and incorporated into SARS-CoV particles

Scott R Schaecher  1 , Jason M Mackenzie, Andrew Pekosz
Affiliations

The ORF7b protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is expressed in virus-infected cells and incorporated into SARS-CoV particles

Scott R Schaecher et al. J Virol. 2007 Jan.

Abstract

Coronavirus replication is facilitated by a number of highly conserved viral proteins. The viruses also encode accessory genes, which are virus group specific and believed to play roles in virus replication and pathogenesis in vivo. Of the eight putative accessory proteins encoded by the severe acute respiratory distress syndrome associated coronavirus (SARS-CoV), only two-open reading frame 3a (ORF3a) and ORF7a-have been identified in virus-infected cells to date. The ORF7b protein is a putative viral accessory protein encoded on subgenomic (sg) RNA 7. The ORF7b initiation codon overlaps the ORF7a stop codon in a -1 shifted ORF. We demonstrate that the ORF7b protein is expressed in virus-infected cell lysates and from a cDNA encoding the gene 7 coding region, indicating that the sgRNA7 is bicistronic. The translation of ORF7b appears to be mediated by ribosome leaky scanning, and the protein has biochemical properties consistent with that of an integral membrane protein. ORF7b localizes to the Golgi compartment and is incorporated into SARS-CoV particles. We therefore conclude that the ORF7b protein is not only an accessory protein but a structural component of the SARS-CoV virion.

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Figures

FIG. 1.
FIG. 1.
SARS-CoV genomic organization and expression of the accessory protein ORF7b. (A) Genomic organization and sgRNAs of SARS-CoV. The ∼29-kb genome is transcribed into nine 3′-coterminal subgenomic RNAs in virus-infected cells. The sgRNAs encode replicase proteins (1a and 1b, white boxes) structural proteins (S, E, M, and N, black boxes), as well as eight proteins of unknown function (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8a, ORF8b, and ORF9b, gray boxes). The ORF7a and ORF7b proteins are encoded by sgRNA 7, with the ORF7a stop codon and the ORF7b initiation codon overlapping. The nucleotide and amino acid sequences for the ORF7a/ORF7b overlap region are shown. (B) Amino acid sequence of the ORF7b protein. The ORF7b protein is 44 amino acids in length with a single transmembrane domain (underlined) predicted by TMPred. (C) Vero cells were either mock infected or infected with SARS-CoV at an MOI of 5.0 for 18 h. The expression of ORF7b, N, and β-actin was determined by Western blotting. The band at ∼75 kDa is an unidentified cellular protein detected by the ORF7b sera. (D) Vero cells were either mock infected or infected with SARS-CoV at an MOI of 5.0 for 18 h. The cells were then processed for flow cytometry using antibodies specific for the ORF7a (horizontal axis) or ORF7b (vertical axis) proteins.
FIG. 2.
FIG. 2.
The ORF7b protein is translated via ribosome leaky scanning. (A). Schematic diagram of the cDNA constructs used to assess the translation of ORF7b. Gene 7 constructs included wild-type sequence (7ab), an optimal translation initiation consensus sequence at the ORF7a initiation codon (7ab 7a Kozak), a point mutation within the ORF7a coding region creating an out-of-frame ATG that does not alter the ORF7a amino acid sequence (7ab ATG), two point mutations within the ORF7a ORF resulting in two in-frame stop codons (7ab 7a Stop), a point mutation eliminating the ORF7a stop codon (7ab 7a Stop KO), a point mutation eliminating the ORF7a initiation codon (7ab 7a Start KO), and a point mutation eliminating the ORF7b initiation codon (7ab 7b Start KO). All ATG sequences are boxed, and stop codons are italicized. (B) 293T cells were transfected with plasmids encoding the indicated cDNAs and analyzed for ORF7a (horizontal axis) and ORF7b (vertical axis) expression by flow cytometry at 18 h posttransfection. (C) 293T cells were transfected with plasmids encoding the indicated cDNAs, lysed 18 h posttransfection, and analyzed for ORF7b and β-actin expression by Western blotting. (D) The ORF7b protein band intensities from panel C were quantified and normalized to β-actin expression by using phosphorimager analysis. The quantified data are from five independent experiments.
FIG. 3.
FIG. 3.
The ORF7b protein is an integral membrane protein. 293T cells were transfected with plasmids encoding the cDNAs for GFP-Rab11, SARS-CoV GFP-N, M2 from influenza A virus, or SARS-CoV ORF7b. At 18 h posttransfection, cells were collected, lysed in hypotonic lysis buffer, and homogenized. The lysate was separated into pellet (P) and supernatant (S) fractions by centrifugation at 100,000 × g. Pellets containing cellular membranes were subsequently treated with 4 M urea, 50 mM Na2CO3 (pH 11.3), or 2 M KCl to disrupt peripheral membrane proteins, and the samples were centrifuged at 100,000 × g. The resultant pellet (P) and supernatant (S) were analyzed by Western blotting with rabbit anti-GFP polyclonal sera, anti-M2 MAb, or rabbit anti-ORF7b polyclonal sera.
FIG. 4.
FIG. 4.
The ORF7b protein does not form disulfide-linked oligomers. 293T cells were transfected with either control plasmid (mock) or plasmids expressing the cDNAs for the influenza A virus M2 protein (A) or SARS-CoV ORF7b (B). The cells were lysed 18 h posttransfection, separated by SDS-PAGE under reducing (+DTT) or nonreducing (−DTT) conditions, and analyzed for protein expression by Western blotting. The M2 protein formed characteristic disulfide-linked dimers (d) and tetramers (t) under nonreducing conditions and monomers (m) in reducing conditions.
FIG. 5.
FIG. 5.
The ORF7b protein is not expressed on the cell surface of transfected cells. (A) Vero cells were transfected with plasmids encoding the cDNAs for the surface-expressed influenza A virus M2 protein (a to d), a C-terminal myc-epitope tagged M2 protein (e to h), ORF7a (i to l), ORF7b (m to p), or an ORF7b protein containing a C-terminal myc epitope tag (q to t). Transfected cells were fixed and permeabilized with 0.1% saponin (left columns) or stained with primary antibody at 4°C and fixed immediately poststaining (nonpermeabilized). Cells were incubated with antibodies specific for the indicated proteins, with the exception of the myc epitope-tagged proteins, which were detected with an anti-myc MAb. Only influenza virus M2 transfected cells stained with the 14C2 MAb (targeting the N-terminal extracellular domain of M2) demonstrated surface staining. (B) Vero cells were cotransfected with cDNAs expressing ORF7b and a GFP construct that localizes to the plasma membrane of transfected cells (S15-GFP). At 18 h posttransfection, the cells were immunostained with antibodies that recognize the ORF7b protein (red). The lack of colocalization between the proteins further suggests little or no ORF7b is present at the cell surface. All images were obtained with a ×63 oil immersion objective and represent a z-stack projection of 0.5-μm slices obtained by confocal microscopy. Nuclei were counterstained with TO-PRO-3 (blue).
FIG. 6.
FIG. 6.
The C terminus of ORF7b is exposed to the cytoplasm. Vero cells were transfected with plasmids expressing the cDNA for ORF7a-GFP (A, D, E, and H), M2-myc (B and F), or ORF7b-myc (C and G). At 18 h posttransfection, the cells were fixed with 2% paraformaldehyde, washed extensively, and permeabilized with PBS containing 0.1% saponin to disrupt all cellular membranes (A to D) or 25 μg of digitonin/ml to selectively permeabilize the plasma membrane (E to H). The cells were incubated with the 2E11 MAb, which recognizes the luminal domain of ORF7a (A, D, E, and H), or the 9E10 MAb, which recognizes the myc epitope tag (B, C, F, and G). The primary antibodies were detected using a goat anti-mouse IgG conjugated to Alexa Fluor 594 and mounted, and the cells were visualized by confocal microscopy using a ×63 oil immersion objective.
FIG. 7.
FIG. 7.
The ORF7b protein localizes to the Golgi apparatus in cDNA transfected or SARS-CoV-infected cells. (A) Vero cells grown on coverslips were transfected with a plasmid expressing the ORF7b cDNA. At 18 h posttransfection, the cells were immunostained with antibodies against ORF7b (green) or the cis-Golgi marker protein GM130 (red) by using confocal microscopy. Nuclei were counterstained with TO-PRO-3 (blue). (B) Vero cells were plated onto coverslips and then infected at an MOI of 5.0 with SARS-CoV. At the indicated times postinfection, the cells were fixed, and colocalization of ORF7b (green) with the cis-Golgi marker GM130 (red) was assessed by confocal microscopy. (C) Vero cells grown on coverslips were transfected with a plasmid expressing the ORF7b protein containing a C-terminal myc-epitope tag and then assessed by confocal microscopy for colocalization with cellular proteins that reside in various subcellular compartments (red) including the cis-Golgi network (GM130), the trans-Golgi network (Golgin-97), cis- and medial-Golgi (α-mannosidase II), ER-to-Golgi intermediate compartment (ERGIC53), ER (Calnexin), and lysosomes/multivesicular body (CD63). The ORF7b and ORF7b-myc antigen (green) colocalized with all three Golgi markers. All images were obtained with a ×63 oil immersion objective lens and represent a z-stack projection of 0.7-μm slices obtained by confocal microscopy. (D to F) Three-dimensional confocal images were generated by using Volocity software (Improvision) and analyzed for colocalization between ORF7b (green) and GM130, Golgin-97, or CD63 (red). (G) Shown is a cross-section along the y axis of the cell. Nuclei were counterstained with TO-PRO-3 (blue). The extent of colocalization was quantified for five cells in each condition, and the measure of fluorophore colocalization is expressed as a Pearson correlation coefficient (39).
FIG. 8.
FIG. 8.
The ORF7b protein is present in purified virus particles. (A) SARS-CoV particles were inactivated with paraformaldehyde, the supernatant was precleared by centrifugation at 1,000 × g and layered on a 20% sucrose cushion, and virus particles were pelleted at 100,000 × g. The virus pellet was resuspended in 2× Laemmli SDS-PAGE buffer, and protein expression was detected by Western blotting. The virus lysate contains no β-actin or nsp8 but is enriched for both S (the asterisk indicating a highly glycosylated virus associated form of S) and ORF7b. (B) The inactivated, pelleted virus particles were loaded onto a 20 to 60% continuous sucrose gradient and centrifuged for 18 h at 100,00 × g, and 1-ml fractions were collected from the top. The fractions were analyzed for the presence of SARS-CoV N or ORF7b by Western blotting. (C) The density of each fraction of the 20 to 60% sucrose gradient was determined by weighing 100 μl of the fraction.
FIG. 9.
FIG. 9.
The ORF7b protein is not secreted from transfected cells. 293T cells were transfected with plasmids encoding the cDNAs for the influenza A virus M2 protein, the WNV NS1 protein, or the SARS-CoV ORF7b protein. Mock samples were transfected with the plasmid vector containing no cDNA insert. At 18 h posttransfection, supernatants were collected and centrifuged at 1,000 × g to remove cellular debris, and an equal volume of 2× Laemmli SDS-PAGE buffer was added. Samples were analyzed by Western blotting for the indicated proteins. NS1 was present in cell lysates (l) and secreted (s) as a higher-molecular-weight glycosylated form, whereas the M2 and ORF7b proteins were present only in the cell lysates.
FIG. 10.
FIG. 10.
Immunogold staining for ORF7b in SARS-CoV particles. Cryosections from SARS-CoV-infected Vero cells were immunolabeled with antibodies raised against ORF7b (A and B) or the SARS-CoV N protein (B) and visualized with 10-nm or 5-nm protein A-gold, respectively. Localization of the ORF7b protein to intracellular virions is highlighted with arrows in panels A and B, and colocalization with N protein (arrowheads) is shown in panel B. (C) Immunolabeling of ORF7b on purified SARS-CoV particles. Magnification bars represent 200 nm in panels A and B and 100 nm in panel C.
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