Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients

Abstract

Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.

Figure 1

Sequence analysis of the 3a protein of SARS-CoV. The ORF 3 gene encodes a putative protein of 274 amino acid residues. Predicted transmembrane regions are demarcated by arrows and the direction is from the outer surface to the interior of virion envelope. Cysteine residues are indicated with triangles and the cysteine-rich region is underlined. Amino acid residues in italics have mutations among the isolates of SARS-CoV, especially G marked with dots has two different mutations.

Figure 2

Formation of interchain disulfide bonds between the 3a protein and spike protein. A, Identification of the 3a protein in the cytosol of SARS-CoV-infected Vero E6 cells. Vero E6 cells (lane E6) and the cytosol of infected Vero E6 cells (lane E6+V) were subjected to Western blotting with anti-3a1 antibody (against the 3a protein). An arrow indicates the band of the 3a protein. B, The 3a protein forms interchain disulfide linkages with S protein. The crude SARS-CoV virions were lysed in a sample loading buffer either with 100 mM DTT (reducing sample, lane R) or without DTT (non-reducing sample, lane NR), and then subjected to Western blotting with anti-S2 antibody (against S protein) (left panel) or anti-3a1 antibody (right panel).

Figure 3

Detection of ORF3a peptide-specific IgG antibodies in SARS patients. The IgG antibodies in the sera of SARS patients were measured by ELISA with: A, 3a1 peptide; B, 3a2 peptide; C, S1 peptide. The low-response group consists of individual patients who had low A values, which were below the average of the normal control, whereas the high-response group had A values that were above the average of the normal control. D, The correlation of antibodies induced by the 3a protein and S protein in the same patient in response to 3a2 and S1-peptides with SigmaPlot software.

Figure 4

Analysis of SNE locus in the coronavirus family. A, Comparison of genome organization between S-gene and E-gene of coronaviruses. The predicted ORFs between the gene encoding the spike (S) protein and the gene encoding the small envelope (E) protein of seven species from three groups of coronaviruses were compared. The transmembrane (TM) region was also analyzed. B, Phylogenetic analysis of SNE locus. Unrooted phylogenetic tree of SNE region was generated by using CLUSTAL W 1.74 with the BLOSUM comparison matrix, and was drawn with the Phylip Drawtree program 3.6a3. Branch lengths indicate the number of substitutions per residue. Numbers indicate bootstrap replicates supporting each node.