The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism

Wang, Yi; Liu, Li

The SARS-CoV M gene stimulates beta interferon gene expression in the human embryonic kidney 293T cell line. The overexpression of the SARS-CoV M gene has been shown to upregulate the transcriptional level of IFN-␤ (26) . To further confirm the result, using either enhanced green fluorescent protein (EGFP) or poly(I:C) as a negative or positive control, respectively, we demonstrated that M gene products specifically promoted IFN-␤ production, since cotransfection with M small interfering RNA (siRNA) completely abolished M-mediated IFN-␤ induction at both protein (Fig. 1A , comparing lanes 3 and 4) and mRNA ( Fig. 1B ) levels. Moreover, after a 48-h transfection, cell supernatants were collected and assayed for the presence of IFN-␤ by enzyme-linked immunosorbent assay (ELISA). Figure 1C clearly demonstrated that delivering pCMV-Myc-M into HEK293T cells specifically and significantly promoted the secretion of IFN-␤ into cell culture medium. In addition, the promoter sequence of IFN-␤ was placed upstream of the firefly luciferase reporter to generate the pGL3-IFN-␤-Luc construct. To test the specificity of M-mediated IFN-␤ induction, other viral envelope-associated genes such as the spike (S) and envelope (E) protein genes as well as the M mutant [M(V68A)] from the GZ50 isolate were also included (27) . The result of a dual-luciferase assay using the Renilla luciferase gene as a transfection control demonstrated that the SARS-CoV M gene rather than the S and E genes markedly increased IFN-␤ promoter activity (Fig. 1D) , whereas the valineto-alanine alteration at residue 68 of M protein completely abolished this induction, indicating that the specificity of M gene products played a role in this process. Consistent with these results, Western blotting and ELISA further validated the above observation ( Fig. 1E and F). To detect if the SARS-CoV M gene has a direct effect on NF-B activation, pCMV-Myc-M was cotransfected with pNFB-luc, which contained five copies of NF-B recognition sites, into HEK293T cells. The results of the dualluciferase assay revealed that the SARS-CoV M gene specifically and dramatically upregulated NF-B activity compared with the controls (Fig. 1G) . Moreover, M could mediate IFN-␤ induction in both dose-and time-dependent manner ( Fig. 1H and I) . Overall, the data strongly indicated that the SARS-CoV M gene product was sufficient to promote IFN-␤ gene expression.