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 product promotes IFN-␤ production through Toll-like-receptor-related signaling pathways in immortalized murine bone marrow-derived macrophage cells. To further confirm the above results, the pCMV-Myc-M construct was also transiently transfected into J2-M cells, an immortalized murine bone marrow-derived macrophage cell line established with J2 virus (30, 31) . The delivery of the M gene product is effec-tive in stimulating the activation of both IFN-␤ and NF-B in murine J2-M cells (Fig. 4A , B, and C). Figure 4D demonstrates that increased delivery of M gene product into J2-M cells indeed promotes IFN-␤ induction through the phosphorylation of IRF3, NF-B p65, and TBK1. In accord with the results in HEK293ET cells, the increased delivery of pCMV-Myc-M into J2-M cells markedly enhanced TRAF6 but not TRAF2 and TRAF3 expression (Fig. 4E) . Moreover, the M gene product did not significantly increase the protein levels of RIG-I, MAVS, STING, and MDA5 ( Fig. 4F) , indicating that the RIG-I signaling pathway might not be activated in responding to exogenous delivery of the M gene product into J2-M cells. In contrast, the increased delivery of the M gene into J2-M cells markedly enhanced MyD88 and TRAM/ TICAM2 but not TRIF expression (Fig. 4G ), indicating that TLRrelated signaling pathways might be mainly associated with M-mediated IFN-␤ induction. Overall, our data in both HEK293ET and J2-M cells strongly indicate that M-mediated induction of IFN-␤ expression is likely associated with the activation of TLR-related signaling pathways.