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

rosis factor (TNF) receptor-associated factors (TRAFs) including TRAF2/TRAF5, TRAF3, and TRAF6 (28, 29) . To test the effect of M on RLR and TLR signaling as well as TRAF expression, an increased dose of pCMV-Myc-M constructs was first transiently transfected into HEK293ET cells. The results in Fig. 3A demonstrate that the increased delivery of pCMV-Myc-M into HEK293ET cells markedly enhanced TRAF6 but not TRAF2 and TRAF3 expression, indicating that TRAF6-mediated signaling transduction might contribute to the upregulation of IFN-␤ production. To further address how TRAF expression is associated with RLR and/or TLR signaling pathways, the M genetransfected HEK293ET cells were also assayed for the expression of upstream sensors and/or signaling molecules of TRAFs. Figure 3B demonstrates that no significant alteration was observed in the expression of RIG-I, MDA5, and MAVS after exogenously delivering M genes into HEK293ET cells, indicating that the RLR signaling pathway might not be targeted by M gene products. In contrast, three adaptor proteins (MyD88, TRAM/TICAM2, and TIRAP) associated with TLRs were all upregulated (Fig. 3C) , while the adaptor protein TRIF failed to be upregulated in responding to M gene overexpression (Fig. 3D) . Overall, the results indicate that TLR signaling pathways are mainly targeted by the SARS-CoV M gene product for the induction of IFN-␤ expression.