These two pieces of data are generally consistent with a previous report,22 and they further strengthen the current model of the IFN antagonism of MERS-CoV M protein. Open in a separate window Figure 1 MERS-CoV M protein inhibits IFN- expression stimulated by Sendai virus infection or poly(I:C) induction. be specific for the activation of IFN regulatory factor 3 (IRF3) but not nuclear factor-B. MERS-CoV M protein interacted with TRAF3 and disrupted TRAF3CTBK1 association leading to reduced IRF3 activation. M proteins from MERS-CoV and SARS-CoV have three highly similar conserved N-terminal transmembrane domains and a C-terminal region. Using chimeric and truncation mutants, the N-terminal transmembrane domains of the MERS-CoV M protein were found to be sufficient for its inhibitory effect on IFN expression, whereas the C-terminal domain was unable to induce this suppression. Collectively, our findings suggest a common and conserved mechanism through which highly pathogenic MERS-CoV and SARS-CoV harness their M proteins to suppress type I IFN expression at the level of TBK1-dependent phosphorylation and activation of IRF3 resulting in evasion of the host innate antiviral response. Alox5 and is most phylogenetically related to two bat coronaviruses, HKU4 and HKU5, providing insight on its evolutionary origin.11, 12 MERS-CoV is a polycistronic positive-sense single-stranded RNA virus with a genome of ~30?Kb in size. The 5 most two-thirds of MERS-CoV genome encodes polyproteins 1a and 1ab, which are further cleaved to yield 16 nonstructural proteins, whereas the 3 end of the genome encodes several structural or lineage-specific proteins.13 Upon infection, these proteins are expressed to facilitate viral replication and propagation in the host.14 MERS-CoV infection has been widely reported to mildly induce type I interferons (IFNs), including IFN- and -, in patients as well as in animal and cellular infection models.15, 16, 17, 18, 19, 20, 21 This has been attributed to the IFN-antagonizing property of some MERS-CoV-encoded proteins, which directly perturb the host IFN production mechanisms,22, 23, 24, 25, 26 lending support to the notion that MERS-CoV uses multiple strategies to evade the innate immune response. In non-specialized epithelial cells as well as a subset of specialized immune cells that are susceptible to MERS-CoV infection,16, 18, 27 type I IFN production is an important part of the host innate immune response and is initiated by ubiquitously expressed cytoplasmic viral CMP3a sensors in the retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) family in response to the detection of viral pathogen-associated molecular patterns such as double-stranded RNA (dsRNA).28, 29 Stimulated RLRs mobilize downstream signal transducers that lead to the activation of the transcription factors IFN regulatory factor 3 (IRF3) and nuclear factor-B (NF-B) that drive IFN- expression.28 The transduction events within this signaling cascade are prone to negative regulation by many MERS-CoV proteins. In a comparative analysis of MERS-CoV structural and accessory proteins, it has been shown that M, ORF4a, ORF4b and ORF5 possess IFN-antagonizing properties.22 We, and others, have characterized the ORF4a protein as a dsRNA-binding protein that interferes with the activation of RLR by either a dsRNA ligand or the protein co-activator PACT.24, 25 However, the molecular mechanisms through which other MERS-CoV proteins manipulate the RLR signaling pathway to disrupt IFN- expression have not been elucidated. In this study, we focused on the characterization of the MERS-CoV M protein in IFN antagonism. Coronavirus M protein is a transmembrane glycoprotein localized predominantly to the Golgi complex and is required for virion assembly.30, 31, 32 MERS-CoV M protein is of particular CMP3a interest because SARS-CoV M protein also inhibits IFN production through a mechanism by which the formation of TRAF3TANKTBK1/IKK-? complex is impeded to ablate the activation of IRF3 transcription factor.30 In contrast, M protein encoded by human coronavirus HKU1 associated with common cold has no influence on IFN production.32 Here we reported that the MERS-CoV M protein also specifically inhibited IRF3 activation but not NF-B signaling. MERS-CoV M protein was capable of interacting with TRAF3 adapter protein and hampered TRAF3CTBK1 interaction leading to diminished IRF3 activation. Using a chimeric protein containing the MERS-CoV CMP3a M protein N-terminal transmembrane domains and a dormant SARS-CoV M protein C-terminal domain, we confirmed that the N-terminal transmembrane domains of MERS-CoV M protein sufficiently account for its inhibitory effect. Although another chimera containing SARS-CoV M protein N-terminal transmembrane domains and a MERS-CoV M protein C-terminal domain was fully competent in IFN antagonism, a truncation mutant lacking the functional first transmembrane domain of SARS-CoV M was not, suggesting that the C-terminal domain of.