The Membrane Protein Of SARS-CoV Functions As A Novel Cytosolic PAMP To Promote IFNβ Induction

The innate immune response is the first line of host immune response against the invading pathogens such as viruses. Most of the intracellular pattern recognition receptors (PRRs) resides at either endolysosome or cytoplasm to sense the pathogen derived RNAs, DNAs or synthetic analog of double stranded RNA (dsRNA) such as polyI:C. However, it remains elusive whether or not a pathogen derived protein can function as a cytosolic PAMP. Our initial study indicates that the membrane gene of severe acute respiratory syndrome associated coronavirus (SARS-CoV) induces type I interferon (IFN-I) production in HEK293 cells, however the mechanism is not clear. In this study, we demonstrate that delivering the membrane gene of severe acute respiratory syndrome associated coronavirus (SARS-CoV) into HEK293T, HEK293ET and mobilized murine bone marrow derived macrophage cells (J2-M(?)) significantly up-regulates IFNβ production. Moreover, SARS-CoV M gene failed to upregulate the IFNβpromoter in A549 and Vero cells, it indicated that the upregulation of IFNP by M gene may exist in some tissues and cells, not all kinds of tissues and cells. Both NFκB and TBK1-IRF3 signaling cascades are activated by M gene products. M-stop translation was constructed, and then compared with wild M protein by its function of the induction of IFNβ. The results show, M protein rather than M mRNA is responsible for M-mediated IFNβ induction that is preferentially associated with the activation of the TLR adaptor proteins MyD88, TIRAP and TICAM2 but not RIG-I signaling cascade. What’s more, CO-IP assay was used to detect the interaction between M protein with adaptor proteins Myd88, TRAM in TLR pathway, however we failed to get the positive results. This may indicate that the M protein of SARS-CoV upregulate the two adaptor proteins indirectly. The band of M protein was smeared in the western blotting assay, so we try to detect whether the M protein exists ubiquitin modification. Through the mutation of its predictional ubiquitin lysine locations, the detection with M co-transfection with HA-Ub, and the mass spectrometry detection of immune precipitation M protein, while the results are both negative. It indicated that the smear band of M protein in Western blotting assay is not ubiquitin modification, it may be other kinds of modification after translation. Blocking the secretion of M protein by brefeldin A (BFA) failed to reverse the M mediated IFNβ induction. The antagonist of both TLR2 and TLR4 did not impede M mediated IFNβ induction, indicating that the driving force for the activation of IFNβ production was generated from the inside of the cells. Inhibiting TRAF3 expression by specific siRNA did not prevent M mediated IFNβ induction. SARS-CoV pseudovirus could induce IFNβ production in an M dependent manner since Valineâ†'Alanine substitution at the residue 68 in M protein markedly inhibited IFNβ production. At last, we constructed the M-deletions plasmids according to the hydrophilic region and the transmembrane region of M protein, and dual luciferase assay showed that the the transmembrane region of M protein may play important role in the upregulation of IFNP promoter.Overall, our study indicates for the first time that pathogen-derived protein is able to function as a cytosolic PAMP to ignite the type I interferon production by activating a non-canonical TLR signaling cascade via a TRAF3 independent manner. This study may exemplify the general effect of viral protein that could potentially serve as cytosolic PAMP to directly promote IFNP production.