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

SARS-CoV M protein has been shown to destabilize the functional TRAF3-TBK1 complex formation (34) . However, it remains unknown whether or not M protein is able to associate with the key components of this complex. To clarify this issue directly, a coimmunoprecipitation (co-IP) experiment was conducted. Plasmid TBK1-Flag, TRAF3-HA, and Myc-M DNAs were transiently cotransfected into HEK293T cells. The cell lysates were first immunoprecipitated with anti-Flag antibody conjugated with an affinity gel and then subsequently probed with antihemagglutinin (anti-HA) and anti-Myc antibodies. The left panel of Fig. 7G indicates that M protein was able to disrupt the physical interaction between TBK1 and TRAF3, but M protein itself could not form a complex with TBK1. In the reverse immunoprecipitation (IP) experiment as shown in the right panel of Fig. 7G , M protein was unable to interact with both TBK1 and TRAF3 directly, indicating that M protein may modulate the TBK1-TRAF3 complex formation indirectly. Interestingly, in contrast to the inhibitory effect of M(V68A) on poly(I:C)-mediated IFN-␤ induction, the M gene product did not affect the IFN-␤ induction stimulated by poly(I:C) (Fig. 7H) , indicating that M has no effect on poly(I:C)induced IFN-␤ production while retaining the ability to destabilize the complex formation.