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 functions at the protein level to induce IFN-␤ production. The next question that we tried to ask was at which level (mRNA or protein) the M-mediated IFN-␤ induction occurred. To address this issue directly, we created an M-stop construct by replacing the start codon AUG with three in-frame tandem stop codons at the 5= end of the M gene (Fig. 5A ). This expression construct can generate only mRNA and no protein due to the translation failure of the mRNA substrates. Western blot analysis shows that the M protein synthesis was completely blocked in the M-stop construct but not the wild-type M construct (Fig. 5B ). Real-time quantitative reverse transcription PCR (qRT-PCR) analysis shows that the M-stop construct did not induce IFN-␤ production in HEK293T cells, indicating that M-mediated IFN-␤ production is dependent on M protein rather than M mRNA (Fig. 5C) .