Figure S1 ERK1/2 phosphorylation is enhanced by hPIV3 infection in A549 cells. A549 cells were infected with hPIV3 (MOI = 3) and after 24 h, cells were starved for 12 h before stimulation with 100 ng/ml of EGF. Phosphorylation of ERK1/2 was determined by western blot analysis at 10 min, 30 min and 2 h post stimulation. hPIV3 infection was confirmed by anti-hPIV3 hemagglutinin-neuraminidase (hPIV3-HN) immunoblotting. Found at: doi:10.1371/journal.ppat.1000587.s001 (7.34 MB TIF) hPIV3-C Impact on IFN-a/b and EGF Pathways Figure S2 MEK1/2 inhibitor U0126 has no effect on MV protein synthesis. HEK-293T cells were left untreated (A) or treated with 20 mM of U0126 for 2 h (B). Then, cells were mocktreated or infected with a recombinant MV strain expressing EGFP (MOI = 1) and cultured with or without U0126 (A and B, respectively). 48 h after infection, EGFP expression was quantified by flow cytometry analysis. Found at: doi:10.1371/journal.ppat.1000587.s002 (7.34 MB TIF)  A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor κB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid–sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand. To sense infection, the innate immune system preferentially responds to conserved molecular signatures of microbes that are absent from normal host cells. There is currently great interest in determining what molecular features of pathogens are detected, and how these features are sensed by the innate immune system. A better understanding of the fundamental principles of how immune responses are stimulated is critical for the design of more effective vaccines, adjuvants, and immune therapeutics.