Enhancement of type I interferon anti-viral responses through activation of non-canonical NOD2 signaling Andras K. Ponti, Megan T. Zangara, Christine M. O’Connor, Erin E. Johnson, & Christine McDonald Departments of Inflammation & Immunity and Genomic Medicine, Lerner Research Institute, Cleveland Clinic Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine at Case Western Reserve University Department of Biology, John Carroll University Type I interferon production and the expression of interferon-stimulated genes (ISGs) are key components of an innate immune response to many microbial pathogens. Dysregulation of this response can result in uncontrolled infection, inflammation, and autoimmune disease. Understanding the molecular mechanisms shaping the strength of type I interferon signaling may provide critical insights into infection control strategies and autoimmune disease therapies. Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular pattern recognition receptor that acts as both a bacterial sensor protein and a mediator of antiviral responses. Antibacterial functions of NOD2 are enhanced by treatment with the small molecule inhibitor of pyrimidine biosynthesis N-phosphonacetyl-L-aspartate (PALA). In this study, PALA was tested for its ability to enhance NOD2-dependent antiviral responses. Alone, PALA treatment of macrophages was not sufficient to induce interferon β (IFNβ) production or ISG expression. Instead, PALA synergized with IFNβ stimulation to enhance expression and activation of interferon-stimulated gene factor 3 (ISGF3) and induce the upregulation of a subset of ISGs in co-treated cells. PALA treatment of epithelial cells resulted in impaired viral replication of human cytomegalovirus. Induction of the PALA enhanced antiviral response required activation of non-canonical NOD2 signaling mediated by mitochondrial antiviral-signaling protein (MAVS) and interferon response factor 1 (IRF1), rather than the classical receptor-interacting serine/threonine protein kinase 2 (RIP2) pathway or other IRFs previously reported to mediate NOD2 antiviral responses. These findings highlight pyrimidine metabolism enzymes as controllers of antimicrobial responses and suggest novel mechanisms for the modulation of type I interferon responses and antiviral activity.