NOD2 modulates MDA5 signaling to promote coxsackievirus B3 replication

Coxsackievirus is an enteric virus that encounters intestinal epithelial cells (IECs) after ingestion and can cause a range of illnesses from mild to severe, including gastroenteritis, pancreatitis, central nervous system diseases and myocarditis. Sensing of CVB3 RNA by melanoma differentiation-associated gene (MDA5) induces a Type 1 Interferon (T1IFN) response that is critical for reducing viral replication and dissemination within cells and the host. The cytosolic nucleotide oligomerization domain-containing protein 2 (NOD2), however, has been implicated in a proviral role in CVB3-induced myocarditis, but the underlying mechanism is not well understood. Because NOD2 is especially important in maintaining intestinal immune homeostasis and CVB3 is an enteric virus that initially infects the intestinal tract leading to virus dissemination to other tissues, we sought to determine whether NOD2 expressed in IECs would impact CVB3 infection. Here, we demonstrate that IEC NOD2 promotes CVB3 replication. Replication of a related Enterovirus, poliovirus, was also enhanced by NOD2. We discovered that NOD2-/- IECs have increased T1IFNs and interferon stimulated gene (ISG) expression, including IFIH1, the gene encoding for MDA5. Replication of CVB3 was rescued when MDA5 expression was reduced in NOD2-/- IECs. CVB3 replication was further increased, and the T1IFN response reduced, in NOD2+/+ IECs treated with the mitophagy inducer carbonyl cyanide m-chlorphenyl hydrazone (CCCP). Mitophagy induction in NOD2-/- IECs, however, had no effect on CVB3 replication, or the T1IFN response, suggesting a role for NOD2 in mitophagy. Overall, our data demonstrate that NOD2 promotes CVB3 replication in IECs by suppressing T1IFN expression and MDA5 activation via NOD2-mediated mitophagy. Since T1IFN restricts enteroviruses in vivo, CVB3 and other enteroviruses might exploit NOD2 activation in the intestinal tract to evade antiviral T1IFN responses. This evasion could enhance their replication in the intestine and promote their spread to other organs, potentially leading to diseases in the pancreas, heart, and central nervous system.