Porcine MARCH8 negatively regulates the RLR signaling pathway by targeting MAVS for protein degradation

Abstract

Membrane-associated RING-CH-type finger 8 (MARCH8) protein is a 'really interesting new gene' (RING) type E3 ubiquitin ligase that mediates interferon-beta (IFN-β) production via regulating ubiquitination of multiple adaptor proteins in innate immune signaling pathways. Here, we cloned the porcine MARCH8 (porMARCH8) gene and analyzed its involvement in type I IFN expression. The full-length porMARCH8 gene encoded 289 amino acids, which shares high sequence similarity (87.7 %–88.9 %) with its orthologues from human, mouse and monkey. The porMARCH8 consists of a RING-CH domain and two TM domains, and is ubiquitously expressed in various tissues in pigs. porMARCH8 was found to be involved in IFN-β expression in response to poly(I:C) stimulation. Exogenous overexpression of porMARC8 significantly inhibited the mRNA expression level of IFN-β in swine testicular (ST) cell. Mechanically, porMARCH8 directly interacted with porcine mitochondrial antiviral-signaling protein (MAVS) and catalyzed its K48-linked poly-ubiquitination at residue K286 for degradation through the ubiquitin-proteasome pathway, thereby down-regulating IFN-β expression. Meanwhile, we demonstrated that the RING-CH domain was essential for the E3 ligase activity of porMARCH8. Together, these data demonstrated the importance roles of porMARCH8 in MAVS-mediated signaling pathways.