Cullin 3-mediated ubiquitination restricts enterovirus D68 replication and is counteracted by viral protease 3C.

Enterovirus D68 (EV-D68) has emerged as a significant threat to public health because of its association with respiratory illnesses and neurological complications, including acute flaccid myelitis. However, the molecular mechanisms underlying EV-D68 replication and pathogenesis remain unclear. Here, we revealed a novel interaction between EV-D68 and the host Cullin-RING E3 ligase system, specifically Cullin 3, which was reported to restrict viral replication. We initially demonstrated that proteasome inhibition enhanced EV-D68 replication, suggesting an important role for the ubiquitin-proteasome system in viral restriction. Cullin 3 was further identified as a key factor that inhibits EV-D68 replication, and the downregulation of its expression increased viral titers. Mechanistically, Cullin 3 was observed to target the viral capsid protein VP1 for ubiquitination and degradation. However, EV-D68 was determined to utilize its protease 3C to cleave Cullin 3 at the Q681 residue, thereby inhibiting E3 ligase activity and facilitating resistance to Cullin 3-mediated VP1 degradation. This study uncovered a host-virus arms race, wherein the ubiquitin-proteasome system of the host actively targets viral proteins for degradation, and viral proteases counteract this defense mechanism. Accordingly, these findings could lead to more effective antiviral treatments. IMPORTANCE: The ubiquitin-proteasome system (UPS) is a critical cellular pathway involved in the regulation of protein stability and has been implicated in the regulation of viral infections. However, its role in EV-D68 infection has not been extensively explored. Our study proves that the host UPS, through the scaffold protein Cullin 3, can restrict EV-D68 replication, representing a previously unrecognized antiviral mechanism. Furthermore, we describe a viral strategy used to evade this host defense mechanism comprising Cullin 3 cleavage, which has broad implications for understanding virus-host interactions and could inform the development of novel therapeutic strategies against EV-D68 and other enteroviruses.