Enterovirus-induced cleavage of Mitofusin 2 generates mitophagosomes for enveloped virion release

Enterovirus D68 (EV-D68) is a plus-strand RNA virus that primarily causes respiratory infections in infants but, in rare cases, has been associated with the pediatric paralytic disease acute flaccid myelitis. We previously demonstrated that EV-D68 induces nonselective autophagy for its benefit. Here, we demonstrate that the 3C protease of EV-D68 cleaves the mitochondrial fusion protein Mitofusin 2 near its C-terminal HR2 domain, inducing fragmentation of the mitochondrial network. This, in turn, triggers the formation of mitophagosomes, a hallmark of mitophagy, a selective form of autophagy that recycles mitochondria. Multiple hallmarks of mitophagy are observed during infection, including loss of mitochondrial membrane potential and Parkin translocation to the mitochondria, but mitochondrial degradation is blocked during infection. While autophagy plays multiple roles in enterovirus infection, depleting Mitofusin 2 or transiently overexpressing Mitofusin 2, particularly the cleavage-resistant mutant, specifically reduces EV-D68 release from cells without affecting intracellular titers. Our results show that enteroviruses induce mitophagosomes as vectors for nonlytic release of virions from cells.