Transcription factor EB (TFEB) interaction with RagC is disrupted during enterovirus D68 infection

Abstract

Enterovirus D68 (EV-D68) is a picornavirus associated with severe respiratory illness and a paralytic disease called acute flaccid myelitis in infants. Currently, no protective vaccines or antivirals are available to combat this virus. Like other enteroviruses, EV-D68 uses components of the cellular autophagy pathway to rewire membranes for its replication. Here, we show that transcription factor EB (TFEB), the master transcriptional regulator of autophagy and lysosomal biogenesis, is crucial for EV-D68 infection. Knockdown of TFEB attenuated EV-D68 genomic RNA replication but did not impact viral binding or entry into host cells. The 3C protease of EV-D68 cleaves TFEB at the N-terminus at glutamine 60 (Q60) immediately post-peak viral RNA replication, disrupting TFEB-RagC interaction and restricting TFEB transport to the surface of the lysosome. Despite this, TFEB remained mostly cytosolic during EV-D68 infection. Overexpression of a TFEB mutant construct lacking the RagC-binding domain, but not the wild-type construct, blocks autophagy and increases EV-D68 nonlytic release in H1HeLa cells but not in autophagy-defective ATG7 KO H1HeLa cells. Our results identify TFEB as a vital host factor regulating multiple stages of the EV-D68 lifecycle and suggest that TFEB could be a promising target for antiviral development against EV-D68. IMPORTANCE Enteroviruses are among the most significant causes of human disease. Some enteroviruses are responsible for severe paralytic diseases such as poliomyelitis or acute flaccid myelitis. The latter disease is associated with multiple non-polio enterovirus species, including enterovirus D68 (EV-D68), enterovirus 71, and coxsackievirus B3 (CVB3). Here, we demonstrate that EV-D68 interacts with a host transcription factor, transcription factor EB (TFEB), to promote viral RNA(vRNA) replication and regulate the egress of virions from cells. TFEB was previously implicated in the viral egress of CVB3, and the viral protease 3C cleaves TFEB during infection. Here, we show that EV-D68 3C protease also cleaves TFEB after the peak of vRNA replication. This cleavage disrupts TFEB interaction with the host protein RagC, which changes the localization and regulation of TFEB. TFEB lacking a RagC-binding domain inhibits autophagic flux and promotes virus egress. These mechanistic insights highlight how common host factors affect closely related, medically important viruses differently.