Several enteroviruses, such as CVB3, PV, and EV71, were reported to escape cells enwrapped in EV containing not only well-known EV marker proteins like CD63, CD81, and flotillin-1 [
30,
32,
33], but also the autophagy-related protein LC3 [
9,
30,
32,
33]. This finding triggered the idea that autophagy could be involved in formation of EV-virus. Autophagy is a cellular catabolic process by which cells under stress conditions engulf and break down damaged or unnecessary cytoplasmic constituents and recycle the building blocks to fuel processes that are crucial to survival. It is well established that autophagy plays an important role in virus infection. Many different picornaviruses, including PV, rhinovirus, EV71, CVB3 (genus enterovirus), and FMDV (genus aphthovirus) actively induce autophagy to sustain infection [
38‐
42]. During autophagy, LC3 is involved in selective cargo sequestration, as well as elongation and closure of the double membrane phagophore to form autophagosomal compartments (reviewed in [
43]). Unlike other components of the autophagy regulatory machinery, LC3 in its lipidated form (LC3-II) decorates both the inner and outer membrane of autophagosomes. The reported presence of LC3 on EV containing naked virus particles suggests that autophagosomes may provide the membrane for EV-virus formation. In support of this idea, it was shown that disruption or stimulation of autophagy initiation appeared to respectively inhibit or boost the non-lytic spread of PV, without affecting virus replication [
1]. These data support the idea that autophagy plays a multifaceted role in picornavirus replication and release. Interestingly, CVB3 infection was shown to not only initiate autophagy but also to block the autophagy flux towards lysosomal degradation [
9,
44]. This suggests that virion-containing autophagosomes may not follow conventional routing towards lysosomes, but rather fuse with the plasma membrane to expel their contents to the external milieu. This route, coined “secretory autophagy” (reviewed in [
45]), is an alternative disposal pathway for aggregated, defective, or non-functional cytoplasmic constituents to alleviate stress caused by these products under conditions of lysosomal dysfunction. Via this secretory autophagy pathway, membrane-bound vesicles decorated with LC3 could be released, provided that the autophagosomal inner membrane is not degraded. EV release via the secretory autophagy pathway has also been described in various non-infectious conditions. In neurodegenerative disorders, for example, this pathway was shown to drive the release of α-synuclein and other prion-like proteins in EV that display LC3 and tetraspanins CD9, CD63, and CD81 [
46‐
51]. A role for secretory autophagy is also recognized in controlling the EV-mediated release of members of the IL-1 family of cytokines, including IL-1β and IL-18, which lack an N-terminal signal peptide needed to enter conventional protein secretion pathways [
52,
53]. To further delineate the relationship between autophagy and EV-virus release, it is important to know whether and how picornaviruses that are proposedly released in LC3-decorated EV actively steer the autophagy pathway towards a secretory rather than degradative process. In this light, it is interesting to note that syntaxin 17, a factor required for fusion of autophagosomes with lysosomes, was sequestered away from autophagosome-like organelles that contained virions in CVB3-infected cells [
9,
54].