So Many Roads: the Multifaceted Regulation of Autophagy Induction.

Autophagy is an evolutionary conserved, degradative process from single-cell eukaryotes, such as Saccharomyces cerevisiae, to higher mammals, such as humans. The regulation of autophagy has been elucidated through the combined study of yeast, Caenorhabditis elegans, mice, Drosophila melanogaster, and humans. MTOR, the major negative regulator of autophagy, and activating nutrient kinases, such as 5'-AMP-activated protein kinase (AMPK), interact with the autophagy regulatory complex: ULK1/2, RB1CC1, ATG13, and ATG101. The ULK1/2 complex induces autophagy by phosphorylating downstream autophagy complexes, such as the BECN1 PIK3 signaling complex that leads to the creation of LC3+ autophagosomes. We highlight in this review various reports of autophagy induction that are independent of these regulators. We discuss reports of MTOR-independent, AMPK-independent, ULK1/2-independent, and BECN1-PIK3C3-independent autophagy. We illustrate that autophagy induction and the components required vary by the nature of the induction signal and type of cell and do not always require canonical members of the autophagy signaling pathway. We illustrate that rather than thinking of autophagy as a linear pathway, it is better to think of autophagy induction as an interconnecting web of key regulators, many of which can induce autophagy through different requirements depending on the type and length of induction signals.

Enteroviruses Remodel Autophagic Trafficking through Regulation of Host SNARE Proteins to Promote Virus Replication and Cell Exit.

Enterovirus D68 (EV-D68) is a medically important respiratory plus-strand RNA virus of children that has been linked to acute flaccid myelitis. We have determined that EV-D68 induces autophagic signaling and membrane formation. Autophagy, a homeostatic degradative process that breaks down protein aggregates and damaged organelles, promotes replication of multiple plus-strand viruses. Induction of autophagic signals promotes EV-D68 replication, but the virus inhibits the downstream degradative steps of autophagy in multiple ways. EV-D68 proteases cleave a major autophagic cargo adaptor and the autophagic SNARE SNAP29, which reportedly regulates fusion between autophagosome to amphisome/autolysosome. Although the virus inhibits autophagic degradation, SNAP29 promotes virus replication early in infection. An orphan SNARE, SNAP47, is shown to have a previously unknown role in autophagy, and SNAP47 promotes the replication of EV-D68. Our study illuminates a mechanism for subversion of autophagic flux and redirection of the autophagic membranes to benefit EV-D68 replication.

Poliovirus induces autophagic signaling independent of the ULK1 complex.

Poliovirus (PV), like many positive-strand RNA viruses, subverts the macroautophagy/autophagy pathway to promote its own replication. Here, we investigate whether the virus uses the canonical autophagic signaling complex, consisting of the ULK1/2 kinases, ATG13, RB1CC1, and ATG101, to activate autophagy. We find that the virus sends autophagic signals independent of the ULK1 complex, and that the members of the autophagic complex are not required for normal levels of viral replication. We also show that the SQSTM1/p62 receptor protein is not degraded in a conventional manner during infection, but is likely cleaved in a manner similar to that shown for coxsackievirus B3. This means that SQSTM1, normally used to monitor autophagic degradation, cannot be used to accurately monitor degradation during poliovirus infection. In fact, autophagic degradation may be affected by the loss of SQSTM1 at the same time as autophagic signals are being sent. Finally, we demonstrate that ULK1 and ULK2 protein levels are greatly reduced during PV infection, and ATG13, RB1CC1, and ATG101 protein levels are reduced as well. Surprisingly, autophagic signaling appears to increase as ULK1 levels decrease. Overexpression of wild-type or dominant-negative ULK1 constructs does not affect virus replication, indicating that ULK1 degradation may be a side effect of the ULK1-independent signaling mechanism used by PV, inducing complex instability. This demonstration of ULK1-independent autophagic signaling is novel and leads to a model by which the virus is signaling to generate autophagosomes downstream of ULK1, while at the same time, cleaving cargo receptors, which may affect cargo loading and autophagic degradative flux. Our data suggest that PV has a finely-tuned relationship with the autophagic machinery, generating autophagosomes without using the primary autophagy signaling pathway. ABBREVIATIONS: ACTB - actin beta; ATG13 - autophagy related 13; ATG14 - autophagy related 14; ATG101 - autophagy related 101; BECN1 - beclin 1; CVB3 - coxsackievirus B3; DMV - double-membraned vesicles; EM - electron microscopy; EMCV - encephalomyocarditis virus; EV-71 - enterovirus 71; FMDV - foot and mouth disease virus; GFP - green fluorescent protein; MAP1LC3B/LC3B - microtubule associated protein 1 light chain 3 beta; MOI - multiplicity of infection; MTOR - mechanistic target of rapamycin kinase; PIK3C3 - phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAA2 - protein kinase AMP-activated catalytic subunit alpha 2; PSMG1 - proteasome assembly chaperone 1; PSMG2 - proteasome assembly chaperone 2PV - poliovirus; RB1CC1 - RB1 inducible coiled-coil 1; SQSTM1 - sequestosome 1; ULK1 - unc-51 like autophagy activating kinase 1; ULK2 - unc-51 like autophagy activating kinase 2; WIPI1 - WD repeat domain, phosphoinositide interacting 1.