Cellular autophagy is an intracellular membrane trafficking pathway which delivers cytoplasmic material to lysosomes where the material is degraded thereby removing not only damaged organelles -as in the case of mitophagy- but also as part of the unfolded protein response misfolded proteins (aggrephagy) or the replication process of viruses (xenophagy). Generally, autophagy is induced under conditions of cellular stress such as nutrient withdrawal, DNA damage, or viral infections. During this process, isolation membranes (the phagosome) derived from the ER are formed and expanded into double-membrane autophagosomes that engulf the cellular cargo. Autophagosomes then fuse either with late Endosomes or Lysosomes forming the autolysosome where the cargo is degraded by lysosomal enzymes. The induction of autophagy is regulated by mammalian target of rapamycin complex-1 (mTORC1) kinase, that regulates a complex consisting of unc-51-like kinase (ULK-1), focal adhesion kinase family interacting protein of 200 kDa (FIP200), Atg13, and Atg101. ULK-1 activates a downstream complex couch as the phsophatidylinositol 3-kinase (PI3-kinase) complex (Atg6/beclin1-Atg14-Vps15-Vps34) and the Atg12 (Atg12-Atg5-Atg16) system as well as the LC-3 ubiquitin-like conjugation system that converts LC3-I to LC3-II in a PI3-kinase dependent manner by recruiting the Atg12-Atg5-Atg16 complex to the phagosome.
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| Outline of autophagy, its enzymes and inhibitors |
In the absence of starvation, mTORC1 inhibitors such as rapamycin or torin can activate the formation of the autophagosome, whereas PI-3 kinase inhibitors such as Wortmannin can inhibit the recruitment of Beclin1/Atg14/Vps15/Vps34. In general, the engulfment of cytoplasmic components is non-specific but ubiquitinylated proteins can be recruited to the autophagosomes specifically by nuclear or cytosolic p62/Sequestosome-1 (SQSTM1) thus binding polyubiquitinated proteins to membrane-associated LC3-II.
In contrast to intracellular cargo, extracellular cargo that is internalized by endocytosis, endocytosed macromolecules are delivered to lysosomes via endosomes, bypassing the autophagy pathway. This pathway involves a “kiss and run” as well as a complete fusion event, the latter dependent on the presence of a target membrane (t-) SNARE complex and a vesicle membrane (v-) SNARE complex which form a tetrameric transSNARE complex. Late endosomes can either fuse with other late endosomes (“homotypic fusion”) or other lysosomes (“heterotypic fusion”), the former requiring Syntaxin-8, VAMP-8, and Vti-1b and the latter Syntaxin -11, VAMP-7 as well as Vti-1b. Both homotypic and heterotypic fusion are also dependent on Rab5GTPase, Rab7GTPase, N-ethylmaleimide (NEM) sensitive factor (NSF) and its soluble associated proteins (SNAPs).
Both the autophagy and the endocytic pathway are not completely separated but united by a common interlocutor, UV radiation resistance-associated (UVRAG). UVRAG activates autophagy by associating with Beclin-1 (thus extending the phagophore) as well as inducing the maturation of the autophagosome in later stages via binding to C/Vps, and accelerating endocytic transport by activating Rab7 (thus leading to heterotypic fusion of the late endosome with lysosomes). In addition to the role in autophagy and fusion of endosomes with lysosomes, UVRAG also plays a role in the integrity of the ER and the Golgi as well in the DNA damage response.
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| UVRAG as the interlocutor between autophagy and homotypic endosome fusion |
Although the role of various autophagy related proteins in viral infections is well established, the role of UVRAG in particular in mediating viral entry has been elusive. A potential role for UVRAG in mediating viral entry can be postulated from the observation that in UVRAG deficient cells cell surface receptor degradation is downregulated. Since UVRAG overexpression is known to target viral proteins to autophagosomes (and thus lead to potential degradation of viral components) and cell surface receptors to lysosomal degradation, findings that suggest that UVRAG overexpression leads to increased viral replication of two negative strand RNA viruses -Influenza A and Vesicular Stomatitis Virus (VSV) - seem at first counterintuitive. Both viruses however encode proteins that inhibit the autophagy pathway (VSV-G and Influenza Virus M2 protein) thus counteracting the antiviral autophagy response, either by inhibiting Akt kinase mediated activation of mTOR (VSV-G) or the degradation of the autophagosome (Influenza Virus M2). On the other hand, UVRAG seems to be required for the replication of VSV and Influenza Virus, suggesting that UVRAG targets internalized viral particles to structures, that prevent them from being degraded and/or recognised by Pattern Recognition Receptors (PRRs) which as we have seen in a different post are an essential part of the cellular antiviral response. By infecting HeLa cells with DiI-labelled VSV, it was shown that viral particles localize to (acidic) late endosomes in a UVRAG dependent manner. Mutational analysis of UVRAG determined that UVRAG mediated virus entry is dependent on the interaction between the C2 and CDD domain of UVRAG and C/Vps as well as Vps-16 and -18. Successful targeting of VSV to endosomes is in addition dependent on VAMP-8. Indeed, VAMP-8 is recruited to VSV-G and Influenza Virus M protein positive vesicles. Since the endosome is involved in recognizing viral RNA via Toll-like receptors, it might be interesting to determine if these endosomes are positive for TLRs or if viral proteins inhibit the antiviral signaling.
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| VSV-G and UVRAG |
It remains to be seen if the infection of cells with positive strand RNA viruses such as Coronaviruses or Enteroviruses or the infection with DNA viruses induces the formation of similar structures or if these are limited to negative sense RNA viruses. As discussed before, positive strand RNA viruses induce the formation of replication transcription complexes and induce autophagy via viral nsps. Targeting viral particles to late endosomes by recruiting UVRAG however might be required early in the replication cycle prior to the formation of RTCs. In this context it might be possible that these early (hypothetical) structures are transported to the site of replication via the cytoskeleton. Indeed, late endosomes have been shown to be transported towards the perinuclear MTOC in a Rab7GTPase dependent manner and VSV-G co-localises with acidic endosomal vesicles in the perinuclear region in a Nocadozole sensitive manner.
Finally, it remains to be seen if the endosomal structures induced by the interaction between VSV-G and Influenza Virus respectively, can mature into LC3-II positive autophagosomes via ATG9; it might be possible however that both VSV-G and Influenza Virus M2 proteins. The finding that the localisation of VSV and Influenza Virus to
Autophagosomes are known to be induced upon entry of various other viruses. Food Mouth and Disease Virus (FMDV) and Vaccinia Virus (VACV) induces the formation of autophagosomes in a PI3-Kinase independent pathway (maybe UVRAG and/or ATG9 dependent?), and Echovirus 7 utilizes the autophagy pathway for its entry by an yet unidentified mechanism (although ATG16L is required) ditto for Dengue Virus. African Swine Fever Virus (AFSV) particles localize to the late endosome, suggesting that UVRAG might be important as well.
In conclusion, UVRAG might be an universal connector between viral entry the induction of autophagy.
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