The cleavage of p62/SQSTM1 and NBR1 by the Coxsackievirus B3 proteases 2Apro and 3Cpro respectively raises the question if other viruses, in particular positive strand RNA viruses, also target p62/SQSTM1 and inhibit selective autophagy. Furthermore as outlined before the inactivation of p62/SQSTM1 raises the possibility that the subsequent ER stress may favour the formation of EDEMosomes via increased expression of EDEM1 and other components of the ERAD pathway, notably EDEM-2/-3 and OS-9, in addition to preventing the formation of mature lysosome and thus the degradation of viral components.
Before we discuss the potential application regarding to cells infected with Coronavirus’ or expressing coronaviral non-structural proteins (nsp), we have to consider the relationship between EDEMosomes and p62/SQSTM1. As briefly described before, ER degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is involved in the localisation of misfolded glycosylated and non- glycosylated proteins from the ER lumen into the cytoplasm as well potentially involved into the retrotranslocation of proteins from the cytoplasm into the ER. In the case of of inducing degradation of proteins, EDEM-1 and OS-9 are believed to bind misfolded proteins within the ER lumen, followed by the formation of vesicles -the EDEMosome, and subsequent targeting of these vesicles to the lysosome for degradation in a Atg5 dependent manner. Although this process involves components of the autophagy machinery, autophagosomes are not formed since LC3 although being recruited is not lipidated. Closer examination of this process revealed however that the EDEM-1 co-localises with components of the selective autophagy pathway, namely (autophagy-linked) FYVE (Alfy), p62/SQSTM1 and NBR1, and indeed the interaction with p62/SQSTM1 and NBR1 with EDEM-1 has been demonstrated to be required to be necessary for the formation of EDEM-1 positive autophagosomes. If then, selective autophagy via p62/SQSTM1 and/or NBR1 is impaired e.g. by the expression of p62/SQSTM1-C or by cleavage of p62/SQSTM1, this pathway would be blocked. EDEM-1 however can only form aggregates with p62/SQSTM1 if it is deglycosylated by cytosolic N-glycanases and ubiquitinylated.
Activation of the ERAD pathway by ER stress (namely PERK mediated phosphorylation of eIF2-α) and subsequent induction of selective autophagy seems to promote this pathway, which is also referred to as the ERAD-II pathway. In contrast, in unstressed cells, EDEM1 forms “classic”-LC3-II/p62/SQSTM1- negative EDEMOsomes.
This distinction has profound consequences in viral infected cells, in particular if viral proteins cause ER stress. In the case of Coxsackievirus B3, cleavage of p62/SQSTM1 might cause and excaberate ER stress induced by viral proteins, albeit selective autophagy is inhibited precisely because both NBR1 and p62/SQSTM1 are rendered inactive, yet the formation of EDEMosomes might not be inhibited.
In the case of Coronavirus infected cells the situation is complicated by the fact that so far the expression of nsp-3, -4, or -6 has not been shown to induce a ER stress response, although autophagy like vesicles are formed -in the case of nsp-6, omegasomes- but the co-localisation with either p62/SQSTM nor EDEM-1 has not been confirmed. Cells infected with the murine CoV, MHV, co-localise EDEM-1 and OS-9 in LC3-II negative vesicles.
Is it possible that p62/SQSTM1 and NBR1 mediated selective autophagy is inhibited and, if so, how? As part of the replication complex, nsp-3,-4, and -6 co-localise with the main viral protease, nsp-5/3CLpro.
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| Membrane topology of nsp-3/-4/-6 and 3CLpro/nsp5 |
More importantly however, SARS-CoV 3CLpro localises both in the cytoplasm and nucleus of transfected A549 and C145A cells. Does however 3CLpro cleave p62/SQSTM1? Although this has not been shown, this would require a cleavage site.
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| Models for the formation of p62/SQSTM1 positive autophagosomes by nsp-3/-4/-6 |
So far nobody has shown that ubiquitinylated proteins are accumulating at the surface of the ER during Coronavirus infection, making this scenario unlikely. Unless proven otherwise the author of these lines favours therefore a model where EDEM1 recruits LC3-I prior the conversion to LC3-II, followed by recruitment of p62/SQSTM1.
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| Model for the formation of p62/SQSTM1 positive autophagosomes by nsp-3/-4/-6 |
Finally, the role of p62/SQSTM1 in preventing the formation of mature lysosomes needs to be investigated, both under starvation and normal conditions.
In short, in the opinion of the author it is crucial to determine if the expression and the formation of DMVs by nsp-3/-4/-6 induces the ER stress response and to determine the role of EDEM-1 in the formation of these structures.
As always, who is up to the challenge? I am waiting to read the publication…..
Further reading
Bernasconi R, Noack J, & Molinari M (2012). Unconventional roles of nonlipidated LC3 in ERAD tuning and coronavirus infection. Autophagy, 8 (10), 1534-6 PMID: 22895348
Calì T, Galli C, Olivari S, & Molinari M (2008). Segregation and rapid turnover of EDEM1 by an autophagy-like mechanism modulates standard ERAD and folding activities. Biochemical and biophysical research communications, 371 (3), 405-10 PMID: 18452703
Cottam EM, Whelband MC, & Wileman T (2019). Coronavirus NSP6 restricts autophagosome expansion. Autophagy, 10 (8), 1426-41 PMID: 24991833
Fujita E, Kouroku Y, Isoai A, Kumagai H, Misutani A, Matsuda C, Hayashi YK, & Momoi T (2007). Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Human molecular genetics, 16 (6), 618-29 PMID: 17331981
Le Fourn V, Park S, Jang I, Gaplovska-Kysela K, Guhl B, Lee Y, Cho JW, Zuber C, & Roth J (2019). Large protein complexes retained in the ER are dislocated by non-COPII vesicles and degraded by selective autophagy. Cellular and molecular life sciences : CMLS, 70 (11), 1985-2002 PMID: 23338832
Liao HH, Wang YC, Chen MC, Tsai HY, Lin J, Chen ST, Tsay GJ, & Cheng SL (2011). Down-regulation of granulocyte-macrophage colony-stimulating factor by 3C-like proteinase in transfected A549 human lung carcinoma cells. BMC immunology, 12 PMID: 21324206
Oostra M, te Lintelo EG, Deijs M, Verheije MH, Rottier PJ, & de Haan CA (2007). Localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication. Journal of virology, 81 (22), 12323-36 PMID: 17855519
Park S, Jang I, Zuber C, Lee Y, Cho JW, Matsuo I, Ito Y, & Roth J (2019). ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase. Histochemistry and cell biology, 142 (2), 153-69 PMID: 24664425
Reggiori F, Monastyrska I, Verheije MH, Calì T, Ulasli M, Bianchi S, Bernasconi R, de Haan CA, & Molinari M (2010). Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication. Cell host & microbe, 7 (6), 500-8 PMID: 20542253
Shi J, Wong J, Piesik P, Fung G, Zhang J, Jagdeo J, Li X, Jan E, & Luo H (2019). Cleavage of sequestosome 1/p62 by an enteroviral protease results in disrupted selective autophagy and impaired NFKB signaling. Autophagy, 9 (10), 1591-603 PMID: 23989536
Shvets E, Abada A, Weidberg H, & Elazar Z (2011). Dissecting the involvement of LC3B and GATE-16 in p62 recruitment into autophagosomes. Autophagy, 7 (7), 683-8 PMID: 21460636
Weidberg H, Shvets E, Shpilka T, Shimron F, Shinder V, & Elazar Z (2010). LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis. The EMBO journal, 29 (11), 1792-802 PMID: 20418806
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