The accumulation of misfolded proteins in the ER lumen induces a stress response commonly known as the Unfolded Protein Response (UPR) or ER stress response, an adaptive signalling pathway increasing the expression of ER chaperones, inhibiting mRNA translation, and stimulating ER associated degradation (ERAD) of accumulated proteins. The degradation via the ERAD pathway in particular requires the formation of double membrane vesicles -more commonly referred to as autophagosomes - which subsequently fuse with lysosomes to form the autolysosome. The ERAD pathway can be induced by all three branches of the ER stress response -PERK, ATF6, and IRE1- which increase the expression of ER degradation enhancer, mannosidase alpha-like-1/-2 (EDEM1/2) proteins in addition to other components of the ERAD pathway either by ATF4 (in conjunction with sXBP1) by cleaved ATF6α. Binding of cytosolic misfolded proteins to components of the ERAD pathway allows the retrotranslocation of these protein into the ER lumen where ER chaperones may assist these proteins to be folded correctly and/or be glycosylated in a process which involves binding to EDEM1/2/3. Alternatively proteins might however be targeted for degradation; in this case the EDEM/protein complex induces the formation of specific autophagosomes, the EDEMosome. In addition to components of the ERAD pathway, ATF4 also induces the expression of autophagy related genes, including but not limited to LC3, ATG16L, p62/SQSTM1, NBR, and ATG7, thus linking the induction of ER stress to the induction of autophagy. As an alternative to ERAD/EDEMosome mediated degradation, proteins which are ubiquitylated are targeted for the proteasome or autophagy by binding to p62/SQSTM1 or NBR; degradation via the EDEMosome in contrast is exclusively mediated by the formation of autophagosomes.
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| The accumulation of protein aggregates in the ER lumen can induce autophagy dependent and independent pathways of degradation |
In the case of viral infected cells, several viruses have been shown to induce the ER stress response signaling pathway and as a result autophagy rather than apoptosis, either during viral replication or following binding of the virus to its receptor. In these cases, the UPR leads to the induction of the cellular Interferon response and viral induced autophagy can lead to the degradation of viral components in the lysosome as well as being processed in multivesicular bodies and subsequent MHC class I/II presentation of viral antigens. On the other hand, prolonged ER stress induced by viral proteins in the absence of autophagy can and would induce apoptosis or necrosis, both which can be considered as an antiviral response as well.
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| Crosstalk between UPR and induction of autophagy |
In the case of JEV (and other viruses) premature apoptosis and the induction of autophagy induced by the localisation of both the non-structural and structural proteins to the ER would decrease viral replication due to cell death or degradation of viral proteins. Both apoptosis and autophagy can be induced by UPR. In the case of autophagy, both the PERK and ATF6 induced pathways induce the formation of autophagosomes whereas the IRE1 pathway inhibits autophagy via CHOP and promotes DR5 dependent apoptotic pathways. It should be noted however that CHOP has a dual role since at least during short term ER stress CHOP increases the expression of autophagy related genes such as ATG5 and ATG7 required for the formation of autophagic vesicles whereas only during prolonged ER stress autophagy is inhibited. to further complicate the matter, CHOP also releases Beclin1 from cytoplasmic Beclin1-Bcl2 complexes and thus induces the formation of autophagic vesicles as well as facilitating nuclear translocation of Bcl2, the latter forming a complex with ASPP2 and inducing a process referred to as autophagic apoptosis in hepatocellular carcinoma cells via induction CHOP expression.
Since both the PERK and ATF6 dependent pathways are activated prior activation of IRE1, these pathways might benefit JEV especially early in infection by preventing apoptosis. Indeed, following the infection of neuronal cells with a neurotrophic strain of JEV, an induction of autophagy as evidenced by the accumulation of LC3-II positive autophagosomes can be observed as early as 16 hrs p.i., coinciding with the detection of NS5, suggesting that newly synthesised proteins rather than incoming structural proteins are responsible for the induction of autophagy. Since these vesicles are not only positive for LC3-II but also for EDEM1, they represent EDEMosomes and can potentially be degraded by the autolysosomal pathway. Furthermore, the formation of EDEMosomes or autophagosomes is necessary for preventing apoptosis as infected ATG5 or ATG7 deficient cells are highly susceptible to virus induced apoptosis. On the other hand, maturation of the autophagosome also enhances the degradation of viral components via the autolysosome; indeed levels of viral RNA, as measured by qRT-PCR, are increased in ATG5 and ATG7 deficient cells compared to wt cells.
In the case of Coronavirus infected cells, the expression of the nsp-6 protein not only induces the formation of omegasomes but also inhibits the formation of mature autolysosomes via inhibition of the mTORC1 complex. It seems possible therefore, that JEV also inhibits the formation of mature autophagosomes and indeed, late in infection the presence of LC3-II positive autophagosomes decreases. If however this is due to the expression of a viral protein is unclear; to the author of this post it seems most likely that the prolonged ER stress induced by the viral NS and structural proteins activates the IRE1 dependent pathway late in infection and thus inhibits autophagy via CHOP induction. It remains to be seen however, if in cells deficient for CHOP and other components of the IRE1 pathway, viral induced autophagy is affected or not and if these cells are undergoing apoptosis in a PERK/ATF6 dependent manner only. As mentioned before, CHOP plays a dual role so it might be possible that in JEV infected cells CHOP is actually required for the induction of autophagy.
As for the signaling pathway leading to the formation of autophagic vesicles, the Core protein of JEV has been shown to induce p38 MAPK, thus activating PERK and ATF6 dependent signaling pathways and since in JEV infected cells autophagy genes are not unregulated it might be possible that JEV selectively activates ATF6α. In addition to upregulating the expression of DR5, both PERK and ATF6αalso induce the expression of autophagy- and ERAD- related genes as mentioned above. It might therefore be possible that prior the induction of IRE1, JEV infected cells predominantly induce autophagy via ATF6αand only the prolonged induction of ER stress induces IRE1 dependent activation of apoptosis via CHOP and autophagy inhibition. In other words, the induction of EDEMosomes following the localisation of JEV proteins may represent not only a scaffold for viral RNA synthesis but also an antiviral response, which is reflected by the increase of viral RNA in ATG5/7 deficient cell lines. If however other proteins that the Core protein contribute to the formation of EDEMosomes remains to be investigated and I am curious to see the results.
Coronavirus and the ER stress response
As discussed in previous posts, nsps’ derived from both Arteri- and Coronavirus’ induce the formation of EDEMosomes and both nsp-7 from PRRSV and nsp--6 induce the formation of omegasomes; furthermore, both proteins (in addition to others) are localised at the ER and part of the viral RTC. As we have seen, the co-localisation of (viral) proteins with EDEM-1 suggests that the protein underwent a retrotranslocation to the ER thus allowing ER localisation in the absence of a classical localisation sequence. The coronaviral nsp-3/-4/-6 proteins are also N-glycosylated, suggesting that once inside the ER they undergo glycosylation. So far however, none of these proteins has been shown to induce a prolonged ER stress response, raising the question what are the unique properties of JEV that induce a stress response? MHV does induce p38 MAPK, IBV infection does induce the ER stress response, and both SARS-CoV and MHV infected cells exhibit increased mRNA levels of homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 1 (HERPUD1). The induction of the UPR following Coronavirus infection has indeed been proposed to induce apoptosis as well as the induction of chemokines (which can be inhibited by various coronaviral proteins. In contrast to JEV however, where the Core protein has been shown to induce UPR (and as the author hypothesizes that the viral NS2A, NS2B, M, and E proteins do so likewise), in the case of Coronavirus’ it is not known which one of the nsp proteins involved -if any- do indeed cause short term or prolonged ER stress. On the other hand, in the case of CoV we know which proteins are sufficient to induce EDEMosomes/omegasomes - if however the structural proteins play a role in the formation of the RTC remains to be seen.
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| Coronavirus (top) and Japanese Encephalitis Virus (bottom) induce the formation of EDEMosomes and inhibit the formation of autolysosomes |
Further reading
Matsumoto H, Miyazaki S, Matsuyama S, Takeda M, Kawano M, Nakagawa H, Nishimura K, & Matsuo S (2019). Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression. Biology open, 2 (10), 1084-90 PMID: 24167719
Kouroku Y, Fujita E, Tanida I, Ueno T, Isoai A, Kumagai H, Ogawa S, Kaufman RJ, Kominami E, & Momoi T (2007). ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation. Cell death and differentiation, 14 (2), 230-9 PMID: 16794605
Shin YJ, Han SH, Kim DS, Lee GH, Yoo WH, Kang YM, Choi JY, Lee YC, Park SJ, Jeong SK, Kim HT, Chae SW, Jeong HJ, Kim HR, & Chae HJ (2010). Autophagy induction and CHOP under-expression promotes survival of fibroblasts from rheumatoid arthritis patients under endoplasmic reticulum stress. Arthritis research & therapy, 12 (1) PMID: 20122151
Liu K, Shi Y, Guo X, Wang S, Ouyang Y, Hao M, Liu D, Qiao L, Li N, Zheng J, & Chen D (2019). CHOP mediates ASPP2-induced autophagic apoptosis in hepatoma cells by releasing Beclin-1 from Bcl-2 and inducing nuclear translocation of Bcl-2. Cell death & disease, 5 PMID: 25032846
Armstrong, J., Flockhart, R., Veal, G., Lovat, P., & Redfern, C. (2009). Regulation of Endoplasmic Reticulum Stress-induced Cell Death by ATF4 in Neuroectodermal Tumor Cells Journal of Biological Chemistry, 285 (9), 6091-6100 DOI: 10.1074/jbc.M109.014092
Shoulders MD, Ryno LM, Genereux JC, Moresco JJ, Tu PG, Wu C, Yates JR 3rd, Su AI, Kelly JW, & Wiseman RL (2019). Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments. Cell reports, 3 (4), 1279-92 PMID: 23583182
Rzymski T, Milani M, Pike L, Buffa F, Mellor HR, Winchester L, Pires I, Hammond E, Ragoussis I, & Harris AL (2010). Regulation of autophagy by ATF4 in response to severe hypoxia. Oncogene, 29 (31), 4424-35 PMID: 20514020
B'chir W, Chaveroux C, Carraro V, Averous J, Maurin AC, Jousse C, Muranishi Y, Parry L, Fafournoux P, & Bruhat A (2019). Dual role for CHOP in the crosstalk between autophagy and apoptosis to determine cell fate in response to amino acid deprivation. Cellular signalling, 26 (7), 1385-91 PMID: 24657471
Yu Z, Wang AM, Adachi H, Katsuno M, Sobue G, Yue Z, Robins DM, & Lieberman AP (2011). Macroautophagy is regulated by the UPR-mediator CHOP and accentuates the phenotype of SBMA mice. PLoS genetics, 7 (10) PMID: 22022281
Sharma M, Bhattacharyya S, Nain M, Kaur M, Sood V, Gupta V, Khasa R, Abdin MZ, Vrati S, & Kalia M (2019). Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes. Autophagy, 10 (9) PMID: 25046112
Li JK, Liang JJ, Liao CL, & Lin YL (2012). Autophagy is involved in the early step of Japanese encephalitis virus infection. Microbes and infection / Institut Pasteur, 14 (2), 159-68 PMID: 21946213
Banerjee S, Narayanan K, Mizutani T, & Makino S (2002). Murine coronavirus replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells. Journal of virology, 76 (12), 5937-48 PMID: 12021326
Fung TS, & Liu DX (2019). Coronavirus infection, ER stress, apoptosis and innate immunity. Frontiers in microbiology, 5 PMID: 24987391
Cottam EM, Whelband MC, & Wileman T (2019). Coronavirus NSP6 restricts autophagosome expansion. Autophagy, 10 (8) PMID: 24991833
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