内质网应激与自噬及其交互作用影响内皮细胞凋亡

内质网应激是普遍存在于真核细胞中的应激-防御机制。在内环境稳态遭到破坏的情况下,未折叠蛋白质反应的3条信号通路,分别通过增强蛋白质折叠能力、减少蛋白质生成和促进内质网相关蛋白质降解等途径缓解细胞内压力。同时,也通过多种分子信号机制调控细胞凋亡。自噬是一种生理性的降解机制。通过形成自噬泡并与溶酶体结合摄取并水解胞内受损细胞器和蛋白质等,清除代谢废物,维持细胞正常功能。自噬缺陷或过度激活均可导致细胞凋亡或非程序性死亡。自噬的程度和细胞内压力水平有关。内质网应激通过未折叠蛋白质反应和Ca²⁺浓度变化及其相关分子信号调控自噬。自噬又可反馈性调节内质网应激反应,二者相互作用,在内皮细胞凋亡过程中发挥重要作用。未来内质网应激和自噬可作为药物靶点为内皮相关性疾病提供诊疗策略。     

An appetite for destruction: From self-eating to cell cannibalism as a neuronal survival strategy

Autophagy plays an important role in cellular survival by resupplying cells with nutrients during starvation or by clearing misfolded proteins and damaged organelles and thereby preventing degenerative diseases. Conversely, the autophagic process is also recognized as a cellular death mechanism. The circumstances that determine whether autophagy has a beneficial or a detrimental role in cellular survival are currently unclear. We recently showed that autophagy induction is detrimental in neurons that lack a functional AMPK enzyme (AMP-activated protein kinase) and that suffer from severe metabolic stress. We further demonstrated that autophagy and AMPK are interconnected in a negative feedback loop that prevents excessive and destructive stimulation of the autophagic process. Finally, we uncovered a new survival mechanism in AMPK-deficient neurons-cell cannibalism.     

An appetite for destruction

Autophagy plays an important role in cellular survival by resupplying cells with nutrients during starvation or by clearing misfolded proteins and damaged organelles and thereby preventing degenerative diseases. Conversely, the autophagic process is also recognized as a cellular death mechanism. The circumstances that determine whether autophagy has a beneficial or a detrimental role in cellular survival are currently unclear. We recently showed that autophagy induction is detrimental in neurons that lack a functional AMPK enzyme (AMP-activated protein kinase) and that suffer from severe metabolic stress. We further demonstrated that autophagy and AMPK are interconnected in a negative feedback loop that prevents excessive and destructive stimulation of the autophagic process. Finally, we uncovered a new survival mechanism in AMPK-deficient neurons—cell cannibalism.     

Acidic intracellular Ca stores and caveolae in Ca signaling and diabetes

Acidic Ca²⁺ stores, particularly lysosomes, are newly discovered players in the well-orchestrated arena of Ca²⁺ signaling and we are at the verge of understanding how lysosomes accumulate Ca²⁺ and how they release it in response to different chemical, such as NAADP, and physical signals. Additionally, it is now clear that lysosomes play a key role in autophagy, a process that allows cells to recycle components or to eliminate damaged structures to ensure cellular well-being. Moreover, lysosomes are being unraveled as hubs that coordinate both anabolism via insulin signaling and catabolism via AMPK. These acidic vesicles have close contact with the ER and there is a bidirectional movement of information between these two organelles that exquisitely regulates cell survival. Lysosomes also connect with plasma membrane where caveolae are located as specialized regions involved in Ca²⁺ and insulin signaling. Alterations of all these signaling pathways are at the core of insulin resistance and diabetes.     

Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells

The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule-associated protein 1 light-chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.     

Targeting autophagy to fight hematopoietic malignancies

Macroautophagy, referred hereafter to as autophagy is an evolutionary conserved catabolic process for the degradation and recycling of macromolecules, bulk cytoplasm and dammaged organelles. Autophagy is activated under stress conditions induced by nutrient deprivation, hypoxia and drug treatments. Morphologically, autophagic cells are characterized by the accumulation of double membrane cytoplasmic vesicules called autophagosomes that surrounds cytoplasmic proteins and/or organelles. Autophagosomes next fuse with lysosomes to generate autolysosomes, the structures in which the retained constituents are digested before recycling into the cytoplasm. In this context, autophagy promotes cell survival under adverse conditions. In contrast, under certain circumstances autophagic cells may engage a specific mode of cell death called type II cell death or autophagic cell death (ACD). Considering the strategic positionnement of this process at the crossroads of cell death and survival, it is not surprising that defects in autophagy have been linked to a plethora of human diseases, including hematopoietic malignancies. Finally, autophagy induction is repressed by the mammalian target of rapamycin complex 1 (mTORC1) and favored by the adenosine-monophosphate activated-protein kinase (AMPK). In the present review, we focus on the functions of autophagy in normal and malignant hematopoiesis and discuss the opportunity to target the AMPK/mTOR pathways as a new therapeutic strategy to fight hematopoietic malignancies with a special emphasis on Chronic Myelogenous Leukemia (CML).     

Unraveling the role of myosin in forming autophagosomes

Macroautophagy (hereafter autophagy) is a membrane-mediated catabolic process that occurs in response to a variety of intra- and extra-cellular stresses. It is characterized by the formation of specialized double-membrane vesicles, autophagosomes, which engulf organelles and long-lived proteins, and in turn fuse with lysosomes for degradation and recycling. How autophagosomes emerge is still unclear. The Atg1 kinase plays a crucial role in the induction of autophagosome formation. While several Atg (autophagy-related) proteins have been associated with, and have been found to regulate, Atg1 kinase activity, the downstream targets of Atg1 that trigger autophagy remain unknown. Our recent studies have identified a myosin light chain kinase (MLCK)-like kinase as the Atg1 kinase effector that induces the activation of myosin II, and have found it to be required for autophagosome formation during nutrient deprivation. We further demonstrated that Atg1-mediated myosin II activation is crucial for the movement of the Atg9 transmembrane protein between the Golgi and the forming autophagosome, which provides a membrane source for the formation of autophagosomes during starvation.     

Unraveling the role of myosin in forming autophagosomes

Macroautophagy (hereafter autophagy) is a membrane-mediated catabolic process that occurs in response to a variety of intra- and extra-cellular stresses. It is characterized by the formation of specialized double-membrane vesicles, autophagosomes, which engulf organelles and long-lived proteins, and in turn fuse with lysosomes for degradation and recycling. How autophagosomes emerge is still unclear. The Atg1 kinase plays a crucial role in the induction of autophagosome formation. While several Atg (autophagy-related) proteins have been associated with, and have been found to regulate, Atg1 kinase activity, the downstream targets of Atg1 that trigger autophagy remain unknown. Our recent studies have identified a myosin light chain kinase (MLCK)-like kinase as the Atg1 kinase effector that induces the activation of myosin II, and have found it to be required for autophagosome formation during nutrient deprivation. We further demonstrated that Atg1-mediated myosin II activation is crucial for the movement of the Atg9 transmembrane protein between the Golgi and the forming autophagosome, which provides a membrane source for the formation of autophagosomes during starvation.     

Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway

In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.     

Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway

In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.     

Autophagosome formation: core machinery and adaptations

Eukaryotic cells employ autophagy to degrade damaged or obsolete organelles and proteins. Central to this process is the formation of autophagosomes, double-membrane vesicles responsible for delivering cytoplasmic material to lysosomes. In the past decade many autophagy-related genes, ATG, have been identified that are required for selective and/or nonselective autophagic functions. In all types of autophagy, a core molecular machinery has a critical role in forming sequestering vesicles, the autophagosome, which is the hallmark morphological feature of this dynamic process. Additional components allow autophagy to adapt to the changing needs of the cell.     

Ambra1 modulates starvation-induced autophagy through AMPK signaling pathway in cardiomyocytes

Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells and cancer cells. However, whether Ambra1 plays an important role in the autophagy pathway in cardiomyocytes is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in cardiomyocytes. To test this hypothesis, we confirmed autophagic activity in serum-starved cardiomyocytes by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes and LC3 protein levels. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in cardiomyocytes. When Ambra1 expression was reduced by siRNA, the cardiomyocytes were more sensitive to staurosporine-induced apoptosis. In addition, co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated cardiomyocytes, suggesting that Ambra1 regulates autophagy in cardiomyocytes by interacting with Beclin1. Finally, we determined that starvation stress-induced activation of Ambra1 contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signaling. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis through AMPK signaling pathway in cardiomyocytes that maintains the balance between autophagy and apoptosis.     

The role of ESCRT proteins in fusion events involving lysosomes, endosomes and autophagosomes

ESCRT (endosomal sorting complex required for transport) proteins were originally identified for their role in delivering endocytosed proteins to the intraluminal vesicles of late-endosomal structures termed multivesicular bodies. Multivesicular bodies then fuse with lysosomes, leading to degradation of the internalized proteins. Four ESCRT complexes interact to concentrate cargo on the endosomal membrane, induce membrane curvature to form an intraluminal bud and finally pinch off the bud through a membrane-scission event to produce the intraluminal vesicle. Recent work suggests that ESCRT proteins are also required downstream of these events to enable fusion of multivesicular bodies with lysosomes. Autophagy is a related pathway required for the degradation of organelles, long-lived proteins and protein aggregates which also converges on lysosomes. The proteins or organelle to be degraded are encapsulated by an autophagosome that fuses either directly with a lysosome or with an endosome to form an amphisome, which then fuses with a lysosome. A common machinery is beginning to emerge that regulates fusion events in the multivesicular body and autophagy pathways, and we focus in the present paper on the role of ESCRT proteins. These fusion events have been implicated in diseases including frontotemporal dementia, Alzheimer's disease, lysosomal storage disorders, myopathies and bacterial pathogen invasion, and therefore further examination of the mechanisms involved may lead to new insight into disease pathogenesis and treatments.     

The BECN1-USP19 axis plays a role in the crosstalk between autophagy and antiviral immune responses

Macroautophagy/autophagy is a conserved intracellular degradation system that traffics substrates including protein aggregates, defunct or disused organelles and invading pathogens to lysosomes via double-membrane vesicles called autophagosomes. BECN1/Beclin 1 functions as a key protein in autophagy initiation and progression; however, the role of BECN1 in innate immunity has not been fully investigated. Recently, we have found that USP19 affects the ubiquitination of BECN1, hence promoting the formation of autophagosomes and inhibiting DDX58/RIG-I-mediated type I interferon signaling.     

Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3L

Macroautophagy (called autophagy hereafter) is a catabolic process activated by various types of stress, most notably by nutrient deprivation. The autophagic degradation of intracellular macromolecules provides metabolic support for the cell; however, this physiological process can also initiate a form of cell death (type 2 programmed cell death). Here we report that oxygen deprivation can activate the autophagic pathway in human cancer cell lines. We observed that hypoxia induced distinct cellular changes characteristic of autophagy such as an increase in cytoplasmic acidic vesicles, and processing and cellular localization of microtubule-associated protein-1 light chain 3. Oxygen deprivation-induced autophagy did not require nutrient deprivation, hypoxia-inducible factor-1 (HIF-1) activity, or expression of the HIF-1 target gene BNIP3 (Bcl-2 adenovirus E1a nineteen kilodalton interacting protein 3) or BNIP3L (BNIP3 like protein). Hypoxia-induced autophagy involved the activity of 5'-AMP-activated protein kinase (AMPK). Finally, we determined that cells lacking the autophagy gene ATG5 were unable to activate the autophagic machinery in hypoxia, had decreased oxygen consumption and increased glucose uptake under hypoxia, had increased survival in hypoxic environments, and exhibited accelerated growth as xenografted tumors. Together, these findings suggest that the autophagic degradation of cellular macromolecules contributes to the energetic balance governed by AMPK, and that suppression of autophagy in transformed cells can increase both resistance to hypoxic stress and tumorigenicity.     

Depletion of NBR1 in urothelial carcinoma cells enhances rapamycin-induced apoptosis through impaired autophagy and mitochondrial dysfunction

Rapamycin is well-recognized in the clinical therapeutic intervention for patients with cancer by specifically targeting mammalian target of rapamycin (mTOR) kinase. Rapamycin regulates general autophagy to clear damaged cells. Previously, we identified increased expression of messenger RNA levels of NBR1 (the neighbor of BRCA1 gene; autophagy cargo receptor) in human urothelial cancer (URCa) cells, which were not exhibited in response to rapamycin treatment for cell growth inhibition. Autophagy plays an important role in cellular physiology and offers protection against chemotherapeutic agents as an adaptive response required for maintaining cellular energy. Here, we hypothesized that loss of NBR1 sensitizes human URCa cells to growth inhibition induced by rapamycin treatment, leading to interruption of protective autophagic activation. Also, the potential role of mitochondria in regulating autophagy was tested to clarify the mechanism by which rapamycin induces apoptosis in NBR1-knockdown URCa cells. NBR1-knockdown URCa cells exhibited enhanced sensitivity to rapamycin associated with the suppression of autophagosomal elongation and mitochondrial defects. Loss of NBR1 expression altered the cellular responses to rapamycin treatment, resulting in impaired ATP homeostasis and an increase in reactive oxygen species (ROS). Although rapamycin treatment-induced autophagy by adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in NBR1-knockdown cells, it did not process the conjugated form of LC3B-II after activation by unc-51 like autophagy-activating kinase 1 (ULK1). NBR1-knockdown URCa cells exhibited rather profound mitochondrial dysfunctions in response to rapamycin treatment as evidenced by Δψm collapse, ATP depletion, ROS accumulation, and apoptosis activation. Therefore, our findings provide a rationale for rapamycin treatment of NBR1-knockdown human urothelial cancer through the regulation of autophagy and mitochondrial dysfunction by regulating the AMPK/mTOR signaling pathway, indicating that NBR1 can be a potential therapeutic target of human urothelial cancer.     

AMPK:细胞能量中枢

腺苷酸活化蛋白激酶(AMPactivated proteinkinase,AMPK)是真核细胞中高度保守的丝氨酸/苏氨酸蛋白激酶,以异源三聚体的形式广泛存在于真核生物体内,是细胞的能量感受器,在能量代谢调控中起极其重要的作用。肝激酶B1(LKB1)、Ca2+/CaM-依赖蛋白激酶激酶β(CaMKKβ)、AMP/ATP或ADP/ATP比值升高以及诸如运动肌肉收缩等生理刺激均可以激活AMPK,进而调节细胞的能量代谢网络,提高其应对内外环境变化的能力,从而维持细胞水平乃至整个机体的稳定状态。活化的AMPK可以增强分解代谢,抑制合成代谢,上调ATP水平,参与细胞糖代谢、脂肪代谢、蛋白质代谢等能量代谢过程,增加细胞能量储备,应对能量缺乏。同时活化的AMPK参与细胞的生长、增殖、凋亡、自噬等基本生物学过程。AMPK是研究肥胖,糖尿病等能量代谢性疾病的核心。肿瘤细胞存在特殊的能量代谢方式,其发生,生长,转移与能量代谢失衡密切相关。AMPK与肿瘤细胞异常的能量代谢相关,为肿瘤发生、发展机制研究提供新的策略。本文主要探讨AMPK的结构、激活机制、参与的物质能量代谢和细胞的基本生物学过程以及与肿瘤发生的关联。     

Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate

Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defence against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, Infectious Bronchitis Virus (IBV) activates autophagy. A screen of individual IBV non-structural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses Mouse Hepatitis Virus, and Severe Acute Respiratory Syndrome Virus, and the equivalent nsp5-7 of the arterivirus Porcine Reproductive and Respiratory Syndrome Virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II-positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double FYVE-domain containing protein (DFCP) indicating localised concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signalling, activation of sirtuin1 or induction of ER stress.     

AMPK：细胞能量中枢

腺苷酸活化蛋白激酶（AMP activated protein kinase,AMPK）是真核细胞中高度保守的丝氨酸／苏氨酸蛋白激酶,以异源三聚体的形式广泛存在于真核生物体内,是细胞的能量感受器,在能量代谢调控中起极其重要的作用。肝激酶B1（LKB1）、Ca^2＋／CaM-依赖蛋白激酶激酶β（CaMKKβ）、AMP／ATP或ADP／ATP比值升高以及诸如运动肌肉收缩等生理刺激均可以激活AMPK,进而调节细胞的能量代谢网络,提高其应对内外环境变化的能力,从而维持细胞水平乃至整个机体的稳定状态。活化的AMPK可以增强分解代谢,抑制合成代谢,上调ATP水平,参与细胞糖代谢、脂肪代谢、蛋白质代谢等能量代谢过程,增加细胞能量储备,应对能量缺乏。同时活化的AMPK参与细胞的生长、增殖、凋亡、自噬等基本生物学过程。AMPK是研究肥胖,糖尿病等能量代谢性疾病的核心。肿瘤细胞存在特殊的能量代谢方式,其发生,生长,转移与能量代谢失衡密切相关。AMPK与肿瘤细胞异常的能量代谢相关,为肿瘤发生、发展机制研究提供新的策略。本文主要探讨AMPK的结构、激活机制、参与的物质能量代谢和细胞的基本生物学过程以及与肿瘤发生的关联。     

Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate

Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defence against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, Infectious Bronchitis Virus (IBV) activates autophagy. A screen of individual IBV non-structural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses Mouse Hepatitis Virus, and Severe Acute Respiratory Syndrome Virus, and the equivalent nsp5-7 of the arterivirus Porcine Reproductive and Respiratory Syndrome Virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II-positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double FYVE-domain containing protein (DFCP) indicating localised concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signalling, activation of sirtuin1 or induction of ER stress.