Enhancing neuronal reticulophagy: a strategy for combating aging and APP toxicity.

Reticulophagy, which directs the endoplasmic reticulum (ER) to the phagophore for sequestration within an autophagosome and subsequent lysosomal degradation via specific receptors, is essential for ER quality control and is implicated in various diseases. This study utilizes Drosophila to establish an in vivo model for reticulophagy. Starvation-induced reticulophagy is detected across multiple tissues in Drosophila. Whole-body upregulation or downregulation of the expression of reticulophagy receptors, atl and Rtnl1, negatively affects fly health. Notably, moderate upregulation of reticulophagy in neuronal tissues by overexpressing these receptors reduces age-related degeneration. In a Drosophila Alzheimer model expressing human APP (amyloid beta precursor protein), reticulophagy is compromised. Correcting reticulophagy by enhancing atl and Rtnl1 expression in the neurons promotes APP degradation, significantly reducing neurodegenerative symptoms. However, overexpression of mutated atl and Rtnl1, which disrupts the interaction of the corresponding proteins with Atg8, does not alleviate these symptoms, emphasizing the importance of receptor functionality. These findings support modulating reticulophagy as a therapeutic strategy for aging and neurodegenerative diseases associated with ER protein accumulation.

RTN3L and CALCOCO1 function in parallel to maintain proteostasis in the endoplasmic reticulum.

Reticulophagy is mediated by autophagy receptors that function in one of the two domains of the ER, tubules or flat sheets. Three different conserved mammalian receptors mediate autophagy in ER tubules: RTN3L, ATL3 and CALCOCO1. Previous studies have shown that RTN3L maintains proteostasis by targeting mutant aggregation-prone proteins for autophagy at distinct foci in ER tubules that we named ERPHS (ER-reticulophagy sites). The role for ATL3 and CALCOCO1 in proteostasis has not been addressed. Here we analyzed three different misfolded disease-causing RTN3L substrates and show that ATL3 and CALCOCO1 target the same cargoes for autophagy. Colocalization and knock down studies revealed that RTN3L and ATL3 are both required for the formation of RTN3L-containing ERPHS, while CALCOCO1 is not. We propose that RTN3L, ATL3 and CALCOCO1 work in parallel to maintain proteostasis within the ER network by targeting cargoes at different sites in the tubules.Abbreviation ATL3: atlastin GTPase 3; Baf: bafilomycin A1; CALCOCO1: calcium binding and coiled-coil domain 1; Epr1: ER-phagy receptor 1; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERPHS: ER-reticulophagy sites; LAMP1: lysosomal associated membrane protein 1; PGRMC1: progesterone receptor membrane component 1; POMC: proopiomelanocortin; Pro-AVP: pro-arginine vasopressin; RETREG1: reticulophagy regulator 1; reticulophagy: endoplasmic reticulum selective autophagy; RTN3L: reticulon 3 long isoform; VAPA: VAMP associated protein A.

Massive ER protein disposal by reticulophagy receptors and selective disposal by TOLLIP.

Proteostasis of the endoplasmic reticulum (ER) is maintained by coordinated action of two major catabolic pathways: proteasome-dependent ER-associated degradation (ERAD) and less characterized lysosomal pathways. Recent studies on ER-specific autophagy (termed "reticulophagy") have highlighted the importance of lysosomes for ER proteostasis. Key to this process are proteins termed reticulophagy receptors that connect ER fragments and Atg8-family proteins, facilitating the lysosomal degradation of both native and aberrant ER proteins in a relatively nonselective manner. In contrast, our recent work identified TOLLIP as a novel type of cargo receptor specifically dedicated to the lysosomal degradation of aberrant ER membrane proteins. The clients of TOLLIP include an engineered model substrate, which mimics an ER-retained aberrant membrane protein, and motor neuron disease-linked misfolded mutants of VAPB and BSCL2/Seipin. TOLLIP acts as a receptor to connect these aberrant ER membrane proteins and phosphatidylinositol-3-phosphate (PtdIns3P) by recognizing the former through its misfolding-sensing intrinsically disordered region (IDR) and ubiquitin-binding CUE domain, and the latter through its C2 domain. These interactions enable PtdIns3P-dependent vesicular trafficking of aberrant membrane proteins to lysosomes without promoting reticulophagic turnover of bulk ER.

Reticulophagy mediated by the V-ATPase-ATG16L1-LC3C axis.

The lysosomal degradation of the endoplasmic reticulum (ER), known as "reticulophagy", is important for protein quality control and organelle turnover. Here we present a noncanonical reticulophagy occurring at ER exit sites (ERESs) induced by the misfolded SERPINA1/α1-antitrypsin (AAT) mutant, Z-AAT. The accumulation of Z-AAT arrests ER-to-Golgi transport, and recruits V-ATPase and ATG16L1 to mediate LC3C decoration of ERESs. Consequently, the receptor RETREG1/FAM134B-2 is recruited by lipidated LC3C to initiate reticulophagy. Furthermore, the blockade of ER export acts as a universal signal to activate reticulophagy mediated by the V-ATPase-ATG16L1-LC3C axis. This study sheds light on the mechanism of how ERESs switch from ER export to reticulophagy for quality control.

REEPing the harvest of reticulophagy and nucleophagy.

Under stress conditions, the endoplasmic reticulum and nucleus undergo turnover through selective macroautophagy/autophagy processes termed reticulophagy and nucleophagy, respectively. Our recent study has identified the protein Hva22/Rop1/Yep1, a member of the REEP1-REEP4 subfamily of the REEP protein family, as an essential factor for both processes in the fission yeast Schizosaccharomyces pombe. In the absence of Hva22/Yep1, reticulophagy and nucleophagy cargos without surrounding autophagic membranes accumulate in the cytoplasm. Interestingly, human proteins in the REEP1-REEP4 subfamily can functionally substitute for Hva22/Yep1 to facilitate reticulophagy. Phylogenetic and synteny analyses further reveal that the budding yeast reticulophagy receptor Atg40 is also a REEP1-REEP4 subfamily member. Similar to human REEP1-REEP4 subfamily proteins, Atg40 can functionally replace Hva22/Yep1. Based on our findings, we propose that promoting reticulophagy is a conserved function of REEP1-REEP4 subfamily proteins.

The spatiotemporal control of ER membrane fragmentation during reticulophagy.

Reticulophagy is an evolutionarily conserved mechanism essential to maintain the endoplasmic reticulum (ER) homeostasis. A series of studies identified a panel of reticulophagy receptors. However, it remains unclear how these receptors sense upstream signals for spatiotemporal control of reticulophagy and how ER is fragmented into small pieces for sequestration into phagophores. Recently, we and others showed that the oligomerization of RETREG1/FAM134B (reticulophagy regulator 1), an reticulophagy receptor, triggers the scission of ER membrane to facilitate reticulophagy. Furthermore, we demonstrated that upstream signals are transduced by sequential phosphorylation and acetylation of RETREG1, which stimulate its oligomerization, ER fragmentation and reticulophagy. Our work provides further mechanistic insights into how reticulophagy receptor conveys cellular signals to fine-tune of ER homeostasis.Abbreviations: ER, endoplasmic reticulum; MAP1LC3, microtubule-associated protein light chain 3; RETREG1, reticulophagy regulator 1; RHD, reticulon-homology domain.

Endoplasmic reticulum-associated SARS-CoV-2 ORF3a elicits heightened cytopathic effects despite robust ER-associated degradation.

IMPORTANCE: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has tragically claimed millions of lives through coronavirus disease 2019 (COVID-19), and there remains a critical gap in our understanding of the precise molecular mechanisms responsible for the associated fatality. One key viral factor of interest is the SARS-CoV-2 ORF3a protein, which has been identified as a potent inducer of host cellular proinflammatory responses capable of triggering the catastrophic cytokine storm, a primary contributor to COVID-19-related deaths. Moreover, ORF3a, much like the spike protein, exhibits a propensity for frequent mutations, with certain variants linked to the severity of COVID-19. Our previous research unveiled two distinct types of ORF3a mutant proteins, categorized by their subcellular localizations, setting the stage for a comparative investigation into the functional and mechanistic disparities between these two types of ORF3a variants. Given the clinical significance and functional implications of the natural ORF3a mutations, the findings of this study promise to provide invaluable insights into the potential roles undertaken by these mutant ORF3a proteins in the pathogenesis of COVID-19.

UVRAG: orchestrating the initiation of reticulophagy.

Reticulophagy is a selective autophagy of the endoplasmic reticulum (ER) mediated by cargo receptors. It plays a crucial role in ER quality control, yet the mechanisms that initiate reticulophagy remain poorly understood. Our study identified the multifunctional protein UVRAG (UV radiation resistance associated gene) as a novel regulator of reticulophagy. UVRAG interacts with sheet and tubular reticulophagy receptors, regulates the oligomerization of receptors and facilitates their interaction with LC3/GABARAP, critical for ER fragmentation and autophagosome targeting. Remarkably, we found that UVRAG's function in reticulophagy initiation is independent of its traditional role in macroautophagy. Furthermore, UVRAG enhances the degradation of ER-associated mutant proteins linked to diseases like diabetes. Our findings offer insights into the mechanisms of reticulophagy initiation and highlight UVRAG's therapeutic potential in ER-related diseases.

Suberoylanilide hydroxamic acid upregulates reticulophagy receptor expression and promotes cell death in hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common clinical condition with a poor prognosis and few effective treatment options. Potent anticancer agents for treating HCC must be identified. Epigenetics plays an essential role in HCC tumorigenesis. Suberoylanilide hydroxamic acid (SAHA), the most common histone deacetylase inhibitor agent, triggers many forms of cell death in HCC. However, the underlying mechanism of action remains unclear. Family with sequence similarity 134 member B (FAM134B)-induced reticulophagy, a selective autophagic pathway, participates in the decision of cell fate and exhibits anticancer activity. This study focused on the relationship between FAM134B-induced reticulophagy and SAHA-mediated cell death. AIM: To elucidate potential roles and underlying molecular mechanisms of reticulophagy in SAHA-induced HCC cell death. METHODS: The viability, apoptosis, cell cycle, migration, and invasion of SAHA-treated Huh7 and MHCC97L cells were measured. Proteins related to the reticulophagy pathway, mitochondria-endoplasmic reticulum (ER) contact sites, intrinsic mitochondrial apoptosis, and histone acetylation were quantified using western blotting. ER and lysosome colocalization, and mitochondrial Ca2+ levels were characterized via confocal microscopy. The level of cell death was evaluated through Hoechst 33342 staining and propidium iodide colocalization. Chromatin immunoprecipitation was used to verify histone H4 lysine-16 acetylation in the FAM134B promoter region. RESULTS: After SAHA treatment, the proliferation of Huh7 and MHCC97L cells was significantly inhibited, and the migration and invasion abilities were greatly blocked in vitro. This promoted apoptosis and caused G1 phase cells to increase in a concentration-dependent manner. Following treatment with SAHA, ER-phagy was activated, thereby triggering autophagy-mediated cell death of HCC cells in vitro. Western blotting and chromatin immunoprecipitation assays confirmed that SAHA regulated FAM134B expression by enhancing the histone H4 lysine-16 acetylation in the FAM134B promoter region. Further, SAHA disturbed the Ca2+ homeostasis and upregulated the level of autocrine motility factor receptor and proteins related to mitochondria-endoplasmic reticulum contact sites in HCC cells. Additionally, SAHA decreased the mitochondrial membrane potential levels, thereby accelerating the activation of the reticulophagy-mediated mitochondrial apoptosis pathway and promoting HCC cell death in vitro. CONCLUSION: SAHA stimulates FAM134B-mediated ER-phagy to synergistically enhance the mitochondrial apoptotic pathway, thereby enhancing HCC cell death.

Hva22, a REEP family protein in fission yeast, promotes reticulophagy in collaboration with a receptor protein.

The endoplasmic reticulum (ER) undergoes selective autophagy called reticulophagy or ER-phagy. Multiple reticulon- and receptor expression enhancing protein (REEP)-like ER-shaping proteins, including budding yeast Atg40, serve as reticulophagy receptors that stabilize the phagophore on the ER by interacting with phagophore-conjugated Atg8. Additionally, they facilitate phagophore engulfment of the ER by remodeling ER morphology. We reveal that Hva22, a REEP family protein in fission yeast, promotes reticulophagy without Atg8-binding capacity. The role of Hva22 in reticulophagy can be replaced by expressing Atg40 independently of its Atg8-binding ability. Conversely, adding an Atg8-binding sequence to Hva22 enables it to substitute for Atg40 in budding yeast. Thus, the phagophore-stabilizing and ER-shaping activities, both of which Atg40 solely contains, are divided between two separate factors, receptors and Hva22, respectively, in fission yeast.Abbreviations: AIM: Atg8-family interacting motif; Atg: autophagy related; DTT: dithiothreitol; ER: endoplasmic reticulum GFP: green fluorescent protein; NAA: 1-naphthaleneacetic acid; REEP: receptor expression enhancing protein; RFP: red fluorescent protein; UPR: unfolded protein response.

Ufmylation bridges autophagy and ER homeostasis in plants.

The autophagic machinery is highly conserved in eukaryotes. Plants, as sessile organisms, are more susceptible to environmental stresses than animals. Autophagy plays a pivotal role in plant stress responses, but the regulation of autophagic flux in plants remains enigmatic with few autophagic receptors identified. We recently characterized an E3 ligase, the ubiquitin-fold modifier 1 (Ufm1) ligase 1 (Ufl1), as well as its small modifier protein Ufm1, as interactors of the core autophagy-related (ATG) proteins. Mutants of these ufmylation system components are hypersensitive to salt stress and trigger the upregulation of endoplasmic reticulum (ER) stress-responsive genes, as well as the accumulation of ER sheets caused by a defect in reticulophagy. Increased expression of Ufl1, Ufm1 and Ufm1-conjugating enzyme 1 (Ufc1) are also triggered by salt stress in plants. This study identified and demonstrated the participation of ufmylation components in maintaining ER homeostasis by regulating reticulophagy under salt stress in plants.Abbreviations: ATG, autophagy-related; ER, endoplasmic reticulum; LIR, LC3-interacting region; ROS, reactive oxygen species; CDK5RAP3/C53, CDK5 regulatory subunit-associated protein 3; Uba5, Ufm1-activating enzyme 5; Ufc1, Ufm1-conjugating enzyme 1; Ufl1, Ufm1 ligase 1; Ufm1, ubiquitin-fold modifier 1; UPR, unfolded protein response.

The Activation of Reticulophagy by ER Stress through the ATF4-MAP1LC3A-CCPG1 Pathway in Ovarian Granulosa Cells Is Linked to Apoptosis and Necroptosis.

Female infertility is caused by premature ovarian failure (POF), which is triggered by the endoplasmic reticulum (ER) stress-mediated apoptosis of granulosa cells. The ER unfolded protein response (UPRer) is initiated to promote cell survival by alleviating excessive ER stress, but cellular apoptosis is induced by persistent or strong ER stress. Recent studies have reported that reticulophagy is initiated by ER stress. Whether reticulophagy is activated in the ER stress-mediated apoptosis of granulosa cells and which pathway is initiated to activate reticulophagy during the apoptosis of granulosa cells are unknown. Therefore, the role of reticulophagy in granulosa cell death and the relationship between ER stress and reticulophagy were investigated in this work. Our results suggest that the ER stress inducer tunicamycin causes POF in mice, which is attributed to the apoptosis of granulosa cells and is accompanied by the activation of UPRer and reticulophagy. Furthermore, granulosa cells were treated with tunicamycin, and granulosa cell apoptosis was triggered and increased the expression of UPRer and reticulophagy molecules. The expression of ATF4 was then downregulated by RNAi, which decreased the levels of autophagy and the reticulophagy receptor CCGP1. Furthermore, ATF4 targets MAP1LC3A, as revealed by the ChIP sequencing results, and co-IP results demonstrated that MAP1LC3A interacts with CCPG1. Therefore, reticulophagy was activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway to mitigate ER stress. Additionally, the role of reticulophagy in granulosa cells was investigated by the knockdown of CCPG1 with RNAi. Interestingly, only a small number of granulosa cells died by apoptosis, whereas the death of most granulosa cells occurred by necroptosis triggered by STAT1 and STAT3 to impair ER proteostasis and the ER protein quality control system UPRer. Taken together, the results indicate that the necroptosis of granulosa cells is triggered by up- and downregulating the reticulophagy receptor CCPG1 through STAT1/STAT3-(p)RIPK1-(p)RIPK3-(p)MLKL and that reticulophagy is activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway.

Sensitive imaging of Endoplasmic reticulum (ER) autophagy with an acidity-reporting ER-Tracker.

Macroautophagic/autophagic turnover of endoplasmic reticulum (reticulophagy) is critical for cell health. Herein we reported a sensitive fluorescence-on imaging of reticulophagy using a small molecule probe (ER-proRed) comprised of green-emissive fluorinated rhodol for ER targeting and nonfluorescent rhodamine-lactam prone to lysosome-triggered red fluorescence. Partitioned in ER to exhibit green fluorescence, ER-proRed gives intense red fluorescence upon co-delivery with ER into acidic lysosomes. Serving as the signal of reticulophagy, the turning on of red fluorescence enables discernment of reticulophagy induced by starvation, varied levels of reticulophagic receptors, and chemical agents such as etoposide and sodium butyrate. These results show ER probes optically activatable in lysosomes, such as ER-proRed, offer a sensitive and simplified tool for studying reticulophagy in biology and diseases.Abbreviations: Baf-A1, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CQ, chloroquine diphosphate; ER, endoplasmic reticulum; FHR, fluorinated hydrophobic rhodol; GFP, green fluorescent protein; Reticulophagy, selective autophagy of ER; RFP, red fluorescent protein; ROX, X-rhodamine; UPR, unfolded protein response.

Actin assembly at sites of contact between the cortical ER and endocytic pits promotes ER autophagy.

A recent screen of the Saccharomyces cerevisiae deletion library implicated End3 in autophagy of the endoplasmic reticulum (ER). Together with Pan1, End3 coordinates endocytic site initiation with the localized assembly of branching actin filaments that promotes invagination of endocytic pits. Oxysterol binding proteins function as an inter-organelle bridge by interacting with VAP proteins on the cortical ER and type I myosins on the endocytic pit. These proteins not only promote localized actin assembly at contact sites, they are required for ER autophagy as well. We propose that localized actin polymerization can push the edge of an ER sheet from the cell cortex toward the site of autophagosome assembly near the vacuole.

An ER-targeted "reserve-release" fluorogen for topological quantification of reticulophagy.

The endoplasmic reticulum's (ER) dynamic nature, essential for maintaining cellular homeostasis, can be influenced by stress-induced damage, which can be assessed by examining the morphology of ER dynamics and, more locally, ER properties such as hydrophobicity, viscosity, and polarity. Although numerous ER-specific chemical probes have been developed to monitor the ER's physical and chemical parameters, the quantitative detection and super-resolution imaging of its local hydrophobicity have yet to be explored. Here, we describe a photostable ER-targeted probe with high signal-to-noise ratio for super-resolution imaging that can specifically respond to changes in ER hydrophobicity under stress based on a "reserve-release" mechanism. The probe shows an excellent ability to target ER over commercial ER dyes and can be used to track local changes of hydrophobicity by fluorescence intensity and morphology during the selective autophagy of ER (i.e., reticulophagy). By correlating the level and location of ER damage with the distribution of fluorescence intensity, we were able to assess reticulophagy at the subcellular level. Beyond that, we developed a topological analytical tool adaptable to any ER probe for detecting structural changes in ER and thus quantitatively identifying reticulophagy. The algorithm-assisted tool can also be adapted to a wide range of molecular probes and organelles. Altogether, the new probe and analytical strategy described here show promise for the quantitative detection and analysis of subtle ER damage and stress.

Cell Cycle Analysis of ER Stress and Autophagy.

Autophagy and ER stress are most often studied employing a Western blotting approach to the measurement of autophagy by LC3B upregulation and the ER stress sensor signaling proteins PERK (protein kinase R-like endoplasmic reticulum kinase), IRE1, and ATF6 which initiate protein refolding and elongation of the ER until ER homeostasis is returned. If the misfolding of proteins is increased, then ER stress is maintained, and microautophagy of the ER or specifically reticulophagy occurs. However, LC3B, PERK, protein misfolding, and changes in ER mass (reticulophagy) can also be measured in a cell cycle-dependent manner by flow cytometry and the use of antibodies, protein misfolding, and ER tracking fluorescent probes.

Family with sequence similarity 134 member B-mediated reticulophagy ameliorates hepatocyte apoptosis induced by dithiothreitol.

BACKGROUND: Endoplasmic reticulum (ER) stress-related hepatocyte apoptosis is responsible for multiple hepatic diseases. Previous studies have revealed that endoplasmic reticulophagy (ER-phagy) promotes the selective clearance of damaged ER fragments during ER stress, playing a crucial role in maintaining ER homeostasis and inhibiting apoptosis. Family with sequence similarity 134 member B (FAM134B) is a receptor involved in ER-phagy that can form a complex with calnexin (CNX) and microtubule-associated protein 1 light chain 3 (LC3). The complex can mediate the selective isolation of ER fragments to attenuate hepatocyte apoptosis. However, the precise regulatory mechanisms remain unclear. AIM: To elucidate the effect of FAM134B-mediated ER-phagy on ER stress-induced apoptosis in buffalo rat liver 3A (BRL-3A) rat hepatocytes and the potential regulatory mechanisms. METHODS: ER stress-related hepatocyte apoptosis was induced using dithiothreitol (DTT). Proteins related to ER stress and autophagy were measured with western blotting. Protein complex interactions with FAM134B were isolated by co-immunoprecipitation. ER-phagy was evaluated in immunofluorescence experiments. Cell cycle distribution and apoptosis were measured by flow cytometry. Mitochondrial Ca2+ levels were evaluated by the co-localization of intracellular Ca2+-tracker and Mito-tracker. The small interfering RNA against FAM134B was used to knockdown FAM134B in BRL-3A cells. RESULTS: ER stress-related and autophagy-related proteins in BRL-3A cells were elevated by both short and long-term DTT treatment. Furthermore, co-immunoprecipitation confirmed an interaction between FAM134B, CNX, FAM134B, and LC3 in BRL-3A cells. Immunofluorescence assays revealed that autolysosomes significantly decreased following short-term DTT treatment, but increased after long-term treatment. Mitochondrial Ca2+ levels and apoptotic rates were dramatically elevated, and more cells were arrested in the G1 stage after short-term DTT treatment; however, these decreased 48 h later. Moreover, FAM134B downregulation accelerated mitochondrial apoptotic pathway activation and aggravated hepatocyte apoptosis under ER stress. CONCLUSION: FAM134B-mediated ER-phagy attenuates hepatocyte apoptosis by suppressing the mitochondrial apoptotic pathway. Our findings provide new evidence highlighting the importance of FAM134B-mediated ER-phagy in attenuating hepatocyte apoptosis.

Mechanistic Insights into Selective Autophagy Subtypes in Alzheimer's Disease.

Eukaryotic cells possess a plethora of regulatory mechanisms to maintain homeostasis and ensure proper biochemical functionality. Autophagy, a central, conserved self-consuming process of the cell, ensures the timely degradation of damaged cellular components. Several studies have demonstrated the important roles of autophagy activation in mitigating neurodegenerative diseases, especially Alzheimer's disease (AD). However, surprisingly, activation of macroautophagy has not shown clinical efficacy. Hence, alternative strategies are urgently needed for AD therapy. In recent years, selective autophagy has been reported to be involved in AD pathology, and different subtypes have been identified, such as aggrephagy, mitophagy, reticulophagy, lipophagy, pexophagy, nucleophagy, lysophagy and ribophagy. By clarifying the underlying mechanisms governing these various subtypes, we may come to understand how to control autophagy to treat AD. In this review, we summarize the latest findings concerning the role of selective autophagy in the pathogenesis of AD. The evidence overwhelmingly suggests that selective autophagy is an active mechanism in AD pathology, and that regulating selective autophagy would be an effective strategy for controlling this pathogenesis.

SARS-CoV-2 ORF3a induces RETREG1/FAM134B-dependent reticulophagy and triggers sequential ER stress and inflammatory responses during SARS-CoV-2 infection.

SARS-CoV-2 infections have resulted in a very large number of severe cases of COVID-19 and deaths worldwide. However, knowledge of SARS-CoV-2 infection, pathogenesis and therapy remains limited, emphasizing the urgent need for fundamental studies and drug development. Studies have shown that induction of macroautophagy/autophagy and hijacking of the autophagic machinery are essential for the infection and replication of SARS-CoV-2; however, the mechanism of this manipulation and the function of autophagy during SARS-CoV-2 infection remain unclear. In the present study, we identified ORF3a as an inducer of autophagy (in particular reticulophagy) and revealed that ORF3a localizes to the ER and induces RETREG1/FAM134B-related reticulophagy through the HMGB1-BECN1 (beclin 1) pathway. As a consequence, ORF3a induces ER stress and inflammatory responses through reticulophagy and then sensitizes cells to the acquisition of an ER stress-related early apoptotic phenotype and facilitates SARS-CoV-2 infection, suggesting that SARS-CoV-2 ORF3a hijacks reticulophagy and then disrupts ER homeostasis to induce ER stress and inflammatory responses during SARS-CoV-2 infection. These findings reveal the sequential induction of reticulophagy, ER stress and acute inflammatory responses during SARS-CoV-2 infection and imply the therapeutic potential of reticulophagy and ER stress-related drugs for COVID-19.Abbreviations: CQ: chloroquine; DEGs: differentially expressed genes; ER: endoplasmic reticulum; GSEA: gene set enrichment analysis; HMGB1: high mobility group box 1; HMOX1: heme oxygenase 1; MERS-CoV: Middle East respiratory syndrome coronavirus; RETREG1/FAM134B: reticulophagy regulator 1; RTN4: reticulon 4; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TN: tunicamycin.