UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER.

Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour1. UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER)2,3. UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER3,4. Neither the molecular mechanism by which the UFMylation machinery achieves such precise target selection nor how this ribosomal modification promotes 60S rescue is known. Here we show that ribosome UFMylation in vivo occurs on free 60S and we present sequential cryo-electron microscopy snapshots of the heterotrimeric UFM1 E3 ligase (E3(UFM1)) engaging its substrate uL24. E3(UFM1) binds the L1 stalk, empty transfer RNA-binding sites and the peptidyl transferase centre through carboxy-terminal domains of UFL1, which results in uL24 modification more than 150 Å away. After catalysing UFM1 transfer, E3(UFM1) remains stably bound to its product, UFMylated 60S, forming a C-shaped clamp that extends all the way around the 60S from the transfer RNA-binding sites to the polypeptide tunnel exit. Our structural and biochemical analyses suggest a role for E3(UFM1) in post-termination release and recycling of the large ribosomal subunit from the ER membrane.

The UFM1 E3 ligase recognizes and releases 60S ribosomes from ER translocons.

Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24)1,2. This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. 3). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a 'writer' to a 'reader' module that recognizes its product-UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER.

Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

Intracellular organelles remodeling in myocardial endotheliocytes in COVID-19: an autopsy-based study.

It is known that the unfavorable outcome in patients infected with SARS-CoV-2 may be associated with the development of complications caused by heart damage due to the direct virus action. The mechanism of these cardiovascular injuries caused by SARS-CoV-2 infection has not been fully understood; however, the study of COVID-19-associated myocardial microcirculatory dysfunction can represent the useful strategy to solving this challenge. Thus, here we aimed to study the ultrastructural organization of endothelial cells of myocardial capillaries in patients with COVID-19. The morphology of endotheliocytes of the myocardial blood capillaries in patients with COVID-19 was studied on cardiac autopsy material using transmission electron microscopy. The endotheliocytes of myocardial capillaries in patients with COVID-19 were characterized by the abundant rough endoplasmic reticulum (ER) membranes, the Golgi complex, and free polysomal complexes of ribosomes and lipids. The presence of double membrane vesicles with virions and zippered ER was detected in the cytoplasm of endotheliocytes. The revealed endothelial ultrastructural changes indicate the remodeling of intracellular membranes during SARS-CoV-2 infection. Our findings confirm the formation of virus-induced structures in myocardial endothelial cells considered critical for viral replication and assembly. The data may elucidate the mechanisms of endothelial dysfunction development in patients with COVID-19 to provide potential targets for drug therapy.

Ultrastructural Reorganization of Endotheliocytes of Pulmonary Blood Capillaries in COVID-19.

The ultrastructural organization endotheliocytes of pulmonary blood capillaries in COVID-19 was studied on autopsy material using electron microscopy. Swelling of the cytoplasm and mitochondria with destruction of the cristae, dilation of the Golgi complex cisternae, a decrease in the volume density of the luminal and basal caveolae and free transport vesicles, an increase of the rough endoplasmic reticulum, as well as the presence of elements of coronavirus replication (reticulovesicular structures, zippered endoplasmic reticulum, electron-dense particles in the Golgi cisternae, and vacuoles with viral particles) were revealed. Further studies of the intracellular mechanisms used by the virus to replicate could help to develop antiviral drugs for the treatment of the new coronavirus infection.

The nanoscale organization of reticulon 4 shapes local endoplasmic reticulum structure in situ.

The endoplasmic reticulum's (ER's) structure is directly linked to the many functions of the ER, but its formation is not fully understood. We investigate how the ER-membrane curving protein reticulon 4 (Rtn4) localizes to and organizes in the membrane and how that affects the local ER structure. We show a strong correlation between the local Rtn4 density and the local ER membrane curvature. Our data further reveal that the typical ER tubule possesses an elliptical cross-section with Rtn4 enriched at either end of the major axis. Rtn4 oligomers are linear shaped, contain about five copies of the protein, and preferentially orient parallel to the tubule axis. Our observations support a mechanism in which oligomerization leads to an increase of the local Rtn4 concentration with each molecule, increasing membrane curvature through a hairpin wedging mechanism. This quantitative analysis of Rtn4 and its effects on the ER membrane result in a new model of tubule shape as it relates to Rtn4.

EMC chaperone-CaV structure reveals an ion channel assembly intermediate.

Voltage-gated ion channels (VGICs) comprise multiple structural units, the assembly of which is required for function1,2. Structural understanding of how VGIC subunits assemble and whether chaperone proteins are required is lacking. High-voltage-activated calcium channels (CaVs)3,4 are paradigmatic multisubunit VGICs whose function and trafficking are powerfully shaped by interactions between pore-forming CaV1 or CaV2 CaVα1 (ref. 3), and the auxiliary CaVß5 and CaVα2δ subunits6,7. Here we present cryo-electron microscopy structures of human brain and cardiac CaV1.2 bound with CaVß3 to a chaperone-the endoplasmic reticulum membrane protein complex (EMC)8,9-and of the assembled CaV1.2-CaVß3-CaVα2δ-1 channel. These structures provide a view of an EMC-client complex and define EMC sites-the transmembrane (TM) and cytoplasmic (Cyto) docks; interaction between these sites and the client channel causes partial extraction of a pore subunit and splays open the CaVα2δ-interaction site. The structures identify the CaVα2δ-binding site for gabapentinoid anti-pain and anti-anxiety drugs6, show that EMC and CaVα2δ interactions with the channel are mutually exclusive, and indicate that EMC-to-CaVα2δ hand-off involves a divalent ion-dependent step and CaV1.2 element ordering. Disruption of the EMC-CaV complex compromises CaV function, suggesting that the EMC functions as a channel holdase that facilitates channel assembly. Together, the structures reveal a CaV assembly intermediate and EMC client-binding sites that could have wide-ranging implications for the biogenesis of VGICs and other membrane proteins.

Control of mitochondria-associated endoplasmic reticulum membranes by protein S-palmitoylation: Novel therapeutic targets for neurodegenerative diseases.

Mitochondria-associated endoplasmic reticulum membranes (MAMs) are dynamic coupling structures between mitochondria and the endoplasmic reticulum (ER). As a new subcellular structure, MAMs combine the two critical organelle functions. Mitochondria and the ER could regulate each other via MAMs. MAMs are involved in calcium (Ca2+) homeostasis, autophagy, ER stress, lipid metabolism, etc. Researchers have found that MAMs are closely related to metabolic syndrome and neurodegenerative diseases (NDs). The formation of MAMs and their functions depend on specific proteins. Numerous protein enrichments, such as the IP3R-Grp75-VDAC complex, constitute MAMs. The changes in these proteins govern the interaction between mitochondria and the ER; they also affect the biological functions of MAMs. S-palmitoylation is a reversible protein post-translational modification (PTM) that mainly occurs on protein cysteine residues. More and more studies have shown that the S-palmitoylation of proteins is closely related to their membrane localization. Here, we first briefly describe the composition and function of MAMs, reviewing the component and biological roles of MAMs mediated by S-palmitoylation, elaborating on S-palmitoylated proteins in Ca2+ flux, lipid rafts, and so on. We try to provide new insight into the molecular basis of MAMs-related diseases, mainly NDs. Finally, we propose potential drug compounds targeting S-palmitoylation.

Ultrastructural evidence of four types of lipofuscins in the melanomacrophagic centers in hepatocytes of zebrafish (Denio rerio).

Melanomacrophagic centers (MMCs) were studied in the hepatocytes of zebrafish using transmission electron microscope (TEM). The MMCs with irregular or amoeboid nucleus were located in the hepatocytes adjacent to the bile canaliculi. Several engulfed structures were present in the cytoplasm of MMCs. The most frequent observation was the presence of mitochondria, ranging in size from small to giant, with distorted shape and inconspicuous cristae. Occasionally the fragments of erythrocytes were found. The rough endoplasmic reticulum (rER) showed whirling around the mitochondria and lipid droplets, forming membrane-like structures. The damaged mitochondria were invaded by the lysosomes, and this was covered by a membrane led to the formation of lipofuscin. Four different types of lipofuscins were observed; namely, (1) granular with/without vacuoles of high electron-density, (2) homogenous surrounded by indistinct limiting membrane, (3) lamellated structures similar to inner matrix and cristae of mitochondria, and, (4) compound structure made by the combinations of first 3 types, (granular and homogenous, granular and lamellated, homogenous and lamellated). The present evidence suggests that MMCs in the hepatocytes of zebrafish perform continuous functions of removal of the damaged cellular organelles. The lipofuscin formation work in coordination with the cellular players of immune system and remove pathogens and maintain the internal homeostasis of cells.

Deep neural network automated segmentation of cellular structures in volume electron microscopy.

Volume electron microscopy is an important imaging modality in contemporary cell biology. Identification of intracellular structures is a laborious process limiting the effective use of this potentially powerful tool. We resolved this bottleneck with automated segmentation of intracellular substructures in electron microscopy (ASEM), a new pipeline to train a convolutional neural network to detect structures of a wide range in size and complexity. We obtained dedicated models for each structure based on a small number of sparsely annotated ground truth images from only one or two cells. Model generalization was improved with a rapid, computationally effective strategy to refine a trained model by including a few additional annotations. We identified mitochondria, Golgi apparatus, endoplasmic reticulum, nuclear pore complexes, caveolae, clathrin-coated pits, and vesicles imaged by focused ion beam scanning electron microscopy. We uncovered a wide range of membrane-nuclear pore diameters within a single cell and derived morphological metrics from clathrin-coated pits and vesicles, consistent with the classical constant-growth assembly model.

Changes in cortical endoplasmic reticulum clusters in the fertilized mouse oocyte†.

Oocytes from many invertebrate and vertebrate species exhibit unique endoplasmic reticulum (ER) specializations (cortical ER clusters), which are thought to be essential for egg activation. In examination of cortical ER clusters, we observed that they were tethered to previously unreported fenestrae within the cortical actin layer. Furthermore, studies demonstrated that sperm preferentially bind to the plasma membrane overlying the fenestrae, establishing close proximity to underlying ER clusters. Moreover, following sperm-oocyte fusion, cortical ER clusters undergo a previously unrecognized global change in volume and shape that persists through sperm incorporation, before dispersing at the pronuclear stage. These changes did not occur in oocytes from females mated with Izumo1 -/- males. In addition to these global changes, highly localized ER modifications were noted at the sperm binding site as cortical ER clusters surround the sperm head during incorporation, then form a diffuse cloud surrounding the decondensing sperm nucleus. This study provides the first evidence that cortical ER clusters interact with the fertilizing sperm, indirectly through a previous unknown lattice work of actin fenestrae, and then directly during sperm incorporation. These observations raise the possibility that oocyte ER cluster-sperm interactions provide a competitive advantage to the oocyte, which may not occur during assisted reproductive technologies such as intracytoplasmic sperm injection.

The pivotal role of FAM134B in selective ER-phagy and diseases.

FAM134B is also known as the reticulophagy regulator 1 (RETREG1) or JK-1. FAM134B consists of two long hydrophobic fragments with a reticulon-homology domain, an N-terminal cytoplasmic domain, and a C-terminal cytoplasmic domain. FAM134B plays an important role in regulating selective ER-phagy, and is related to the occurrence and development of many diseases. In the present review, we describe theFAM134B molecular structure, subcellular localization, tissue distribution, and review its mechanisms of action during selective ER-phagy. Furthermore, we summarize the relationship between FAM134B and diseases, including neoplastic diseases, degenerative diseases, central nervous system disease, and infectious diseases. Considering the pleiotropic action of FAM134B, targeting FAM134B may be a potent therapeutic avenue for these diseases.

In situ artificial contact sites (ISACS) between synthetic and endogenous organelle membranes allow for quantification of protein-tethering activities.

Membrane contact sites are specialized areas where the membranes of two distinct organelles are physically connected and allow for the exchange of molecules and for signaling processes. Understanding the mechanisms whereby proteins localize to and function in these structures is of special interest; however, methods allowing for reconstitution of these contact sites are few and only based on synthetic membranes and recombinant proteins. Here, we devised a strategy to create in situ artificial contact sites between synthetic and endogenous organelle membranes. Liposomes functionalized with a peptide containing a two phenylalanines in an acidic tract (FFAT) motif were added to adherent cells whose plasma membrane was perforated. Confocal and super-resolution microscopy revealed that these liposomes associated with the endoplasmic reticulum via the specific interaction of the FFAT motif with endoplasmic reticulum-resident vesicle-associated membrane protein-associated proteins. This approach allowed for quantification of the attachment properties of peptides corresponding to FFAT motifs derived from distinct proteins and of a protein construct derived from steroidogenic acute regulatory protein-related lipid transfer domain-3. Collectively, these data indicate that the creation of in situ artificial contact sites represents an efficient approach for studying the membrane-tethering activity of proteins and for designing membrane contact site reconstitution assays in cellular contexts.

Early-onset dietary restriction maintains mitochondrial health, autophagy and ER function in the left ventricle during aging.

Dietary restriction (DR) exerts healthy benefits, including heart functions. However, the cardioprotective role of DR is till controversial among researchers due to the variation of DR conditions. The present study focuses on the protective effect of early-onset DR on cardiac injury using mitochondrial structure and expression of protein associated with mitochondrial homeostasis, autophagy and endoplasmic reticulum (ER) function as measures. 2-month-old mice were fed with a breeding diet ad libitum (AL) or DR (60% of AL) for 3 (Young) or 20 (Aged) months. Body weight increased with aging, whereas DR treatment kept body weight consistent. DR mice exhibited a higher relative heart weight than AL mice. DR mice displayed lower plasma glucose levels, compared with AL groups. Furthermore, Aged-AL, but not Aged-DR mice, had increased collagen content and morphological distortions in the left ventricle (LV). Aged-DR mice had a higher ATP and lower TBARS in the LV than Aged-AL mice. Mitochondrial morphology was detected by electron microscopy; Aged-AL mice had increased abnormal morphology of mitochondria. Treatment with DR reduced abnormal mitochondrial accumulation. Aging elevated the protein expressions of mitochondrial functions and ER-induced apoptosis. Aging downregulated autophagy related proteins and chaperones in the heart. Dietary restriction reversed those protein expressions. The present study demonstrated a beneficial effect of early onset DR on cardiac aging. The age-dependent mitochondrial dysfunction and protein quality control dysregulation was significantly reversed by long-term DR, demonstrating a concordance with the beneficial effect in the heart.

Regulating peroxisome-ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3ß.

Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 (acyl-coenzyme A-binding domain protein 5) and the ER-resident protein VAPB (vesicle-associated membrane protein-associated protein B). ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like (two phenylalanines [FF] in an acidic tract) motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB-and thus peroxisome-ER contact sites-differently. Moreover, we demonstrate that GSK3ß (glycogen synthase kinase-3 ß) regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome-ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.

Mechanism of shaping membrane nanostructures of endoplasmic reticulum.

Recent advances in super-resolution microscopy revealed the previously unknown nanoscopic level of organization of endoplasmic reticulum (ER), one of the most vital intracellular organelles. Membrane nanostructures of 10- to 100-nm intrinsic length scales, which include ER tubular matrices, ER sheet nanoholes, internal membranes of ER exit sites (ERES), and ER transport intermediates, were discovered and imaged in considerable detail, but the physical factors determining their unique geometrical features remained unknown. Here, we proposed and computationally substantiated a common concept for mechanisms of all ER nanostructures based on the membrane intrinsic curvature as a primary factor shaping the membrane and ultra-low membrane tensions as modulators of the membrane configurations. We computationally revealed a common structural motif underlying most of the nanostructures. We predicted the existence of a discrete series of equilibrium configurations of ER tubular matrices and recovered the one corresponding to the observations and favored by ultra-low tensions. We modeled the nanohole formation as resulting from a spontaneous collapse of elements of the ER tubular network adjacent to the ER sheet edge and calculated the nanohole dimensions. We proposed the ERES membrane to have a shape of a super flexible membrane bead chain, which acquires random walk configurations unless an ultra-low tension converts it into a straight conformation of a transport intermediate. The adequacy of the proposed concept is supported by a close qualitative and quantitative similarity between the predicted and observed configurations of all four ER nanostructures.

Targeting FAM134B-mediated reticulophagy activates sorafenib-induced ferroptosis in hepatocellular carcinoma.

Ferroptosis is a kind of cell death closely related to selective autophagy, such as ferritinophagy, lipophagy, clockophagy and chaperone-mediated autophagy. However, the role of reticulophagy, which specifically degrades endoplasmic reticulum (ER) fragments (also known as ER-phagy), in ferroptosis regulation is still unclear. In this study, we found that sorafenib (ferroptosis inducer) can effectively activate the receptor protein FAM134B-mediated ER-phagy, and FAM134B knockdown not only blocked ER-phagy but also significantly strengthened cellular sensitivity to ferroptosis without affecting macroautophagy. In vivo experiments also yielded similar results. These evidences provided new clues for ferroptosis regulation. Subsequently, bioinformatic analysis combined with RNA binding protein immunoprecipitation and polyribosome fractionation preliminarily indicated that PABPC1 can interact with FAM134B mRNA and promote its translation. Taken together, this study revealed the role of the PABPC1-FAM134B-ER-phagy pathway on ferroptosis, providing important evidence for novel anti-cancer strategies.

A cryo-ET survey of microtubules and intracellular compartments in mammalian axons.

The neuronal axon is packed with cytoskeletal filaments, membranes, and organelles, many of which move between the cell body and axon tip. Here, we used cryo-electron tomography to survey the internal components of mammalian sensory axons. We determined the polarity of the axonal microtubules (MTs) by combining subtomogram classification and visual inspection, finding MT plus and minus ends are structurally similar. Subtomogram averaging of globular densities in the MT lumen suggests they have a defined structure, which is surprising given they likely contain the disordered protein MAP6. We found the endoplasmic reticulum in axons is tethered to MTs through multiple short linkers. We surveyed membrane-bound cargos and describe unexpected internal features such as granules and broken membranes. In addition, we detected proteinaceous compartments, including numerous virus-like capsid particles. Our observations outline novel features of axonal cargos and MTs, providing a platform for identification of their constituents.

Cryo-EM structures of the endoplasmic reticulum membrane complex.

The transmembrane α-helices of membrane proteins are in general highly hydrophobic, and they enter the lipid bilayer through a lateral gate in the Sec61 translocon. However, some transmembrane α-helices are less hydrophobic and form membrane channels or substrate-binding pockets. Insertion of these amphipathic transmembrane α-helices into the membrane requires the specific membrane-embedded insertase called the endoplasmic reticulum membrane complex (EMC), which is a multi-subunit chaperone distinct from the GET insertase complex. Four recent cryo-electron microscopy studies on the eukaryotic EMC have revealed their remarkable architectural conservation from yeast to humans; a general consensus on the substrate transmembrane helix-binding pocket; and the evolutionary link to the prokaryotic insertases of the tail-anchored membrane proteins. These structures provide a solid framework for future mechanistic investigation.

The PERK/PKR-eIF2α Pathway Negatively Regulates Porcine Hemagglutinating Encephalomyelitis Virus Replication by Attenuating Global Protein Translation and Facilitating Stress Granule Formation.

The replication of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is closely associated with the endoplasmic reticulum (ER) of infected cells. The unfolded protein response (UPR), which is mediated by ER stress (ERS), is a typical outcome in coronavirus-infected cells and is closely associated with the characteristics of coronaviruses. However, the interaction between virus-induced ERS and coronavirus replication is poorly understood. Here, we demonstrate that infection with the betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) induced ERS and triggered all three branches of the UPR signaling pathway both in vitro and in vivo. In addition, ERS suppressed PHEV replication in mouse neuro-2a (N2a) cells primarily by activating the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) axis of the UPR. Moreover, another eIF2α phosphorylation kinase, interferon (IFN)-induced double-stranded RNA-dependent protein kinase (PKR), was also activated and acted cooperatively with PERK to decrease PHEV replication. Furthermore, we demonstrate that the PERK/PKR-eIF2α pathways negatively regulated PHEV replication by attenuating global protein translation. Phosphorylated eIF2α also promoted the formation of stress granules (SGs), which in turn repressed PHEV replication. In summary, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets (e.g., PERK, PKR, and eIF2α) for antiviral drugs. IMPORTANCE Coronavirus diseases are caused by different coronaviruses of importance in humans and animals, and specific treatments are extremely limited. ERS, which can activate the UPR to modulate viral replication and the host innate response, is a frequent occurrence in coronavirus-infected cells. PHEV, a neurotropic betacoronavirus, causes nerve cell damage, which accounts for the high mortality rates in suckling piglets. However, it remains incompletely understood whether the highly developed ER in nerve cells plays an antiviral role in ERS and how ERS regulates viral proliferation. In this study, we found that PHEV infection induced ERS and activated the UPR both in vitro and in vivo and that the activated PERK/PKR-eIF2α axis inhibited PHEV replication through attenuating global protein translation and promoting SG formation. A better understanding of coronavirus-induced ERS and UPR activation may reveal the pathogenic mechanism of coronavirus and facilitate the development of new treatment strategies for these diseases.