SARS-CoV-2 ORF3A interacts with the Clic-like chloride channel-1 (CLCC1) and triggers an unfolded protein response.

Understanding the interactions between SARS-CoV-2 and host cell machinery may reveal new targets to treat COVID-19. We focused on an interaction between the SARS-CoV-2 ORF3A accessory protein and the CLIC-like chloride channel-1 (CLCC1). We found that ORF3A partially co-localized with CLCC1 and that ORF3A and CLCC1 could be co-immunoprecipitated. Since CLCC1 plays a role in the unfolded protein response (UPR), we hypothesized that ORF3A may also play a role in the UPR. Indeed, ORF3A expression triggered a transcriptional UPR that was similar to knockdown of CLCC1. ORF3A expression in 293T cells induced cell death and this was rescued by the chemical chaperone taurodeoxycholic acid (TUDCA). Cells with CLCC1 knockdown were partially protected from ORF3A-mediated cell death. CLCC1 knockdown upregulated several of the homeostatic UPR targets induced by ORF3A expression, including HSPA6 and spliced XBP1, and these were not further upregulated by ORF3A. Our data suggest a model where CLCC1 silencing triggers a homeostatic UPR that prevents cell death due to ORF3A expression.

The Role of Endoplasmic Reticulum Stress in Differentiation of Cells of Mesenchymal Origin.

Endoplasmic reticulum (ER) is a multifunctional membrane-enclosed organelle. One of the major ER functions is cotranslational transport and processing of secretory, lysosomal, and transmembrane proteins. Impaired protein processing caused by disturbances in the ER homeostasis results in the ER stress. Restoration of normal ER functioning requires activation of an adaptive mechanism involving cell response to misfolded proteins, the so-called unfolded protein response (UPR). Besides controlling protein folding, UPR plays a key role in other physiological processes, in particular, differentiation of cells of connective, muscle, epithelial, and neural tissues. Cell differentiation is induced by the physiological levels of ER stress, while excessive ER stress suppresses differentiation and can result in cell death. So far, it remains unknown whether UPR activation induces cell differentiation or if UPR is initiated by the upregulated synthesis of secretory proteins during cell differentiation. Cell differentiation is an important stage in the development of multicellular organisms and is tightly controlled. Suppression or excessive activation of this process can lead to the development of various pathologies in an organism. In particular, impairments in the differentiation of connective tissue cells can result in the development of fibrosis, obesity, and osteoporosis. Recently, special attention has been paid to fibrosis as one of the major complications of COVID-19. Therefore, studying the role of UPR in the activation of cell differentiation is of both theoretical and practical interest, as it might result in the identification of molecular targets for selective regulation of cell differentiation stages and as well as the potential to modulate the mechanisms involved in the development of various pathological states.

P53 in endothelial function and unfolded protein response regulation.

Vascular barrier dysfunction due to endothelial hyperpermeability has been associated with the pathophysiology of sepsis and severe lung injury, which may inflict acute respiratory distress syndrome (ARDS). Our group is focused on the mechanisms operating towards the regulation of endothelial permeability, to contribute in the development of efficient and targeted countermeasures against ARDS. Unfortunately, the number of ARDS-related deaths in the intensive care units has dramatically increased during the COVID-19 era. The findings described herein inform the corresponding scientific and medical community on the relation of P53 and stress responses in barrier function.

ZAP isoforms regulate unfolded protein response and epithelial- mesenchymal transition.

Human ZAP inhibits many viruses, including HIV and coronaviruses, by binding to viral RNAs to promote their degradation and/or translation suppression. However, the regulatory role of ZAP in host mRNAs is largely unknown. Two major alternatively spliced ZAP isoforms, the constitutively expressed ZAPL and the infection-inducible ZAPS, play overlapping yet different antiviral and other roles that need further characterization. We found that the splicing factors hnRNPA1/A2, PTBP1/2, and U1-snRNP inhibit ZAPS production and demonstrated the feasibility to modulate the ZAPL/S balance by splice-switching antisense oligonucleotides in human cells. Transcriptomic analysis of ZAP-isoform-specific knockout cells revealed uncharacterized host mRNAs targeted by ZAPL/S with broad cellular functions such as unfolded protein response (UPR), epithelial-mesenchymal transition (EMT), and innate immunity. We established that endogenous ZAPL and ZAPS localize to membrane compartments and cytosol, respectively, and that the differential localization correlates with their target-RNA specificity. We showed that the ZAP isoforms regulated different UPR branches under resting and stress conditions and affected cell viability during ER stress. We also provided evidence for a different function of the ZAP isoforms in EMT-related cell migration, with effects that are cell-type dependent. Overall, this study demonstrates that the competition between splicing and IPA is a potential target for the modulation of the ZAPL/S balance, and reports new cellular transcripts and processes regulated by the ZAP isoforms.

Repurposing the Antiplatelet Agent Ticlopidine to Counteract the Acute Phase of ER Stress Condition: An Opportunity for Fighting Coronavirus Infections and Cancer.

Different pathological conditions, including viral infections and cancer, can have a massive impact on the endoplasmic reticulum (ER), causing severe damage to the cell and exacerbating the disease. In particular, coronavirus infections, including SARS coronavirus-2 (SARS-CoV-2), responsible for COVID-19, cause ER stress as a consequence of the enormous amounts of viral glycoproteins synthesized, the perturbation of ER homeostasis and the modification of ER membranes. Therefore, ER has a central role in the viral life cycle, thus representing one of the Achilles' heels on which to focus therapeutic intervention. On the other hand, prolonged ER stress has been demonstrated to promote many pro-tumoral attributes in cancer cells, having a key role in tumor growth, metastasis and response to therapies. In this report, adopting a repurposing approach of approved drugs, we identified the antiplatelet agent ticlopidine as an interferent of the unfolded protein response (UPR) via sigma receptors (SRs) modulation. The promising results obtained suggest the potential use of ticlopidine to counteract ER stress induced by viral infections, such as COVID-19, and cancer.

Induction and modulation of the unfolded protein response during porcine deltacoronavirus infection.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that has the potential for cross-species infection. Many viruses have been reported to induce endoplasmic reticulum stress (ERS) and activate the unfolded protein response (UPR). To date, little is known about whether and, if so, how the UPR is activated by PDCoV infection. Here, we investigated the activation state of UPR pathways and their effects on viral replication during PDCoV infection. We found that PDCoV infection induced ERS and activated all three known UPR pathways (inositol-requiring enzyme 1 [IRE1], activating transcription factor 6 [ATF6], and PKR-like ER kinase [PERK]), as demonstrated by IRE1-mediated XBP1 mRNA cleavage and increased mRNA expression of XBP1s, ATF4, CHOP, GADD34, GRP78, and GRP94, as well as phosphorylated eIF2α expression. Through pharmacologic treatment, RNA interference, and overexpression experiments, we confirmed the negative role of the PERK-eIF2α pathway and the positive regulatory role of the ATF6 pathway, but found no obvious effect of IRE1 pathway, on PDCoV replication. Taken together, our results characterize, for the first time, the state of the ERS response during PDCoV infection and identify the PERK and ATF6 pathways as potential antiviral targets.

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.

Glycosite-deleted mRNA of SARS-CoV-2 spike protein as a broad-spectrum vaccine.

Development of the messenger RNA (mRNA) vaccine has emerged as an effective and speedy strategy to control the spread of new pathogens. After vaccination, the mRNA is translated into the real protein vaccine, and there is no need to manufacture the protein in vitro. However, the fate of mRNA and its posttranslational modification inside the cell may affect immune response. Here, we showed that the mRNA vaccine of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with deletion of glycosites in the receptor-binding domain (RBD) or especially the subunit 2 (S2) domain to expose more conserved epitopes elicited stronger antibody and CD8+ T cell responses with broader protection against the alpha, beta, gamma, delta, and omicron variants, compared to the unmodified mRNA. Immunization of such mRNA resulted in accumulation of misfolded spike protein in the endoplasmic reticulum, causing the up-regulation of BiP/GRP78, XBP1, and p-eIF2α to induce cell apoptosis and strong CD8+ T cell response. In addition, dendritic cells (DCs) incubated with S2-glysosite deleted mRNA vaccine increased class I major histocompatibility complex (MHC I) expression. This study provides a direction for the development of broad-spectrum mRNA vaccines which may not be achieved with the use of expressed proteins as antigens.

Possible Therapeutic Intervention Strategies for COVID-19 by Manipulating the Cellular Proteostasis Network.

INTRODUCTION: The recent outbreak of coronavirus infection by SARS-CoV-2 that started from the Wuhan Province of China in 2019 has spread to most parts of the world infecting millions of people. Although the case fatality rate of SARS-CoV-2 infection is less than the previous epidemics by other closely related coronaviruses, due to its high infectivity, the total number of SARS-CoV-2 infection-associated disease, called Covid-19, is a matter of global concern. Despite drastic preventive measures, the number of Covid-19 cases are steadily increasing, and the future course of this pandemic is highly unpredictable. The most concerning fact about Covid-19 is the absence of specific and effective preventive or therapeutic agents against the disease. Finding an immediate intervention against Covid-19 is the need of the hour. In this chapter, we have discussed the role of different branches of the cellular proteostasis network, represented by Hsp70-Hsp40 chaperone system, Ubiquitin-Proteasome System (UPS), autophagy, and endoplasmic reticulum-Unfolded Protein Response (ER-UPR) pathway in the pathogenesis of coronavirus infections and in the host antiviral defense mechanisms. RESULTS: Based on scientific literature, we present that pharmacological manipulation of proteostasis network can alter the fate of coronavirus infections and may help to prevent the resulting pathologies like Covid-19.

SARS-CoV-2 ORF3a Induces Incomplete Autophagy via the Unfolded Protein Response.

In the past year and a half, SARS-CoV-2 has caused 240 million confirmed cases and 5 million deaths worldwide. Autophagy is a conserved process that either promotes or inhibits viral infections. Although coronaviruses are known to utilize the transport of autophagy-dependent vesicles for the viral life cycle, the underlying autophagy-inducing mechanisms remain largely unexplored. Using several autophagy-deficient cell lines and autophagy inhibitors, we demonstrated that SARS-CoV-2 ORF3a was able to induce incomplete autophagy in a FIP200/Beclin-1-dependent manner. Moreover, ORF3a was involved in the induction of the UPR (unfolded protein response), while the IRE1 and ATF6 pathways, but not the PERK pathway, were responsible for mediating the ORF3a-induced autophagy. These results identify the role of the UPR pathway in the ORF3a-induced classical autophagy process, which may provide us with a better understanding of SARS-CoV-2 and suggest new therapeutic modalities in the treatment of COVID-19.

Unfolded Protein Response Inhibition Reduces Middle East Respiratory Syndrome Coronavirus-Induced Acute Lung Injury.

Tissue- and cell-specific expression patterns are highly variable within and across individuals, leading to altered host responses after acute virus infection. Unraveling key tissue-specific response patterns provides novel opportunities for defining fundamental mechanisms of virus-host interaction in disease and the identification of critical tissue-specific networks for disease intervention in the lung. Currently, there are no approved therapeutics for Middle East respiratory syndrome coronavirus (MERS-CoV) patients, and little is understood about how lung cell types contribute to disease outcomes. MERS-CoV replicates equivalently in primary human lung microvascular endothelial cells (MVE) and fibroblasts (FB) and to equivalent peak titers but with slower replication kinetics in human airway epithelial cell cultures (HAE). However, only infected MVE demonstrate observable virus-induced cytopathic effect. To explore mechanisms leading to reduced MVE viability, donor-matched human lung MVE, HAE, and FB were infected, and their transcriptomes, proteomes, and lipidomes were monitored over time. Validated functional enrichment analysis demonstrated that MERS-CoV-infected MVE were dying via an unfolded protein response (UPR)-mediated apoptosis. Pharmacologic manipulation of the UPR in MERS-CoV-infected primary lung cells reduced viral titers and in male mice improved respiratory function with accompanying reductions in weight loss, pathological signatures of acute lung injury, and times to recovery. Systems biology analysis and validation studies of global kinetic transcript, protein, and lipid data sets confirmed that inhibition of host stress pathways that are differentially regulated following MERS-CoV infection of different tissue types can alleviate symptom progression to end-stage lung disease commonly seen following emerging coronavirus outbreaks. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe atypical pneumonia in infected individuals, but the underlying mechanisms of pathogenesis remain unknown. While much has been learned from the few reported autopsy cases, an in-depth understanding of the cells targeted by MERS-CoV in the human lung and their relative contribution to disease outcomes is needed. The host response in MERS-CoV-infected primary human lung microvascular endothelial (MVE) cells and fibroblasts (FB) was evaluated over time by analyzing total RNA, proteins, and lipids to determine the cellular pathways modulated postinfection. Findings revealed that MERS-CoV-infected MVE cells die via apoptotic mechanisms downstream of the unfolded protein response (UPR). Interruption of enzymatic processes within the UPR in MERS-CoV-infected male mice reduced disease symptoms, virus-induced lung injury, and time to recovery. These data suggest that the UPR plays an important role in MERS-CoV infection and may represent a host target for therapeutic intervention.

Multi-level inhibition of coronavirus replication by chemical ER stress.

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.

Endoplasmic reticulum stress and NF-kB activation in SARS-CoV-2 infected cells and their response to antiviral therapy.

Unfolded protein response (UPR) and endoplasmic reticulum (ER) stress are aspects of SARS-CoV-2-host cell interaction with proposed role in the cytopathic and inflammatory pathogenesis of this viral infection. The role of the NF-kB pathway in these cellular processes remains poorly characterized. When investigated in VERO-E6 cells, SARS-CoV-2 infection was found to markedly stimulate NF-kB protein expression and activity. NF-kB activation occurs early in the infection process (6 hpi) and it is associated with increased MAPK signaling and expression of the UPR inducer IRE-1α. These signal transduction processes characterize the cellular stress response to the virus promoting a pro-inflammatory environment and caspase activation in the host cell. Inhibition of viral replication by the viral protease inhibitor Nelfinavir reverts all these molecular changes also stimulating c-Jun expression, a key component of the JNK/AP-1 pathway with important role in the IRE-1α-mediated transcriptional regulation of stress response genes with anti-inflammatory and cytoprotection function. The present study demonstrates that UPR signaling and its interaction with cellular MAPKs and the NF-kB activity are important aspects of SARS-CoV-2-host cell interaction that deserve further investigation to identify more efficient therapies for this viral infection.

Comparative Host Interactomes of the SARS-CoV-2 Nonstructural Protein 3 and Human Coronavirus Homologs.

Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold-like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, nonstructural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV; and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) data set for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance, nuclear protein import for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.2 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.

Manipulation of the unfolded protein response: A pharmacological strategy against coronavirus infection.

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.

Autophagy, Unfolded Protein Response, and Neuropilin-1 Cross-Talk in SARS-CoV-2 Infection: What Can Be Learned from Other Coronaviruses.

The COVID-19 pandemic is caused by the 2019-nCoV/SARS-CoV-2 virus. This severe acute respiratory syndrome is currently a global health emergency and needs much effort to generate an urgent practical treatment to reduce COVID-19 complications and mortality in humans. Viral infection activates various cellular responses in infected cells, including cellular stress responses such as unfolded protein response (UPR) and autophagy, following the inhibition of mTOR. Both UPR and autophagy mechanisms are involved in cellular and tissue homeostasis, apoptosis, innate immunity modulation, and clearance of pathogens such as viral particles. However, during an evolutionary arms race, viruses gain the ability to subvert autophagy and UPR for their benefit. SARS-CoV-2 can enter host cells through binding to cell surface receptors, including angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1). ACE2 blockage increases autophagy through mTOR inhibition, leading to gastrointestinal complications during SARS-CoV-2 virus infection. NRP1 is also regulated by the mTOR pathway. An increased NRP1 can enhance the susceptibility of immune system dendritic cells (DCs) to SARS-CoV-2 and induce cytokine storm, which is related to high COVID-19 mortality. Therefore, signaling pathways such as mTOR, UPR, and autophagy may be potential therapeutic targets for COVID-19. Hence, extensive investigations are required to confirm these potentials. Since there is currently no specific treatment for COVID-19 infection, we sought to review and discuss the important roles of autophagy, UPR, and mTOR mechanisms in the regulation of cellular responses to coronavirus infection to help identify new antiviral modalities against SARS-CoV-2 virus.

Induction of the Proinflammatory Chemokine Interleukin-8 Is Regulated by Integrated Stress Response and AP-1 Family Proteins Activated during Coronavirus Infection.

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and IL-6. Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, we investigated the roles of the integrated stress response (ISR) and activator protein-1 (AP-1) family proteins in regulating coronavirus-induced IL-8 and IL-6 upregulation. The mRNA expression of IL-8 and IL-6 was significantly induced in cells infected with infectious bronchitis virus (IBV), a gammacoronavirus, and porcine epidemic diarrhea virus, an alphacoronavirus. Overexpression of a constitutively active phosphomimetic mutant of eukaryotic translation initiation factor 2α (eIF2α), chemical inhibition of its dephosphorylation, or overexpression of its upstream double-stranded RNA-dependent protein kinase (PKR) significantly enhanced IL-8 mRNA expression in IBV-infected cells. Overexpression of the AP-1 protein cJUN or its upstream kinase also increased the IBV-induced IL-8 mRNA expression, which was synergistically enhanced by overexpression of cFOS. Taken together, this study demonstrated the important regulatory roles of ISR and AP-1 proteins in IL-8 production during coronavirus infection, highlighting the complex interactions between cellular stress pathways and the innate immune response.

The Unfolded Protein Response and Autophagy on the Crossroads of Coronaviruses Infections.

The ability to sense and adequately respond to variable environmental conditions is central for cellular and organismal homeostasis. Eukaryotic cells are equipped with highly conserved stress-response mechanisms that support cellular function when homeostasis is compromised, promoting survival. Two such mechanisms - the unfolded protein response (UPR) and autophagy - are involved in the cellular response to perturbations in the endoplasmic reticulum, in calcium homeostasis, in cellular energy or redox status. Each of them operates through conserved signaling pathways to promote cellular adaptations that include re-programming transcription of genes and translation of new proteins and degradation of cellular components. In addition to their specific functions, it is becoming increasingly clear that these pathways intersect in many ways in different contexts of cellular stress. Viral infections are a major cause of cellular stress as many cellular functions are coopted to support viral replication. Both UPR and autophagy are induced upon infection with many different viruses with varying outcomes - in some instances controlling infection while in others supporting viral replication and infection. The role of UPR and autophagy in response to coronavirus infection has been a matter of debate in the last decade. It has been suggested that CoV exploit components of autophagy machinery and UPR to generate double-membrane vesicles where it establishes its replicative niche and to control the balance between cell death and survival during infection. Even though the molecular mechanisms are not fully elucidated, it is clear that UPR and autophagy are intimately associated during CoV infections. The current SARS-CoV-2 pandemic has brought renewed interest to this topic as several drugs known to modulate autophagy - including chloroquine, niclosamide, valinomycin, and spermine - were proposed as therapeutic options. Their efficacy is still debatable, highlighting the need to better understand the molecular interactions between CoV, UPR and autophagy.

Glucose-regulated protein (GRP78) is an important cell surface receptor for viral invasion, cancers, and neurological disorders.

The 78 kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum (ER)-resident molecular chaperone. GRP78 is a member of the 70 kDa heat shock family of proteins involved in correcting and clearing misfolded proteins in the ER. In response to cellular stress, GRP78 escapes from the ER and moves to the plasma membrane where it (a) functions as a receptor for many ligands, and (b) behaves as an autoantigen for autoantibodies that contribute to human disease and cancer. Cell surface GRP78 (csGRP78) associates with the major histocompatibility complex class I (MHC-I), and is the port of entry for several viruses, including the predictive binding of the novel SARS-CoV-2. Furthermore, csGRP78 is found in association with partners as diverse as the teratocarcinoma-derived growth factor 1 (Cripto), the melanocortin-4 receptor (MC4R) and the DnaJ-like protein MTJ-1. CsGRP78 also serves as a receptor for a large variety of ligands including activated α2 -macroglobulin (α2 M*), plasminogen kringle 5 (K5), microplasminogen, the voltage-dependent anion channel (VDAC), tissue factor (TF), and the prostate apoptosis response-4 protein (Par-4). In this review, we discuss the mechanisms involved in the translocation of GRP78 from the ER to the cell surface, and the role of secreted GRP78 and its autoantibodies in cancer and neurological disorders.

The chaperone GRP78 is a host auxiliary factor for SARS-CoV-2 and GRP78 depleting antibody blocks viral entry and infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors might also play important roles in SARS-CoV-2 infection, providing new directions for antiviral treatments. GRP78 is a stress-inducible chaperone important for entry and infectivity for many viruses. Recent molecular docking analyses revealed putative interaction between GRP78 and the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (SARS-2-S). Here we report that GRP78 can form a complex with SARS-2-S and ACE2 on the surface and at the perinuclear region typical of the endoplasmic reticulum in VeroE6-ACE2 cells and that the substrate-binding domain of GRP78 is critical for this interaction. In vitro binding studies further confirmed that GRP78 can directly bind to the RBD of SARS-2-S and ACE2. To investigate the role of GRP78 in this complex, we knocked down GRP78 in VeroE6-ACE2 cells. Loss of GRP78 markedly reduced cell surface ACE2 expression and led to activation of markers of the unfolded protein response. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159) selected for its safe clinical profile in preclinical models depleted cell surface GRP78 and reduced cell surface ACE2 expression, as well as SARS-2-S-driven viral entry and SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78 in combination therapy.