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1.
Microbiol Mol Biol Rev ; 85(4): e0003521, 2021 12 15.
Article in English | MEDLINE | ID: covidwho-1854242

ABSTRACT

Viruses are intracellular parasites that subvert the functions of their host cells to accomplish their infection cycle. The endoplasmic reticulum (ER)-residing chaperone proteins are central for the achievement of different steps of the viral cycle, from entry and replication to assembly and exit. The most abundant ER chaperones are GRP78 (78-kDa glucose-regulated protein), GRP94 (94-kDa glucose-regulated protein), the carbohydrate or lectin-like chaperones calnexin (CNX) and calreticulin (CRT), the protein disulfide isomerases (PDIs), and the DNAJ chaperones. This review will focus on the pleiotropic roles of ER chaperones during viral infection. We will cover their essential role in the folding and quality control of viral proteins, notably viral glycoproteins which play a major role in host cell infection. We will also describe how viruses co-opt ER chaperones at various steps of their infectious cycle but also in order to evade immune responses and avoid apoptosis. Finally, we will discuss the different molecules targeting these chaperones and the perspectives in the development of broad-spectrum antiviral drugs.


Subject(s)
Virus Diseases , Calnexin/metabolism , Endoplasmic Reticulum/metabolism , Humans , Molecular Chaperones/metabolism
2.
Virology ; 570: 1-8, 2022 05.
Article in English | MEDLINE | ID: covidwho-1839383

ABSTRACT

Enveloped viruses such as Coronaviridae (CoV) enter the host cell by fusing the viral envelope directly with the plasma membrane (PM) or with the membrane of the endosome. Replication of the CoV genome takes place in membrane compartments formed by rearrangement of the endoplasmic reticulum (ER) membrane network. Budding of these viruses occurs from the ER-Golgi intermediate compartment (ERGIC). The relationship between proteins and various membranes is crucial for the replication cycle of CoVs. The role of transmembrane domains (TMDs) and pre-transmembrane domains (pre-TMD) of viral proteins in this process is gaining more recognition. Here we present a thorough analysis of physico-chemical parameters, such as accessible surface area (ASA), average hydrophobicity (Hav), and contribution of specific amino acids in TMDs and pre-TMDs of single-span membrane proteins of human viruses. We focus on unique properties of these elements in CoV and postulate their role in adaptation to diverse host membranes and regulation of retention of membrane proteins during replication.


Subject(s)
Coronaviridae , Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Domains , Viral Proteins/metabolism
3.
J Virol ; 96(1): e0169521, 2022 01 12.
Article in English | MEDLINE | ID: covidwho-1816694

ABSTRACT

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.


Subject(s)
Betacoronavirus 1/physiology , Coronavirus Infections/metabolism , Eukaryotic Initiation Factor-2/metabolism , Virus Replication/physiology , eIF-2 Kinase/metabolism , Animals , Betacoronavirus 1/metabolism , Cell Line , Coronavirus Infections/virology , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum Stress , Mice , Phosphorylation , Protein Biosynthesis , Signal Transduction , Unfolded Protein Response
4.
Commun Biol ; 5(1): 115, 2022 02 08.
Article in English | MEDLINE | ID: covidwho-1684117

ABSTRACT

ß-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.


Subject(s)
COVID-19/metabolism , Coat Protein Complex I/metabolism , Coatomer Protein/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence , Binding Sites/genetics , COVID-19/genetics , COVID-19/virology , Coat Protein Complex I/chemistry , Coat Protein Complex I/genetics , Coatomer Protein/chemistry , Coatomer Protein/genetics , Computer Simulation , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , HEK293 Cells , Humans , Models, Molecular , Mutation , Phylogeny , Protein Binding , Protein Domains , Protein Transport , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/classification , Spike Glycoprotein, Coronavirus/genetics , WD40 Repeats/genetics
5.
J Phys Chem Lett ; 12(51): 12249-12255, 2021 Dec 30.
Article in English | MEDLINE | ID: covidwho-1586057

ABSTRACT

SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratory's refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics that inhibit coronavirus budding.


Subject(s)
Coronavirus Envelope Proteins/metabolism , Molecular Dynamics Simulation , SARS-CoV-2/physiology , Viral Matrix Proteins/metabolism , COVID-19/pathology , COVID-19/virology , Coronavirus Envelope Proteins/chemistry , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Humans , Protein Multimerization , Protein Transport , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/chemistry
6.
Nat Struct Mol Biol ; 28(7): 614-625, 2021 07.
Article in English | MEDLINE | ID: covidwho-1550333

ABSTRACT

p97 processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Here, we report a series of cryo-EM structures of the substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Å that captured 'power-stroke'-like motions of both the D1 and D2 ATPase rings of p97. A key feature of these structures is the critical conformational changes of the intersubunit signaling (ISS) motifs, which tighten the binding of nucleotides and neighboring subunits and contribute to the spiral staircase conformation of the D1 and D2 rings. In addition, we determined the cryo-EM structure of human p97 in complex with NMS-873, a potent p97 inhibitor, at a resolution of 2.4 Å. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change and thus blocking substrate translocation allosterically.


Subject(s)
Adenosine Triphosphate/chemistry , Protein Folding , Proteostasis/physiology , Signal Transduction/physiology , Valosin Containing Protein/metabolism , Acetanilides/pharmacology , Animals , Benzothiazoles/pharmacology , Cryoelectron Microscopy , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum-Associated Degradation/physiology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Conformation , Ubiquitinated Proteins/metabolism , Valosin Containing Protein/antagonists & inhibitors
7.
Biochem Biophys Res Commun ; 586: 137-142, 2022 01 01.
Article in English | MEDLINE | ID: covidwho-1520712

ABSTRACT

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Host Microbial Interactions/physiology , Membrane Glycoproteins/metabolism , Nuclear Pore Complex Proteins/metabolism , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Active Transport, Cell Nucleus , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Gene Knockdown Techniques , HeLa Cells , Humans , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/genetics , Models, Biological , Nuclear Envelope/metabolism , Nuclear Envelope/virology , Nuclear Pore Complex Proteins/antagonists & inhibitors , Nuclear Pore Complex Proteins/genetics , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factor RelA/metabolism , Viral Nonstructural Proteins/genetics
8.
Cell ; 184(24): 5950-5969.e22, 2021 11 24.
Article in English | MEDLINE | ID: covidwho-1499701

ABSTRACT

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.


Subject(s)
Autophagosomes/virology , COVID-19/virology , Autophagy , COVID-19/metabolism , CRISPR-Cas Systems , Cell Line, Tumor , Endoplasmic Reticulum/metabolism , Endosomes/physiology , Endosomes/virology , Golgi Apparatus/physiology , HEK293 Cells , HeLa Cells , Humans , Membrane Fusion , Microscopy, Confocal , Phagosomes/metabolism , Phagosomes/virology , Qa-SNARE Proteins/biosynthesis , Receptors, sigma/biosynthesis , SARS-CoV-2 , Sarcoplasmic Reticulum Calcium-Transporting ATPases/biosynthesis , Synaptotagmins/biosynthesis
9.
Cells ; 10(10)2021 09 24.
Article in English | MEDLINE | ID: covidwho-1438526

ABSTRACT

Eukaryotic cells contain dynamic membrane-bound organelles that are constantly remodeled in response to physiological and environmental cues. Key organelles are the endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which are interconnected by vesicular traffic through the secretory transport route. Numerous viruses, especially enveloped viruses, use and modify compartments of the secretory pathway to promote their replication, assembly and cell egression by hijacking the host cell machinery. In some cases, the subversion mechanism has been uncovered. In this review, we summarize our current understanding of how the secretory pathway is subverted and exploited by viruses belonging to Picornaviridae, Coronaviridae, Flaviviridae, Poxviridae, Parvoviridae and Herpesviridae families.


Subject(s)
Endoplasmic Reticulum/virology , Golgi Apparatus/virology , Secretory Pathway/physiology , Viruses/isolation & purification , Biological Transport/physiology , Cell Membrane/metabolism , Cell Membrane/virology , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Humans
11.
Nat Commun ; 12(1): 5333, 2021 09 09.
Article in English | MEDLINE | ID: covidwho-1402067

ABSTRACT

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain, and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. Here we report a proteomic screen for cellular factors that interact with the cytoplasmic tail of S. We confirm interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding site promotes exit from the endoplasmic reticulum, and although binding to COPI should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.


Subject(s)
COVID-19/metabolism , Cell Membrane/metabolism , Giant Cells/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , COP-Coated Vesicles/metabolism , Chlorocebus aethiops , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , HEK293 Cells , Humans , Protein Binding , Protein Domains , Proteomics , Vero Cells , Virus Assembly/genetics
12.
Microbiol Spectr ; 9(2): e0119921, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1398600

ABSTRACT

Human angiotensin I-converting enzyme 2 (hACE2) is a type I transmembrane glycoprotein that serves as the major cell entry receptor for SARS-CoV and SARS-CoV-2. The viral spike (S) protein is required for the attachment to ACE2 and subsequent virus-host cell membrane fusion. Previous work has demonstrated the presence of N-linked glycans in ACE2. N-glycosylation is implicated in many biological activities, including protein folding, protein activity, and cell surface expression of biomolecules. However, the contribution of N-glycosylation to ACE2 function is poorly understood. Here, we examined the role of N-glycosylation in the activity and localization of two species with different susceptibility to SARS-CoV-2 infection, porcine ACE2 (pACE2) and hACE2. The elimination of N-glycosylation by tunicamycin (TM) treatment, or mutagenesis, showed that N-glycosylation is critical for the proper cell surface expression of ACE2 but not for its carboxiprotease activity. Furthermore, nonglycosylable ACE2 was localized predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Our data also revealed that binding of SARS-CoV or SARS-CoV-2 S protein to porcine or human ACE2 was not affected by deglycosylation of ACE2 or S proteins, suggesting that N-glycosylation does not play a role in the interaction between SARS coronaviruses and the ACE2 receptor. Impairment of hACE2 N-glycosylation decreased cell-to-cell fusion mediated by SARS-CoV S protein but not that mediated by SARS-CoV-2 S protein. Finally, we found that hACE2 N-glycosylation is required for an efficient viral entry of SARS-CoV/SARS-CoV-2 S pseudotyped viruses, which may be the result of low cell surface expression of the deglycosylated ACE2 receptor. IMPORTANCE Understanding the role of glycosylation in the virus-receptor interaction is important for developing approaches that disrupt infection. In this study, we showed that deglycosylation of both ACE2 and S had a minimal effect on the spike-ACE2 interaction. In addition, we found that the removal of N-glycans of ACE2 impaired its ability to support an efficient transduction of SARS-CoV and SARS-CoV-2 S pseudotyped viruses. Our data suggest that the role of deglycosylation of ACE2 on reducing infection is likely due to a reduced expression of the viral receptor on the cell surface. These findings offer insight into the glycan structure and function of ACE2 and potentially suggest that future antiviral therapies against coronaviruses and other coronavirus-related illnesses involving inhibition of ACE2 recruitment to the cell membrane could be developed.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , SARS-CoV-2/growth & development , Tunicamycin/pharmacology , Virus Attachment/drug effects , Virus Internalization/drug effects , Animals , Antiviral Agents/pharmacology , COVID-19/pathology , Carboxypeptidases/drug effects , Cell Line , Endoplasmic Reticulum/metabolism , Glycosylation/drug effects , HEK293 Cells , Humans , Membrane Proteins/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Swine
13.
Basic Res Cardiol ; 116(1): 42, 2021 07 05.
Article in English | MEDLINE | ID: covidwho-1293364

ABSTRACT

Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.


Subject(s)
Endoplasmic Reticulum/virology , Endothelial Cells/virology , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/metabolism , Calnexin/metabolism , Cells, Cultured , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Endothelial Cells/metabolism , Host-Pathogen Interactions , Humans , Membrane Proteins/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
14.
Cell Metab ; 33(8): 1655-1670.e8, 2021 08 03.
Article in English | MEDLINE | ID: covidwho-1233395

ABSTRACT

How amphipathic phospholipids are shuttled between the membrane bilayer remains an essential but elusive process, particularly at the endoplasmic reticulum (ER). One prominent phospholipid shuttling process concerns the biogenesis of APOB-containing lipoproteins within the ER lumen, which may require bulk trans-bilayer movement of phospholipids from the cytoplasmic leaflet of the ER bilayer. Here, we show that TMEM41B, present in the lipoprotein export machinery, encodes a previously conceptualized ER lipid scramblase mediating trans-bilayer shuttling of bulk phospholipids. Loss of hepatic TMEM41B eliminates plasma lipids, due to complete absence of mature lipoproteins within the ER, but paradoxically also activates lipid production. Mechanistically, scramblase deficiency triggers unique ER morphological changes and unsuppressed activation of SREBPs, which potently promotes lipid synthesis despite stalled secretion. Together, this response induces full-blown nonalcoholic hepatosteatosis in the TMEM41B-deficient mice within weeks. Collectively, our data uncovered a fundamental mechanism safe-guarding ER function and integrity, dysfunction of which disrupts lipid homeostasis.


Subject(s)
Endoplasmic Reticulum , Phospholipids , Animals , Endoplasmic Reticulum/metabolism , Homeostasis , Lipogenesis , Lipoproteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Phospholipids/metabolism
15.
Front Cell Infect Microbiol ; 11: 668034, 2021.
Article in English | MEDLINE | ID: covidwho-1231324

ABSTRACT

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.


Subject(s)
COVID-19 , Autophagy , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Humans , SARS-CoV-2 , Unfolded Protein Response
16.
Sci Adv ; 7(19)2021 05.
Article in English | MEDLINE | ID: covidwho-1226702

ABSTRACT

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of guanosine triphosphate nucleotides. Among posttranslational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER structure remains unclear. Here, we show that exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multiomics and superresolution imaging to characterize the broad effect of EXT1 inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Last, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome structure axis, with implications in biotechnology and medicine.


Subject(s)
Endoplasmic Reticulum Stress , Endoplasmic Reticulum , Animals , Endoplasmic Reticulum/metabolism , Glycosylation , Mammals , Mice , Protein Processing, Post-Translational , Protein Transport
17.
Uirusu ; 70(1): 29-36, 2020.
Article in Japanese | MEDLINE | ID: covidwho-1221885

ABSTRACT

Coronaviruses are pathogens that infect many of animals, resulting in respiratory or enteric diseases. Coronaviruses constitute Nidovirales together with Arteriviridae. Most of human coronaviruses are known to cause mild illness and common cold. However, an epidemic of severe acute respiratory syndrome (SARS) occurred in 2002, ten years after SARS epidemic Middle East respiratory syndrome (MERS) emerged in 2012. Now, we face on a novel coronavirus which emerges in end of 2019. This novel coronavirus is named as SARS-CoV-2. SARS-CoV-2 is spread to worldwide within one to two months and causes coronavirus disease 2019 (COVID-19), respiratory illness. Coronaviruses are enveloped viruses possessing a positive-sense and large single stranded RNA genomes. The 5' two-thirds of the CoV genome consists of two overlapping open reading frames (ORFs 1a and 1b) that encode non-structural proteins (nsps). The other one-third of the genome consists of ORFs encoding structural proteins, including spike (S), membrane (M), envelope (E) and nucleocapsid (N) proteins, and accessory proteins. Upon infection of CoV into host cells, the translation of two precursor polyproteins, pp1a and pp1ab, occurs and these polyproteins are cleaved into 16 nsps by viral proteases. Structural proteins assemble to the vesicles located from ER to Golgi (ER Golgiintermediate compartment) and virions bud into the vesicles. Virions are released from infectedcells via exocytosis.


Subject(s)
COVID-19/virology , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Animals , Endoplasmic Reticulum/metabolism , Genome, Viral/genetics , Golgi Apparatus/metabolism , Humans , Open Reading Frames , Polyproteins/metabolism , RNA, Viral/genetics , Viral Proteases , Viral Proteins/genetics , Viral Proteins/metabolism , Viral Structural Proteins/metabolism , Virion
18.
J Biol Chem ; 296: 100759, 2021.
Article in English | MEDLINE | ID: covidwho-1219049

ABSTRACT

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.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Heat-Shock Proteins/genetics , Host-Pathogen Interactions/genetics , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization/drug effects , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Binding Sites , Chlorocebus aethiops , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Gene Expression Regulation , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/metabolism , Humans , Mutation , Protein Binding , Protein Domains , Protein Multimerization , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Signal Transduction , Spike Glycoprotein, Coronavirus/metabolism , Unfolded Protein Response , Vero Cells
19.
Int Rev Cell Mol Biol ; 363: 203-269, 2021.
Article in English | MEDLINE | ID: covidwho-1212320

ABSTRACT

An increase in intracellular Ca2+ concentration ([Ca2+]i) regulates a plethora of functions in the cardiovascular (CV) system, including contraction in cardiomyocytes and vascular smooth muscle cells (VSMCs), and angiogenesis in vascular endothelial cells and endothelial colony forming cells. The sarco/endoplasmic reticulum (SR/ER) represents the largest endogenous Ca2+ store, which releases Ca2+ through ryanodine receptors (RyRs) and/or inositol-1,4,5-trisphosphate receptors (InsP3Rs) upon extracellular stimulation. The acidic vesicles of the endolysosomal (EL) compartment represent an additional endogenous Ca2+ store, which is targeted by several second messengers, including nicotinic acid adenine dinucleotide phosphate (NAADP) and phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], and may release intraluminal Ca2+ through multiple Ca2+ permeable channels, including two-pore channels 1 and 2 (TPC1-2) and Transient Receptor Potential Mucolipin 1 (TRPML1). Herein, we discuss the emerging, pathophysiological role of EL Ca2+ signaling in the CV system. We describe the role of cardiac TPCs in ß-adrenoceptor stimulation, arrhythmia, hypertrophy, and ischemia-reperfusion injury. We then illustrate the role of EL Ca2+ signaling in VSMCs, where TPCs promote vasoconstriction and contribute to pulmonary artery hypertension and atherosclerosis, whereas TRPML1 sustains vasodilation and is also involved in atherosclerosis. Subsequently, we describe the mechanisms whereby endothelial TPCs promote vasodilation, contribute to neurovascular coupling in the brain and stimulate angiogenesis and vasculogenesis. Finally, we discuss about the possibility to target TPCs, which are likely to mediate CV cell infection by the Severe Acute Respiratory Disease-Coronavirus-2, with Food and Drug Administration-approved drugs to alleviate the detrimental effects of Coronavirus Disease-19 on the CV system.


Subject(s)
COVID-19/complications , COVID-19/drug therapy , Calcium Signaling/physiology , Cardiovascular Diseases/etiology , Cardiovascular Diseases/metabolism , Cardiovascular System/metabolism , Lysosomes/metabolism , SARS-CoV-2 , ADP-ribosyl Cyclase 1/metabolism , Animals , Brain/blood supply , Brain/metabolism , COVID-19/metabolism , Calcium Channels/metabolism , Cardiovascular Diseases/drug therapy , Endoplasmic Reticulum/metabolism , Endothelial Cells/metabolism , Humans , Models, Cardiovascular , Myocytes, Cardiac/metabolism , NADP/analogs & derivatives , NADP/metabolism , Receptors, Adrenergic, beta/metabolism , Sarcoplasmic Reticulum/metabolism , Transient Receptor Potential Channels/metabolism
20.
Eur Rev Med Pharmacol Sci ; 25(7): 3136-3144, 2021 04.
Article in English | MEDLINE | ID: covidwho-1194855

ABSTRACT

OBJECTIVE: Disruption of intracellular Ca2+ homeostasis via excessive and pathological Ca2+ release from the endoplasmic reticulum (ER) and/or sarcoplasmic reticulum (SR) through ryanodine receptor (RyRs) Ca2+ channels play a critical role in the pathology of systemic inflammatory response syndrome (SIRS) and associated multiple organ dysfunction syndrome (MODS) in sepsis or septic shock. Dantrolene, a potent inhibitor of RyRs, is expected to ameliorate SIRS and MODS and decrease mortality in sepsis or septic shock patients. This review summarized the potential mechanisms of therapeutic effects of dantrolene in sepsis or septic shock at molecular, cell, and organ levels and provided suggestions and strategies for future clinical studies.


Subject(s)
COVID-19/drug therapy , Calcium Channel Blockers/therapeutic use , Dantrolene/therapeutic use , Sepsis/drug therapy , COVID-19/metabolism , Calcium/metabolism , Drug Repositioning , Endoplasmic Reticulum/metabolism , Humans , Mortality , Multiple Organ Failure , Ryanodine Receptor Calcium Release Channel/metabolism , SARS-CoV-2 , Sarcoplasmic Reticulum/metabolism , Sepsis/metabolism , Shock, Septic/drug therapy , Shock, Septic/metabolism
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