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1.
Viruses ; 14(5)2022 05 15.
Article in English | MEDLINE | ID: covidwho-1884377

ABSTRACT

In this study, we investigated the correlation between the mechanism involved in porcine epidemic diarrhea virus (PEDV) replication and autophagic flux. In this study, we found that as PEDV replicated, production of LC3-II was significantly induced up to 24 h post-infection (hpi). Interestingly, although there was significant production of LC3-II, greater p62 accumulation was simultaneously found. Pretreatment with rapamycin significantly induced PEDV replication, but autolysosome formation was reduced. These results were confirmed by the evaluation of ATG5/ATG12 and LAMP1/LAMP2. Taken together, we conclude that PEDV infection induces autophagosome formation but inhibits autolysosome formation during replication.


Subject(s)
Autophagosomes/metabolism , Porcine epidemic diarrhea virus , Animals , Autophagosomes/genetics , Chlorocebus aethiops , Lysosomes/genetics , Lysosomes/metabolism , Macroautophagy , Porcine epidemic diarrhea virus/immunology , Swine , Vero Cells
2.
J Cell Biol ; 221(6)2022 06 06.
Article in English | MEDLINE | ID: covidwho-1806200

ABSTRACT

The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.


Subject(s)
Autophagy , Extracellular Vesicles , Lysosomes , Proteostasis , Autophagosomes/metabolism , Extracellular Vesicles/metabolism , Humans , Lysosomes/metabolism , SARS-CoV-2 , Sequestosome-1 Protein , Viroporin Proteins , rab27 GTP-Binding Proteins
3.
Nat Commun ; 12(1): 7276, 2021 12 14.
Article in English | MEDLINE | ID: covidwho-1575708

ABSTRACT

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV and SARS-CoV-2 replication and proper DMV formation. An intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.


Subject(s)
Hepacivirus/genetics , Phosphatidic Acids/metabolism , SARS-CoV-2/genetics , Virus Replication/physiology , 1-Acylglycerol-3-Phosphate O-Acyltransferase , Acyltransferases , Autophagosomes/metabolism , Autophagy , COVID-19/virology , Cell Line , Cell Survival , Dengue Virus , HEK293 Cells , Humans , Membrane Proteins , Spike Glycoprotein, Coronavirus , Viral Nonstructural Proteins , Viral Proteins , Zika Virus
4.
Cell Rep ; 37(8): 110049, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1509642

ABSTRACT

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles.


Subject(s)
Autophagy/physiology , Hepacivirus/physiology , SARS-CoV-2/physiology , Viral Replication Compartments/metabolism , Autophagosomes/metabolism , Carrier Proteins/metabolism , Class III Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Class III Phosphatidylinositol 3-Kinases/metabolism , Humans , Phosphatidylinositol Phosphates/metabolism , RNA, Viral/biosynthesis , Viral Nonstructural Proteins/metabolism , Virus Replication
5.
J Virol ; 95(19): e0085121, 2021 09 09.
Article in English | MEDLINE | ID: covidwho-1403028

ABSTRACT

Uncoordinated 51-like kinase 1 (ULK1) is a well-characterized initiator of canonical autophagy under basal or pathological conditions. Porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus (ß-CoV), impairs ULK1 kinase but hijacks autophagy to facilitate viral proliferation. However, the machinery of PHEV-induced autophagy initiation upon ULK1 kinase deficiency remains unclear. Here, the time course of PHEV infection showed a significant accumulation of autophagosomes (APs) in nerve cells in vivo and in vitro. Utilizing ULK1-knockout neuroblastoma cells, we have identified that ULK1 is not essential for productive AP formation induced by PHEV. In vitro phosphorylation studies discovered that mTORC1-regulated ULK1 activation stalls during PHEV infection, whereas AP biogenesis was controlled by AMPK-driven BECN1 phosphorylation. A lack of BECN1 is sufficient to block LC3 lipidation and disrupt recruitment of the LC3-ATG14 complex. Moreover, BECN1 acts as a bona fide substrate for ULK1-independent neural autophagy, and ectopic expression of BECN1 somewhat enhances PHEV replication. These findings highlight a novel machinery of noncanonical autophagy independent of ULK1 that bypasses the conserved initiation circuit of AMPK-mTORC1-ULK1, providing new insights into the interplay between neurotropic ß-CoV and the host. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic alongside the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) pose Betacoronavirus (ß-CoV) as a global public health challenge. Coronaviruses subvert, hijack, or utilize autophagy to promote proliferation, and thus, exploring the cross talk between ß-CoV and autophagy is of great significance in confronting future ß-CoV outbreaks. Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic ß-CoV that invades the central nervous system (CNS) in pigs, but understanding of the pathogenesis for PHEV-induced neurological dysfunction is yet limited. Here, we discovered a novel regulatory principle of neural autophagy initiation during PHEV infection, where productive autophagosome (AP) biogenesis bypasses the multifaceted regulation of ULK1 kinase. The PHEV-triggered noncanonical autophagy underscores the complex interactions of virus and host and will help in the development of therapeutic strategies targeting noncanonical autophagy to treat ß-CoV disease.


Subject(s)
Autophagy-Related Protein-1 Homolog/genetics , Autophagy-Related Protein-1 Homolog/metabolism , Autophagy/physiology , Betacoronavirus 1/metabolism , Animals , Autophagosomes/metabolism , Beclin-1/metabolism , COVID-19 , Cell Line , Gene Knockout Techniques , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Inbred BALB C , Neurons/metabolism , Phosphorylation , SARS-CoV-2
6.
Autophagy ; 17(8): 2048-2050, 2021 08.
Article in English | MEDLINE | ID: covidwho-1393103

ABSTRACT

TMEM41B and VMP1, two endoplasmic reticulum (ER)-resident transmembrane proteins, play important roles in regulating the formation of lipid droplets (LDs), autophagy initiation, and viral infection. However, the biochemical functions of TMEM41B and VMP1 are unclear. A lipids distribution screen suggested TMEM41B and VMP1 are critical to the normal distribution of cholesterol and phosphatidylserine. Biochemical analyses unveiled that TMEM41B and VMP1 have scramblase activity. These findings shed light on the mechanism by which TMEM41B and VMP1 regulate LD formation, lipids distribution, macroautophagy, and viral infection.


Subject(s)
Autophagy/physiology , Membrane Proteins/metabolism , Phospholipid Transfer Proteins/metabolism , Animals , Autophagosomes/metabolism , Humans , Macroautophagy/physiology
7.
Nat Cell Biol ; 23(8): 846-858, 2021 08.
Article in English | MEDLINE | ID: covidwho-1309445

ABSTRACT

The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity.


Subject(s)
Autophagy-Related Proteins/metabolism , Cell Membrane/metabolism , Membrane Proteins/metabolism , Vesicular Transport Proteins/metabolism , Autophagosomes/metabolism , Autophagy-Related Proteins/genetics , HEK293 Cells , HeLa Cells , Humans , Immunoblotting , Immunoprecipitation , Membrane Proteins/genetics , Microscopy, Confocal , Protein Transport/physiology , Vesicular Transport Proteins/genetics
8.
Nat Commun ; 12(1): 3818, 2021 06 21.
Article in English | MEDLINE | ID: covidwho-1279876

ABSTRACT

Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC50 values of 136.7, 7.67, 0.11, and 0.13 µM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19.


Subject(s)
COVID-19/metabolism , COVID-19/virology , SARS-CoV-2/metabolism , Animals , Antinematodal Agents/pharmacology , Autophagosomes/metabolism , Autophagy , Autophagy-Related Proteins/metabolism , COVID-19/drug therapy , COVID-19/pathology , Cells, Cultured , Chlorocebus aethiops , Cricetinae , Disease Models, Animal , Humans , Lung/metabolism , Lung/pathology , Lung/virology , Metabolome , Niclosamide/pharmacology , Organoids , SARS-CoV-2/isolation & purification , Spermidine/pharmacology , Spermine/pharmacology
10.
Dev Cell ; 56(4): 427-442.e5, 2021 02 22.
Article in English | MEDLINE | ID: covidwho-978254

ABSTRACT

Autophagy acts as a cellular surveillance mechanism to combat invading pathogens. Viruses have evolved various strategies to block autophagy and even subvert it for their replication and release. Here, we demonstrated that ORF3a of the COVID-19 virus SARS-CoV-2 inhibits autophagy activity by blocking fusion of autophagosomes/amphisomes with lysosomes. The late endosome-localized ORF3a directly interacts with and sequestrates the homotypic fusion and protein sorting (HOPS) component VPS39, thereby preventing HOPS complex from interacting with the autophagosomal SNARE protein STX17. This blocks assembly of the STX17-SNAP29-VAMP8 SNARE complex, which mediates autophagosome/amphisome fusion with lysosomes. Expression of ORF3a also damages lysosomes and impairs their function. SARS-CoV-2 virus infection blocks autophagy, resulting in accumulation of autophagosomes/amphisomes, and causes late endosomal sequestration of VPS39. Surprisingly, ORF3a from the SARS virus SARS-CoV fails to interact with HOPS or block autophagy. Our study reveals a mechanism by which SARS-CoV-2 evades lysosomal destruction and provides insights for developing new strategies to treat COVID-19.


Subject(s)
Autophagosomes/metabolism , COVID-19/metabolism , Lysosomes/metabolism , SNARE Proteins/metabolism , Viroporin Proteins/metabolism , Autophagy , Autophagy-Related Proteins/metabolism , COVID-19/virology , HEK293 Cells , HeLa Cells , Humans , Protein Binding , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Vesicular Transport Proteins/metabolism , Viroporin Proteins/genetics
11.
Exp Cell Res ; 396(1): 112276, 2020 11 01.
Article in English | MEDLINE | ID: covidwho-752714

ABSTRACT

Autophagy is an evolutionary conserved catabolic process devoted to the removal of unnecessary and harmful cellular components. In its general form, autophagy governs cellular lifecycle through the formation of double membrane vesicles, termed autophagosomes, that enwrap and deliver unwanted intracellular components to lysosomes. In addition to this omniscient role, forms of selective autophagy, relying on specialized receptors for cargo recognition, exert fine-tuned control over cellular homeostasis. In this regard, xenophagy plays a pivotal role in restricting the replication of intracellular pathogens, thus acting as an ancient innate defense system against infections. Recently, selective autophagy of the endoplasmic reticulum (ER), more simply ER-phagy, has been uncovered as a critical mechanism governing ER network shape and function. Six ER-resident proteins have been characterized as ER-phagy receptors and their orchestrated function enables ER homeostasis and turnover overtime. Unfortunately, ER is also the preferred site for viral replication and several viruses hijack ER machinery for their needs. Thus, it is not surprising that some ER-phagy receptors can act to counteract viral replication and minimize the spread of infection throughout the organism. On the other hand, evolutionary pressure has armed pathogens with strategies to evade and subvert xenophagy and ER-phagy. Although ER-phagy biology is still in its infancy, the present review aims to summarize recent ER-phagy literature, with a special focus on its role in counteracting viral infections. Moreover, we aim to offer some hints for future targeted approaches to counteract host-pathogen interactions by modulating xenophagy and ER-phagy pathways.


Subject(s)
Autophagosomes/immunology , Bacterial Infections/immunology , Endoplasmic Reticulum/immunology , Host-Pathogen Interactions/immunology , Macroautophagy/immunology , Virus Diseases/immunology , Autophagosomes/metabolism , Bacteria/immunology , Bacterial Infections/genetics , Bacterial Infections/microbiology , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/microbiology , Endoplasmic Reticulum/virology , Endoplasmic Reticulum Stress/genetics , Endoplasmic Reticulum Stress/immunology , Homeostasis/genetics , Homeostasis/immunology , Host-Pathogen Interactions/genetics , Humans , Immunity, Innate , Lysosomes/immunology , Lysosomes/metabolism , Macroautophagy/genetics , Virus Diseases/genetics , Virus Diseases/virology , Viruses/immunology
12.
Front Immunol ; 11: 1337, 2020.
Article in English | MEDLINE | ID: covidwho-687353

ABSTRACT

Autophagy is a cellular recycling system found in almost all types of eukaryotic organisms. The system is made up of a variety of proteins which function to deliver intracellular cargo to lysosomes for formation of autophagosomes in which the contents are degraded. The maintenance of cellular homeostasis is key in the survival and function of a variety of human cell populations. The interconnection between metabolism and autophagy is extensive, therefore it has a role in a variety of different cell functions. The disruption or dysfunction of autophagy in these cell types have been implicated in the development of a variety of inflammatory diseases including asthma. The role of autophagy in non-immune and immune cells both lead to the pathogenesis of lung inflammation. Autophagy in pulmonary non-immune cells leads to tissue remodeling which can develop into chronic asthma cases with long term effects. The role autophagy in the lymphoid and myeloid lineages in the pathology of asthma differ in their functions. Impaired autophagy in lymphoid populations have been shown, in general, to decrease inflammation in both asthma and inflammatory disease models. Many lymphoid cells rely on autophagy for effector function and maintained inflammation. In stark contrast, autophagy deficient antigen presenting cells have been shown to have an activated inflammasome. This is largely characterized by a TH17 response that is accompanied with a much worse prognosis including granulocyte mediated inflammation and steroid resistance. The cell specificity associated with changes in autophagic flux complicates its targeting for amelioration of asthmatic symptoms. Differing asthmatic phenotypes between TH2 and TH17 mediated disease may require different autophagic modulations. Therefore, treatments call for a more cell specific and personalized approach when looking at chronic asthma cases. Viral-induced lung inflammation, such as that caused by SARS-CoV-2, also may involve autophagic modulation leading to inflammation mediated by lung resident cells. In this review, we will be discussing the role of autophagy in non-immune cells, myeloid cells, and lymphoid cells for their implications into lung inflammation and asthma. Finally, we will discuss autophagy's role viral pathogenesis, immunometabolism, and asthma with insights into autophagic modulators for amelioration of lung inflammation.


Subject(s)
Asthma/complications , Asthma/pathology , Autophagy/immunology , Betacoronavirus , Coronavirus Infections/complications , Coronavirus Infections/pathology , Pneumonia, Viral/complications , Pneumonia, Viral/pathology , Animals , Asthma/immunology , Autophagosomes/metabolism , Autophagy/drug effects , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/immunology , Dendritic Cells/metabolism , Humans , Lymphocytes/metabolism , Lysosomes/metabolism , Myeloid Cells/metabolism , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/immunology , Respiratory Mucosa/metabolism , SARS-CoV-2 , Signal Transduction/immunology
13.
Mol Med ; 26(1): 69, 2020 07 08.
Article in English | MEDLINE | ID: covidwho-635101

ABSTRACT

BACKGROUND: We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. METHODS: C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. RESULTS: CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. CONCLUSION: Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.


Subject(s)
Caspase 1/metabolism , Caspases, Initiator/metabolism , Chloroquine/pharmacology , Coronavirus Infections/pathology , Ischemia/pathology , Muscle, Skeletal/pathology , Pneumonia, Viral/pathology , Animals , Autophagosomes/metabolism , Autophagy/drug effects , Betacoronavirus , COVID-19 , Coronavirus Infections/drug therapy , Glycolysis/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microtubule-Associated Proteins/metabolism , Muscle Cells/metabolism , Muscle Development , Muscle, Skeletal/metabolism , Neovascularization, Physiologic , Oxidative Phosphorylation , Pandemics , Peripheral Arterial Disease/pathology , Pneumonia, Viral/drug therapy , Regeneration , SARS-CoV-2 , Signal Transduction
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