scholarly journals Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators

2020 ◽  
Vol 16 (12) ◽  
pp. e1009127
Author(s):  
Suelen Silva Gomes Dias ◽  
Vinicius Cardoso Soares ◽  
André C. Ferreira ◽  
Carolina Q. Sacramento ◽  
Natalia Fintelman-Rodrigues ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Intracellular Transport ◽  
Energy Homeostasis ◽  
Lipid Synthesis ◽  
Vero Cells ◽  
Multiple Roles ◽  
Host Cells ◽  
Intracellular Parasites ◽  
Viral Particles

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

scholarly journals Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators

2020 ◽  
Author(s):  
Suelen da Silva Gomes Dias ◽  
Vinicius Cardoso Soares ◽  
André C. Ferreira ◽  
Carolina Q. Sacramento ◽  
Natalia Fintelman-Rodrigues ◽  
...  
Keyword(s):  
Inflammatory Mediators ◽  
Lipid Droplets ◽  
Energy Homeostasis ◽  
Lipid Synthesis ◽  
Multiple Roles ◽  
Host Cells ◽  
Intracellular Parasites ◽  
Infected Cells

AbstractViruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism and energy homeostasis, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection modulates pathways of lipid synthesis and uptake, including CD36, SREBP-1, PPARγ and DGAT-1 in monocytes and triggered LD formation in different human cells. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected cells. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of pro-inflammatory mediators. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

scholarly journals LIPID SYNTHESIS, INTRACELLULAR TRANSPORT, AND STORAGE

1968 ◽  
Vol 36 (1) ◽  
pp. 63-77 ◽  
Author(s):  
Olga Stein ◽  
Yechezkiel Stein
Keyword(s):  
Fatty Acid ◽  
Free Fatty Acid ◽  
Sarcoplasmic Reticulum ◽  
Lipid Droplets ◽  
Intracellular Transport ◽  
Specific Activity ◽  
Lipid Synthesis ◽  
A Value ◽  
Rat Hearts

Rat hearts pulse-labeled by perfusion in vitro with 9,10-oleic acid-3H for 15 or 30 sec were shown to take up the fatty acid extensively. In hearts postperfused with unlabeled medium for 15 sec or more, 90% of the radioactivity was recovered in esterified lipids. The radioautographic reaction was localized initially over elements of the sarcoplasmic reticulum and mitochondria. After longer periods of postperfusion (2–20 min), there was concentration of silver grains over lipid droplets. In mitochondria and sarcoplasmic reticulum isolated from hearts postperfused for 1 min or more, most of the esterified lipid was in the form of triglyceride. The ratio of the specific activity of isolated sarcoplasmic reticulum triglyceride to mitochondrial triglyceride changed from a value of 3.2 to 1.3 during 5 min of postperfusion. Under conditions of hypothermia, considerable uptake of free fatty acid occurred. The radioactivity recovered in the heart was mostly in the form of free fatty acid, and the radioautographic reaction was seen over sarcoplasmic reticulum and mitochondria, but not over lipid droplets or myofibrils. The results are interpreted to show that intracellular transport of free fatty acid, which occurs also when esterification is repressed, proceeds through intracellular channels, i.e. the sarcoplasmic reticulum. Esterification of fatty acid into triglycerides occurs mostly in the sarcoplasmic reticulum, especially in the region of the dyad, in the vicinity of which lipid is stored in the form of droplets.

scholarly journals In Vitro Induction of Neospora caninum Bradyzoites in Vero Cells Reveals Differential Antigen Expression, Localization, and Host-Cell Recognition of Tachyzoites and Bradyzoites

2004 ◽  
Vol 72 (1) ◽  
pp. 576-583 ◽  
Author(s):  
Nathalie Vonlaufen ◽  
Nicole Guetg ◽  
Arunasalam Naguleswaran ◽  
Norbert Müller ◽  
Camilla Björkman ◽  
...  
Keyword(s):  
Host Cell ◽  
Cyst Wall ◽  
Neospora Caninum ◽  
Antigen Expression ◽  
Vero Cells ◽  
Dense Granule ◽  
Host Cells ◽  
Transmission Electron ◽  
Intracellular Parasites

ABSTRACT We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 μM sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residues from either the host cell or the parasite surface increased the invasion of Vero cells by bradyzoites, but not tachyzoites.

scholarly journals Fibroblasts as Host Cells in Latent Leishmaniosis

2000 ◽  
Vol 191 (12) ◽  
pp. 2121-2130 ◽  
Author(s):  
Christian Bogdan ◽  
Norbert Donhauser ◽  
Reinhard Döring ◽  
Martin Röllinghoff ◽  
Andreas Diefenbach ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Leishmania Major ◽  
Chronic Phase ◽  
Host Cells ◽  
Confocal Laser ◽  
Intracellular Parasites ◽  
Important Host ◽  
Mechanisms Of Persistence ◽  
Activated Fibroblasts

Intracellular parasites are known to persist lifelong in mammalian hosts after the clinical cure of the disease, but the mechanisms of persistence are poorly understood. Here, we show by confocal laser microscopy that in the draining lymph nodes of mice that had healed a cutaneous infection with Leishmania major, 40% of the persisting parasites were associated with fibroblasts forming the reticular meshwork of the lymph nodes. In vitro, both promastigotes and amastigotes of L. major infected primary skin or lymph node fibroblasts. Compared with macrophages, cytokine-activated fibroblasts had a reduced ability to express type 2 nitric oxide synthase and to kill intracellular L. major. These data identify fibroblasts as an important host cell for Leishmania during the chronic phase of infection and suggest that they might serve as safe targets for the parasites in clinically latent disease.

scholarly journals Attachment and invasion ofToxoplasma gondiiandNeospora caninumto epithelial and fibroblast cell linesin vitro

Parasitology ◽  
2005 ◽  
Vol 131 (5) ◽  
pp. 583-590 ◽  
Author(s):  
YING LEI ◽  
M. DAVEY ◽  
J. T. ELLIS
Keyword(s):  
Cell Lines ◽  
Neospora Caninum ◽  
Fibroblast Cell ◽  
Cell Types ◽  
Vero Cells ◽  
Host Cells ◽  
Trypsin Treatment ◽  
Epithelial Cell Lines ◽  
Pre Treatment

Attachment and invasion ofToxoplasma gondiiandNeospora caninumto a cat and a dog fibroblast cell line and 2 epithelial cell lines (a cat kidney and Vero) were comparedin vitrousing fluorescence antibody methodology. In addition, trypsin treatment of tachyzoites was used to determine whether protein molecules were essential to the process of invasion. The results show that bothT. gondiiandN. caninuminvaded all 4 cell lines, and that pre-treatment ofT. gondiitachyzoites with trypsin caused an increase in the ability of the parasite to invade these host cells. FurthermoreT. gondii, in comparison toN. caninum, invaded all 4 cell lines at greater levels. The results here support the conclusion that bothT. gondiiandN. caninumhave the ability to invade a variety of cell types including both dog and cat cells, and questions the utility of Vero cells as an appropriate host cell forin vitrostudies on the biology of these taxa.

scholarly journals A Polyphenol Rich Extract from Solanum melongena L. DR2 Peel Exhibits Antioxidant Properties and Anti-Herpes Simplex virus Type 1 Activity In Vitro

2018 ◽  
Author(s):  
Antonella Di Sotto ◽  
Silvia Di Giacomo ◽  
Donatella Amatore ◽  
Marcello Locatelli ◽  
Annabella Vitalone ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Antioxidant Properties ◽  
Solanum Melongena ◽  
Vero Cells ◽  
Host Cells ◽  
Reducing Conditions ◽  
Simplex Virus ◽  
Hsv 1

DR2B and DR2C extracts, from peel of commercially and physiologically ripe eggplants, were studied for the antioxidative cytoprotective properties and anti-HSV-1 activity, in line with the evidence that several antioxidants can impair viral replication by maintaining reducing conditions into the host cells. The antioxidative cytoprotective effects against tBOOH-induced damage was assessed in Caco2 cells, while the antiviral activity was studied in Vero cells; phenolic and anthocyanin fingerprint was characterized by integrated phytochemical methods. Results highlighted different compositions of the extracts, with chlorogenic acid and delphinidin-3-rutinoside as the major constituents; other peculiar phytochemicals were also identified. DR2C resulted able to partly counteract the tBOOH-induced cytotoxicity, with a remarkable lowering of lactate metabolism under both normoxia and hypoxia. DR2B and DR2C reduced ROS production, possessed scavenging and chelating properties. Interestingly, DR2C increased intracellular GSH levels. Furthermore, DR2C inhibited the HSV-1 replication when added for 24 h after viral adsorption, as also confirmed by the reduction of many viral proteins expression. Since DR2C was able to reduce NOX4 expression during HSV-1 infection, its antiviral activity may be correlated to its antioxidant properties. Although further studies are needed to better characterize DR2C activity, the results suggest this extract as a promising new anti-HSV-1 agent.

scholarly journals Antiviral Effectivity of Favipiravir Against Peste Des Petits Ruminants Virus Is Mediated by the JAK/STAT and PI3K/AKT Pathways

2021 ◽  
Vol 8 ◽  
Author(s):  
Weifeng Zhang ◽  
Hualong Deng ◽  
Yanfen Liu ◽  
Shaohong Chen ◽  
You Liu ◽  
...  
Keyword(s):  
Western Blotting ◽  
Effective Means ◽  
Small Ruminants ◽  
Antiviral Agent ◽  
Vero Cells ◽  
Host Cells ◽  
Peste Des Petits Ruminants ◽  
Expression Levels ◽  
Qrt Pcr

Peste des petits ruminants virus (PPRV), belonging to the genus Morbillivirus in the family Paramyxoviridae, causes severe infectious disease in small ruminants and has been rapidly spreading in many parts of Africa, the Middle East, and Asia. Although vaccination is considered to be an effective means of controlling PPR, the heat-sensitive nature of the vaccines against PPRV greatly limits their application in areas with a hot climate. In the present study, we investigated the anti-PPRV effects of favipiravir and sought to identify the underlying mechanisms in vitro using the Vero cell line. MTT assays, Western blotting, indirect immunofluorescence assays, virus plaque formation assays, and qRT-PCR were used to assess the effects of favipiravir on the life cycle of PPRV and the expression of RNA-dependent RNA polymerase (RdRp). Additionally, the expression levels of JAK1, STAT1, phosphorylated (p)-STAT1, PI3K, AKT, and p-AKT, as well as those of signaling molecules acting downstream of the JAK/STAT and PI3K/AKT signaling pathways, were determined by Western blotting and qRT-PCR. The results indicated that, in PPRV-infected, favipiravir-treated Vero cells, the attachment, invasion, replication, and release of PPRV were significantly inhibited, as was the expression of RdRp, when compared with that in untreated PPRV-infected cells. Furthermore, in favipiravir-treated cells, the expression of JAK1 and STAT1 was downregulated, whereas that of p-STAT1 was significantly upregulated. Similarly, the expression levels of PKR, IRF9, ISG54, and MxA proteins that are associated with innate antiviral activity in host cells were also markedly increased. Moreover, with favipiravir treatment, the expression of PI3K and p-AKT and the p-AKT/AKT ratio were significantly decreased, whereas the expression of AKT was noticeably upregulated. The expression of GSK3, NF-κB p65, p-NF-κB p65, and BAD was also increased with favipiravir treatment, while the expression of CREB, p-CREB, p-GSK3, and Bcl-2 was slightly decreased. In addition, all the p-GSK3/GSK3, p-CREB/CREB, p-NF-κB/NF-κB, and p-BAD/BAD ratios were significantly reduced in favipiravir-treated cells. These results implied that the antiviral effectivity of favipiravir against PPRV is mediated by the JAK/STAT and PI3K/AKT pathways and that favipiravir has potential for use as an effective antiviral agent against PPRV.

scholarly journals Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

Parasitology ◽  
2013 ◽  
Vol 140 (8) ◽  
pp. 1033-1050 ◽  
Author(s):  
FERIAL ALAEDDINE ◽  
ANDREW HEMPHILL ◽  
KARIM DEBACHE ◽  
CHRISTOPHE GUIONAUD
Keyword(s):  
Host Cell ◽  
Family Member ◽  
Vaccine Candidate ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
General Belief ◽  
Intracellular Parasites ◽  
Promising Vaccine Candidate

SUMMARYRecent publications demonstrated that a fragment of aNeospora caninumROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein,N. caninumROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of ‘cysts’ producedin vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasionin vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

scholarly journals Evidences for lipid involvement in SARS-CoV-2 cytopathogenesis

2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Roberta Nardacci ◽  
Francesca Colavita ◽  
Concetta Castilletti ◽  
Daniele Lapa ◽  
Giulia Matusali ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Vero Cells ◽  
Blood Cholesterol ◽  
Ultrastructural Changes ◽  
Type Ii ◽  
Cytopathic Effects ◽  
Type Ii Pneumocytes ◽  
Infected Cells ◽  
Accumulation Of Lipids

AbstractThe pathogenesis of SARS-CoV-2 remains to be completely understood, and detailed SARS-CoV-2 cellular cytopathic effects requires definition. We performed a comparative ultrastructural study of SARS-CoV-1 and SARS-CoV-2 infection in Vero E6 cells and in lungs from deceased COVID-19 patients. SARS-CoV-2 induces rapid death associated with profound ultrastructural changes in Vero cells. Type II pneumocytes in lung tissue showed prominent altered features with numerous vacuoles and swollen mitochondria with presence of abundant lipid droplets. The accumulation of lipids was the most striking finding we observed in SARS-CoV-2 infected cells, both in vitro and in the lungs of patients, suggesting that lipids can be involved in SARS-CoV-2 pathogenesis. Considering that in most cases, COVID-19 patients show alteration of blood cholesterol and lipoprotein homeostasis, our findings highlight a peculiar important topic that can suggest new approaches for pharmacological treatment to contrast the pathogenicity of SARS-CoV-2.

scholarly journals Identification of Potential Dual-Targets Anti-Toxoplasma gondii Compounds Through Structure-Based Virtual Screening and In-Vitro Studies

2019 ◽  
Author(s):  
Nurul Hanim Salin ◽  
Rahmah Noordin ◽  
Belal Omar Al Najjar ◽  
Ezatul Ezleen Kamarulzaman ◽  
Muhammad Hafiznur Yunus ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Targets ◽  
Active Sites ◽  
Vero Cells ◽  
Etiologic Agent ◽  
Adenosine Kinase ◽  
Host Cells ◽  
Cell Viability Assay ◽  
Limited Effectiveness

AbstractToxoplasma gondii is the etiologic agent of toxoplasmosis, a disease which can lead to morbidity and mortality of the fetus and immunocompromised individuals. Due to the limited effectiveness or side effects of existing drugs, the search for better drug can didates is still ongoing. In this study, we performed structure-based screening of potential dual-targets inhibitors of active sites of T. gondii drug targets such as uracil phosphoribosyltransferase (UPRTase) and adenosine kinase (AK). First screening of virtual compounds from the National Cancer Institute (NCI) was performed via molecular docking. Subsequently, the hit compounds were tested in-vitro for anti-T. gondii effect using cell viability assay with Vero cells as host to determine cytotoxicity effects and drug selectivities. Clindamycin, as positive control, showed a selectivity index (SI) of 10.9, thus compounds with SI > 10.9 specifically target T. gondii proliferation with no significant effect on the host cells. Good anti-T. gondii effects were observed with NSC77468 (7-ethoxy-4-methyl-6,7-dihydro-5H-thiopyrano[2,3-d] pyrimidin-2-amine) which showed SI values of 25. This study showed that in-silico selection can serve as an effective way to discover potentially potent and selective compounds against T. gondii.

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