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1.
Dokl Biochem Biophys ; 506(1): 206-209, 2022 Oct.
Article in English | MEDLINE | ID: covidwho-2088454

ABSTRACT

In the present manuscript we analyzed the influence of hypoxic response in Caco-2 cells on the expression of genes and miRNAs involved in the mechanisms of intracellular transport of SARS-CoV-2 viral particles, especially endocytosis and transcytosis. With the use of RNA sequencing of Caco-2 cells treated with hypoxia-inducing oxyquinoline derivative, we showed two-fold increase in the expression of the main SARS-CoV-2 receptor ACE2. Expression of the non-canonical receptor TFRC was also elevated. We also observed a significant increase in the expression levels of genes from the low-density lipoprotein (LDL) receptor family, which play a crucial role in the transcytosis: LDLR, LRP1, LRP4, and LRP5. Upregulation of LDLR was coupled with the downregulation of hsa-miR-148a-3p, which can directly bind to LDLR mRNA. Thus, the hypoxic response in Caco-2 cells includes upregulation of genes involved in the mechanisms of endocytosis and transcytosis of SARS-CoV-2 viral particles.


Subject(s)
COVID-19 , Cell Hypoxia , Endocytosis , Transcytosis , Humans , Caco-2 Cells , MicroRNAs/genetics , SARS-CoV-2
2.
J Biol Chem ; 298(11): 102500, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2041895

ABSTRACT

Coronavirus disease represents a real threat to the global population, and understanding the biological features of the causative virus, that is, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is imperative for mitigating this threat. Analyses of proteins such as primary receptors and coreceptors (cofactors), which are involved in the entry of SARS-CoV-2 into host cells, will provide important clues to help control the virus. Here, we identified host cell membrane protein candidates present in proximity to the attachment sites of SARS-CoV-2 spike proteins, using proximity labeling and proteomic analysis. The identified proteins represent key candidate factors that may be required for viral entry. We found SARS-CoV-2 host protein DPP4, cell adhesion protein Cadherin 17, and glycoprotein CD133 colocalized with cell membrane-bound SARS-CoV-2 spike proteins in Caco-2 cells and thus showed potential as candidate factors. Additionally, our analysis of the experimental infection of HEK293T cells with a SARS-CoV-2 pseudovirus indicated a 2-fold enhanced infectivity in the CD133-ACE2-coexpressing HEK293T cells compared to that in HEK293T cells expressing ACE-2 alone. The information and resources regarding these coreceptor labeling and analysis techniques could be utilized for the development of antiviral agents against SARS-CoV-2 and other emerging viruses.


Subject(s)
COVID-19 , Membrane Proteins , Spike Glycoprotein, Coronavirus , Virus Attachment , Humans , Angiotensin-Converting Enzyme 2 , Caco-2 Cells , HEK293 Cells , Membrane Proteins/metabolism , Protein Binding , Proteomics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Receptors, Virus/metabolism
3.
Int J Mol Sci ; 23(18)2022 Sep 09.
Article in English | MEDLINE | ID: covidwho-2032982

ABSTRACT

The systemic nature of COVID-19 with multiple extrapulmonary manifestations of disease, largely due to the wide tissue expression of SARS-CoV-2 major entry factors, as well as the patient-specific features of COVID-19 pathobiology, determine important directions for basic and translational research. In the current study, we addressed the questions of singularities and commonalities in cellular responses to SARS-CoV-2 and related SARS-CoV on the basis of compendium-wide analysis of publicly available transcriptomic datasets as part of the herein implemented multi-modular UNCOVIDING approach. We focused on cellular models attributed to the epithelial cells of the respiratory system, the Calu-3 cell line, and epithelial cells of the gastrointestinal tract, the Caco-2 cell line, infected with either SARS-CoV-2 or SARS-CoV. Here, we report the outcome of a comparative analysis based on differentially expressed genes in terms of perturbations and diseases, Canonical pathways, and Upstream Regulators. We furthermore performed compendium-wide analysis across more than 19,000 mRNASeq datasets and dissected the condition-specific gene signatures. Information was gained with respect to common and unique cellular responses and molecular events. We identified that in cell lines of colon or lung origin, both viruses show similarities in cellular responses; by contrast, there are cell type-specific regulators that differed for Calu-3 and Caco-2 cells. Among the major findings is the impact of the interferon system for lung Calu-3 cells and novel links to the liver- and lipid-metabolism-associated responses for colon Caco-2 cells as part of the extrapulmonary pathomechanisms in the course of COVID-19. Among differently expressed genes, we specifically dissected the expression pattern of the APOBEC family members and propose APOBEC3G as a promising intrinsic antiviral factor of the host response to SARS-CoV-2. Overall, our study provides gene expression level evidence for the cellular responses attributed to pulmonary and gastrointestinal manifestations of COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents , COVID-19/genetics , Caco-2 Cells , Colon , Humans , Interferons , Lipids , Lung
4.
Sci Rep ; 12(1): 14972, 2022 Sep 13.
Article in English | MEDLINE | ID: covidwho-2028722

ABSTRACT

During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure.


Subject(s)
COVID-19 , RNA Editing , APOBEC Deaminases/genetics , APOBEC Deaminases/metabolism , COVID-19/genetics , Caco-2 Cells , Cytidine Deaminase , Humans , Mutation , Pandemics , Proteins , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics
5.
Front Immunol ; 13: 966236, 2022.
Article in English | MEDLINE | ID: covidwho-2022749

ABSTRACT

Class 1 and 2 monoclonal antibodies inhibit SARS-CoV-2 entry by blocking the interaction of the viral receptor-binding domain with angiotensin-converting enzyme 2 (ACE2), while class 3 antibodies target a highly conserved epitope outside the ACE2 binding site. We aimed to investigate the plasticity of the spike protein by propagating wild-type SARS-CoV-2 in the presence of class 3 antibody S309. After 12 weeks, we obtained a viral strain that was completely resistant to inhibition by S309, due to successively evolving amino acid exchanges R346S and P337L located in the paratope of S309. The antibody lost affinity to receptor-binding domains carrying P337L or both amino acid exchanges, while ACE2 binding was not affected. The resistant strain replicated efficiently in human CaCo-2 cells and was more susceptible to inhibition of fusion than the original strain. Overall, SARS-CoV-2 escaped inhibition by class 3 antibody S309 through a slow, but targeted evolution enabling immune escape and altering cell entry. This immune-driven enhancement of infectivity and pathogenicity could play an important role in the future evolution of SARS-CoV-2, which is under increasing immunological pressure from vaccination and previous infections.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Amino Acids , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Antibodies, Neutralizing , Caco-2 Cells , Humans , Peptidyl-Dipeptidase A/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism
6.
Microbiol Spectr ; 10(5): e0160422, 2022 Oct 26.
Article in English | MEDLINE | ID: covidwho-2019791

ABSTRACT

The Delta variant of SARS-CoV-2 has caused more severe infections than its previous variants. We studied the host innate immune response to Delta, Alpha, and two earlier variants to map the evolution of the recent ones. Our biochemical and transcriptomic studies in human colon epithelial cell line Caco2 reveal that Alpha and Delta have progressively evolved over the ancestral variants by silencing the innate immune response, thereby limiting cytokine and chemokine production. Though Alpha silenced the retinoic acid-inducible gene (RIG)-I-like receptor (RLR) pathway just like Delta did, it failed to persistently silence the innate immune response, unlike Delta. Both Alpha and Delta have evolved to resist interferon (IFN) treatment, while they are still susceptible to RLR activation, further highlighting the importance of RLR-mediated, IFN-independent mechanisms in restricting SARS-CoV-2. Our studies reveal that SARS-CoV-2 Delta has integrated multiple mechanisms to silence the host innate immune response and evade the IFN response. We speculate that Delta's silent replication and sustained suppression of the host innate immune response, thereby resulting in delayed or reduced intervention by the adaptive immune response, could have potentially contributed to the severe symptoms and poor recovery index associated with it. It is likely that this altered association with the host would play an important role in the coevolution of SARS-CoV-2 with humans. IMPORTANCE Viruses generally learn to coexist with the host during the process of evolution. It is expected that SARS-CoV-2 would also evolve to coexist in humans by trading off its virulence for longer persistence, causing milder disease. Clinically, the fatality associated with COVID-19 has been declining due to vaccination and preinfections, but the Delta variant caused the most severe disease and fatality across several parts of the world. Our study identified an evolving trend of SARS-CoV-2 variants where the variants that emerged during early parts of the pandemic caused a more robust innate immune response, while the later emerging variant Delta showed features of suppression of the response. The features that Delta has acquired could have strongly influenced the distinct pathophysiology associated with its infection. How these changed associations with the host influence the long-term evolution of the virus and the disease outcome should be closely studied to understand the process of viral evolution.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Interferons/genetics , Caco-2 Cells , Immunity, Innate , Antiviral Agents , Epithelial Cells , Cytokines , Chemokines , Colon , Tretinoin
7.
Life Sci ; 308: 120930, 2022 Nov 01.
Article in English | MEDLINE | ID: covidwho-2007929

ABSTRACT

AIMS: This study evaluated SARS-CoV-2 replication in human cell lines derived from various tissues and investigated molecular mechanisms related to viral infection susceptibility and replication. MAIN METHODS: SARS-CoV-2 replication in BEAS-2B and A549 (respiratory tract), HEK-293 T (kidney), HuH7 (liver), SH-SY5Y (brain), MCF7 (breast), Huvec (endothelial) and Caco-2 (intestine) was evaluated by RT-qPCR. Concomitantly, expression levels of ACE2 (Angiotensin Converting Enzyme) and TMPRSS2 were assessed through RT-qPCR and western blot. Proteins related to autophagy and mitochondrial metabolism were monitored in uninfected cells to characterize the cellular metabolism of each cell line. The effect of ACE2 overexpression on viral replication in pulmonary cells was also investigated. KEY FINDINGS: Our data show that HuH7, Caco-2 and MCF7 presented a higher viral load compared to the other cell lines. The increased susceptibility to SARS-CoV-2 infection seems to be associated not only with the differential levels of proteins intrinsically related to energetic metabolism, such as ATP synthase, citrate synthase, COX and NDUFS2 but also with the considerably higher TMPRSS2 mRNA expression. The two least susceptible cell types, BEAS-2B and A549, showed drastically increased SARS-CoV-2 replication capacity when ACE2 was overexpressed. These modified cell lines are relevant for studying SARS-CoV-2 replication in vitro. SIGNIFICANCE: Our data not only reinforce that TMPRSS2 expression and cellular energy metabolism are important molecular mechanisms for SARS-CoV-2 infection and replication, but also indicate that HuH7, MCF7 and Caco-2 are suitable models for mechanistic studies of COVID-19. Moreover, pulmonary cells overexpressing ACE2 can be used to understand mechanisms associated with SARS-CoV-2 replication.


Subject(s)
COVID-19 , Neuroblastoma , Adenosine Triphosphate , Angiotensin-Converting Enzyme 2/genetics , Autophagy , Caco-2 Cells , Citrate (si)-Synthase , HEK293 Cells , Humans , Peptidyl-Dipeptidase A/metabolism , RNA, Messenger/genetics , SARS-CoV-2
8.
Nat Genet ; 54(8): 1090-1102, 2022 08.
Article in English | MEDLINE | ID: covidwho-1960393

ABSTRACT

CRISPR knockout (KO) screens have identified host factors regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Here, we conducted a meta-analysis of these screens, which showed a high level of cell-type specificity of the identified hits, highlighting the necessity of additional models to uncover the full landscape of host factors. Thus, we performed genome-wide KO and activation screens in Calu-3 lung cells and KO screens in Caco-2 colorectal cells, followed by secondary screens in four human cell lines. This revealed host-dependency factors, including AP1G1 adaptin and ATP8B1 flippase, as well as inhibitors, including mucins. Interestingly, some of the identified genes also modulate Middle East respiratory syndrome coronavirus (MERS-CoV) and seasonal human coronavirus (HCoV) (HCoV-NL63 and HCoV-229E) replication. Moreover, most genes had an impact on viral entry, with AP1G1 likely regulating TMPRSS2 activity at the plasma membrane. These results demonstrate the value of multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential targets for therapeutic interventions.


Subject(s)
COVID-19 , Middle East Respiratory Syndrome Coronavirus , COVID-19/genetics , Caco-2 Cells , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , SARS-CoV-2/genetics , Seasons
9.
Int J Biol Sci ; 18(12): 4744-4755, 2022.
Article in English | MEDLINE | ID: covidwho-1954694

ABSTRACT

Viruses exploit the host lipid metabolism machinery to achieve efficient replication. We herein characterize the lipids profile reprogramming in vitro and in vivo using liquid chromatography-mass spectrometry-based untargeted lipidomics. The lipidome of SARS-CoV-2-infected Caco-2 cells was markedly different from that of mock-infected samples, with most of the changes involving downregulation of ceramides. In COVID-19 patients' plasma samples, a total of 54 lipids belonging to 12 lipid classes that were significantly perturbed compared to non-infected control subjects' plasma samples were identified. Among these 12 lipid classes, ether-linked phosphatidylcholines, ether-linked phosphatidylethanolamines, phosphatidylcholines, and ceramides were the four most perturbed. Pathway analysis revealed that the glycerophospholipid, sphingolipid, and ether lipid metabolisms pathway were the most significantly perturbed host pathways. Phosphatidic acid phosphatases (PAP) were involved in all three pathways and PAP-1 deficiency significantly suppressed SARS-CoV-2 replication. siRNA knockdown of LPIN2 and LPIN3 resulted in significant reduction of SARS-CoV-2 load. In summary, these findings characterized the host lipidomic changes upon SARS-CoV-2 infection and identified PAP-1 as a potential target for intervention for COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , Caco-2 Cells , Ceramides , Ethers , Glycerophospholipids , Humans , Lipid Metabolism , Phosphatidate Phosphatase/genetics , Phosphatidate Phosphatase/metabolism , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism
10.
Proc Natl Acad Sci U S A ; 119(30): e2123065119, 2022 07 26.
Article in English | MEDLINE | ID: covidwho-1947760

ABSTRACT

SARS-CoV-2, the causative agent of the COVID-19 pandemic, undergoes continuous evolution, highlighting an urgent need for development of novel antiviral therapies. Here we show a quantitative mass spectrometry-based succinylproteomics analysis of SARS-CoV-2 infection in Caco-2 cells, revealing dramatic reshape of succinylation on host and viral proteins. SARS-CoV-2 infection promotes succinylation of several key enzymes in the TCA, leading to inhibition of cellular metabolic pathways. We demonstrated that host protein succinylation is regulated by viral nonstructural protein (NSP14) through interaction with sirtuin 5 (SIRT5); overexpressed SIRT5 can effectively inhibit virus replication. We found succinylation inhibitors possess significant antiviral effects. We also found that SARS-CoV-2 nucleocapsid and membrane proteins underwent succinylation modification, which was conserved in SARS-CoV-2 and its variants. Collectively, our results uncover a regulatory mechanism of host protein posttranslational modification and cellular pathways mediated by SARS-CoV-2, which may become antiviral drug targets against COVID-19.


Subject(s)
Antiviral Agents , COVID-19 , Host-Pathogen Interactions , Molecular Targeted Therapy , Protein Processing, Post-Translational , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/metabolism , COVID-19/virology , Caco-2 Cells , Exoribonucleases/metabolism , Host-Pathogen Interactions/drug effects , Humans , Protein Processing, Post-Translational/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Sirtuins/metabolism , Succinates/metabolism , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
11.
PLoS One ; 17(7): e0271112, 2022.
Article in English | MEDLINE | ID: covidwho-1933379

ABSTRACT

The outbreak of the coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 triggered a global pandemic where control is needed through therapeutic and preventive interventions. This study aims to identify natural compounds that could affect the fusion between the viral membrane (receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 spike protein) and the human cell receptor angiotensin-converting enzyme 2. Accordingly, we performed the enzyme-linked immunosorbent assay-based screening of 10 phytochemicals that already showed numerous positive effects on human health in several epidemiological studies and clinical trials. Among these phytochemicals, epigallocatechin gallate, a polyphenol and a major component of green tea, could effectively inhibit the interaction between the receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 spike protein and the human cell receptor angiotensin-converting enzyme 2. Alternately, in silico molecular docking studies of epigallocatechin gallate and angiotensin-converting enzyme 2 indicated a binding score of -7.8 kcal/mol and identified a hydrogen bond between R393 and angiotensin-converting enzyme 2, which is considered as a key interacting residue involved in binding with the severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain, suggesting the possible blocking of interaction between receptor-binding domain and angiotensin-converting enzyme 2. Furthermore, epigallocatechin gallate could attenuate severe acute respiratory syndrome coronavirus 2 infection and replication in Caco-2 cells. These results shed insight into identification and validation of severe acute respiratory syndrome coronavirus 2 entry inhibitors.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Catechin , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/drug therapy , COVID-19/metabolism , COVID-19/virology , Caco-2 Cells , Catechin/analogs & derivatives , Catechin/pharmacology , Humans , Molecular Docking Simulation , Peptidyl-Dipeptidase A/metabolism , Protein Binding , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
12.
Biochem Cell Biol ; 100(4): 338-348, 2022 08 01.
Article in English | MEDLINE | ID: covidwho-1932794

ABSTRACT

Bovine lactoferrin (bLF) is a naturally occurring glycoprotein with antibacterial and antiviral activities. We evaluated whether bLF can prevent viral infections in the human intestinal epithelial cell line Caco-2. To assess antiviral responses, we measured the levels of interferon (IFN) expression, IFN-stimulated gene expression, and infection with a pseudotyped virus bearing either severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or vesicular stomatitis virus (VSV)-G protein after treatment of cells with both bLF and polyinosinic-polycytidylic acid, an analog of double-stranded RNA that mimics viral infection. Combination treatment of cells with both bLF and polyinosinic-polycytidylic acid increased mRNA and protein expression of several IFN genes (IFNB, IFNL1, and IFNL2) and IFN-stimulated genes (ISG15, MX1, IFITM1, and IFITM3) in Caco-2 cells. However, treatment with bLF alone did not induce an antiviral response. Furthermore, combination treatment suppressed infection of the SARS-CoV-2 pseudotyped virus more efficiently than did bLF treatment alone, even though combination treatment increased the expression of mRNA encoding ACE2. These results indicate that bLF increases the antiviral response associated with the double-stranded RNA-stimulated signaling pathway. Our results also suggest that bLF and double-stranded RNA analogs can be used to treat viral infections, including those caused by SARS-CoV-2.


Subject(s)
COVID-19 , Lactoferrin , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Caco-2 Cells , Humans , Lactoferrin/metabolism , Membrane Proteins/metabolism , Poly I-C , RNA, Double-Stranded , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , SARS-CoV-2
13.
Molecules ; 27(11)2022 May 26.
Article in English | MEDLINE | ID: covidwho-1869715

ABSTRACT

Impaired autophagy, responsible for increased inflammation, constitutes a risk factor for the more severe COVID-19 outcomes. Spermidine (SPD) is a known autophagy modulator and supplementation for COVID-19 risk groups (including the elderly) is recommended. However, information on the modulatory effects of eugenol (EUG) is scarce. Therefore, the effects of SPD and EUG, both singularly and in combination, on autophagy were investigated using different cell lines (HBEpiC, SHSY5Y, HUVEC, Caco-2, L929 and U937). SPD (0.3 mM), EUG (0.2 mM) and 0.3 mM SPD + 0.2 mM EUG, significantly increased autophagy using the hallmark measure of LC3-II protein accumulation in the cell lines without cytotoxic effects. Using Caco-2 cells as a model, several crucial autophagy proteins were upregulated at all stages of autophagic flux in response to the treatments. This effect was verified by the activation/differentiation and migration of U937 monocytes in a three-dimensional reconstituted intestinal model (Caco-2, L929 and U937 cells). Comparable benefits of SPD, EUG and SPD + EUG in inducing autophagy were shown by the protection of Caco-2 and L929 cells against lipopolysaccharide-induced inflammation. SPD + EUG is an innovative dual therapy capable of stimulating autophagy and reducing inflammation in vitro and could show promise for COVID-19 risk groups.


Subject(s)
COVID-19 , Syzygium , Aged , Autophagy , COVID-19/drug therapy , Caco-2 Cells , Eugenol/pharmacology , Humans , Inflammation , Monocytes , Plant Oils , Spermidine/pharmacology , Triticum
14.
Front Cell Infect Microbiol ; 12: 798767, 2022.
Article in English | MEDLINE | ID: covidwho-1862592

ABSTRACT

COVID-19 is the biggest pandemic the world has seen this century. Alongside the respiratory damage observed in patients with severe forms of the disease, gastrointestinal symptoms have been frequently reported. These symptoms (e.g., diarrhoea), sometimes precede the development of respiratory tract illnesses, as if the digestive tract was a major target during early SARS-CoV-2 dissemination. We hypothesize that in patients carrying intestinal SARS-CoV-2, the virus may trigger epithelial barrier damage through the disruption of E-cadherin (E-cad) adherens junctions, thereby contributing to the overall gastrointestinal symptoms of COVID-19. Here, we use an intestinal Caco-2 cell line of human origin which expresses the viral receptor/co-receptor as well as the membrane anchored cell surface adhesion protein E-cad to investigate the expression of E-cad after exposure to SARS-CoV-2. We found that the expression of CDH1/E-cad mRNA was significantly lower in cells infected with SARS-CoV-2 at 24 hours post-infection, compared to virus-free Caco-2 cells. The viral receptor ACE2 mRNA expression was specifically down-regulated in SARS-CoV-2-infected Caco-2 cells, while it remained stable in HCoV-OC43-infected Caco-2 cells, a virus which uses HLA class I instead of ACE2 to enter cells. It is worth noting that SARS-CoV-2 induces lower transcription of TMPRSS2 (involved in viral entry) and higher expression of B0AT1 mRNA (that encodes a protein known to co-express with ACE2 on intestinal cells). At 48 hours post-exposure to the virus, we also detected a small but significant increase of soluble E-cad protein (sE-cad) in the culture supernatant of SARS-CoV-2-infected Caco-2 cells. The increase of sE-cad release was also found in the intestinal HT29 cell line when infected by SARS-CoV-2. Beside the dysregulation of E-cad, SARS-CoV-2 infection of Caco-2 cells also leads to the dysregulation of other cell adhesion proteins (occludin, JAMA-A, zonulin, connexin-43 and PECAM-1). Taken together, these results shed light on the fact that infection of Caco-2 cells with SARS-CoV-2 affects tight-, adherens-, and gap-junctions. Moreover, intestinal tissues damage was associated to the intranasal SARS-CoV-2 infection in human ACE2 transgenic mice.


Subject(s)
COVID-19 , Cadherins , Gastrointestinal Diseases , Angiotensin-Converting Enzyme 2/genetics , Animals , Antigens, CD/genetics , Caco-2 Cells , Cadherins/genetics , Gene Expression , Humans , Mice , RNA, Messenger , Receptors, Virus/genetics , SARS-CoV-2/genetics
15.
Biomed Pharmacother ; 151: 113104, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1850705

ABSTRACT

The Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) has continuously evolved, resulting in the emergence of several variants of concern (VOCs). To study mechanisms of viral entry and potentially identify specific inhibitors, we pseudotyped lentiviral vectors with different SARS-CoV-2 VOC spike variants (D614G, Alpha, Beta, Delta, Omicron/BA.1), responsible for receptor binding and membrane fusion. These SARS-CoV-2 lentiviral pseudoviruses were applied to screen 774 FDA-approved drugs. For the assay we decided to use CaCo2 cells, since they equally allow cell entry through both the direct membrane fusion pathway mediated by TMPRSS2 and the endocytosis pathway mediated by cathepsin-L. The active molecules which showed stronger differences in their potency to inhibit certain SARS-CoV-2 VOCs included antagonists of G-protein coupled receptors, like phenothiazine-derived antipsychotic compounds such as Chlorpromazine, with highest activity against the Omicron pseudovirus. In general, our data showed that the various VOCs differ in their preferences for cell entry, and we were able to identify synergistic combinations of inhibitors. Notably, Omicron singled out by relying primarily on the endocytosis pathway while Delta preferred cell entry via membrane fusion. In conclusion, our data provide new insights into different entry preferences of SARS-CoV-2 VOCs, which might help to identify new drug targets.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/drug therapy , Caco-2 Cells , Drug Evaluation, Preclinical , Humans , Spike Glycoprotein, Coronavirus/metabolism
16.
Methods Mol Biol ; 2452: 111-129, 2022.
Article in English | MEDLINE | ID: covidwho-1844263

ABSTRACT

In late 2019, the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. Since its emergence, SARS-CoV-2 has been responsible for a world-wide pandemic resulting in over 80 million infections and over 1.8 million deaths. The severity of the pandemic has prompted widespread research efforts to more fully understand SARS-CoV-2 and the disease it causes, COVID-19. Research into this novel virus will be facilitated by the availability of clearly described and effective protocols that enable the propagation and quantification of infectious virus. Here, we describe protocols for the propagation of SARS-CoV-2 in Vero E6 cells as well as two human cells lines, the intestinal epithelial Caco-2 cell line and the respiratory epithelial Calu-3 cell line. Additionally, we provide protocols for the quantification of SARS-CoV-2 by plaque assays and immunofocus forming assays in Vero E6 cells utilizing liquid overlays. These protocols provide a foundation for laboratories acquiring the ability to study SARS-CoV-2 to address this ongoing pandemic.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Caco-2 Cells , Chlorocebus aethiops , Humans , Pandemics , Vero Cells
17.
Viruses ; 14(4)2022 03 29.
Article in English | MEDLINE | ID: covidwho-1820405

ABSTRACT

Coronavirus disease 19 (COVID-19) clinical manifestations include the involvement of the gastrointestinal tract, affecting around 10% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected children. In the present work, the consequence of a short time of viral absorption (5, 15, 30 and 60 min) was tested on the Caco-2 intestinal epithelial cell line. Our findings show that Caco-2 cells are highly permissive to SARS-CoV-2 infection, even after 5 min of viral inoculation at a multiplicity of infection of 0.1. No cytopathic effect was evident during the subsequent 7 days of monitoring; nevertheless, the immunofluorescence staining for the viral nucleocapsid confirmed the presence of intracellular SARS-CoV-2. Our findings highlight the very short time during which SARS-CoV-2 is able to infect these cells in vitro.


Subject(s)
COVID-19 , Caco-2 Cells , Child , Cytopathogenic Effect, Viral , Gastrointestinal Tract , Humans , SARS-CoV-2
18.
Virol J ; 19(1): 76, 2022 04 26.
Article in English | MEDLINE | ID: covidwho-1817229

ABSTRACT

BACKGROUND: During the ongoing Covid-19 pandemic caused by the emerging virus SARS-CoV-2, research in the field of coronaviruses has expanded tremendously. The genome of SARS-CoV-2 has rapidly acquired numerous mutations, giving rise to several Variants of Concern (VOCs) with altered epidemiological, immunological, and pathogenic properties. METHODS: As cell culture models are important tools to study viruses, we investigated replication kinetics and infectivity of SARS-CoV-2 in the African Green Monkey-derived Vero E6 kidney cell line and the two human cell lines Caco-2, a colon epithelial carcinoma cell line, and the airway epithelial carcinoma cell line Calu-3. We assessed viral RNA copy numbers and infectivity of viral particles in cell culture supernatants at different time points ranging from 2 to 96 h post-infection. RESULTS: We here describe a systematic comparison of growth kinetics of the five SARS-CoV-2 VOCs Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/B.1.617.2, and Omicron/B.1.1.529 and a non-VOC/B.1.1 strain on three different cell lines to provide profound information on the differential behaviour of VOCs in different cell lines for researchers worldwide. We show distinct differences in viral replication kinetics of the SARS-CoV-2 non-VOC and five VOCs on the three cell culture models Vero E6, Caco-2, and Calu-3. CONCLUSION: This is the first systematic comparison of all SARS-CoV-2 VOCs on three different cell culture models. This data provides support for researchers worldwide in their experimental design for work on SARS-CoV-2. It is recommended to perform virus isolation and propagation on Vero E6 while infection studies or drug screening and antibody-based assays should rather be conducted on the human cell lines Caco-2 and Calu-3.


Subject(s)
COVID-19 , Carcinoma , Caco-2 Cells , Cell Culture Techniques , Chlorocebus aethiops , Humans , Kinetics , Pandemics , SARS-CoV-2/genetics
19.
Front Immunol ; 13: 832223, 2022.
Article in English | MEDLINE | ID: covidwho-1809390

ABSTRACT

Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) using Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analyzed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~101 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.


Subject(s)
COVID-19 , Animals , COVID-19/genetics , Caco-2 Cells , Chlorocebus aethiops , Humans , Polyadenylation , RNA, Messenger/metabolism , Ribosomal Proteins/metabolism , SARS-CoV-2 , Sequence Analysis, RNA , Vero Cells
20.
Front Immunol ; 13: 870787, 2022.
Article in English | MEDLINE | ID: covidwho-1785352

ABSTRACT

Cannabidiol (CBD) can prevent the inflammatory response of SARS-CoV-2 spike protein in Caco-2-cells. This action is coupled with the inhibition of IL-1beta, IL-6, IL-18, and TNF-alpha, responsible for the inflammatory process during SARS-CoV-2 infection. CBD can act on the different proteins encoded by SARS-CoV-2 and as an antiviral agent to prevent the viral infection. Furthermore, recent studies have shown the possible action of CBD as an antagonist of cytokine release syndromes. In the SARS-CoV-2 pathophysiology, the angiotensin-converting enzyme 2 (ACE2) seems to be the key cell receptor for SARS-CoV-2 infection. The WNT/ß-catenin pathway and PPARγ interact in an opposite manner in many diseases, including SARS-CoV-2 infection. CBD exerts its activity through the interaction with PPARγ in SARS-CoV-2 infection. Thus, we can hypothesize that CBD may counteract the inflammatory process of SARS-CoV-2 by its interactions with both ACE2 and the interplay between the WNT/ß-catenin pathway and PPARγ. Vaccines are the only way to prevent COVID-19, but it appears important to find therapeutic complements to treat patients already affected by SARS-CoV-2 infection. The possible role of CBD should be investigated by clinical trials to show its effectiveness.


Subject(s)
COVID-19 , Cannabidiol , Angiotensin-Converting Enzyme 2 , COVID-19/drug therapy , Caco-2 Cells , Cannabidiol/pharmacology , Cannabidiol/therapeutic use , Humans , PPAR gamma , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , beta Catenin
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