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1.
Cell Mol Life Sci ; 79(1): 65, 2022 Jan 10.
Article in English | MEDLINE | ID: covidwho-1616112

ABSTRACT

Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air-liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Fenamates/pharmacology , MAP Kinase Signaling System/drug effects , Protein Kinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Adult , Animals , COVID-19/metabolism , Cell Line , Cells, Cultured , Chlorocebus aethiops , Cytokines/metabolism , Humans , Inflammation/drug therapy , Inflammation/metabolism , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/antagonists & inhibitors , MAP Kinase Kinase 2/metabolism , SARS-CoV-2/physiology , Vero Cells , Virus Replication/drug effects
2.
Front Immunol ; 12: 781352, 2021.
Article in English | MEDLINE | ID: covidwho-1613552

ABSTRACT

After the outburst of the SARS-CoV-2 pandemic, a worldwide research effort has led to the uncovering of many aspects of the COVID-19, among which we can count the outstanding role played by inflammatory cytokine milieu in the disease progression. Despite that, molecular mechanisms that regulate SARS-CoV-2 pathogenesis are still almost unidentified. In this study, we investigated whether the pro-inflammatory milieu of the host affects the susceptibility of SARS-CoV-2 infection by modulating ACE2 and TMPRSS2 expression. Our results indicated that the host inflammatory milieu favors SARS-CoV-2 infection by directly increasing TMPRSS2 expression. We unveiled the molecular mechanism that regulates this process and that can be therapeutically advantageously targeted.


Subject(s)
GATA2 Transcription Factor/metabolism , Interleukin-1beta/metabolism , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Virus Internalization , A549 Cells , COVID-19 , Humans , p38 Mitogen-Activated Protein Kinases/metabolism
3.
Signal Transduct Target Ther ; 6(1): 167, 2021 04 24.
Article in English | MEDLINE | ID: covidwho-1585891

ABSTRACT

The ongoing 2019 novel coronavirus disease (COVID-19) caused by SARS-CoV-2 has posed a worldwide pandemic and a major global public health threat. The severity and mortality of COVID-19 are associated with virus-induced dysfunctional inflammatory responses and cytokine storms. However, the interplay between host inflammatory responses and SARS-CoV-2 infection remains largely unknown. Here, we demonstrate that SARS-CoV-2 nucleocapsid (N) protein, the major structural protein of the virion, promotes the virus-triggered activation of NF-κB signaling. After binding to viral RNA, N protein robustly undergoes liquid-liquid phase separation (LLPS), which recruits TAK1 and IKK complex, the key kinases of NF-κB signaling, to enhance NF-κB activation. Moreover, 1,6-hexanediol, the inhibitor of LLPS, can attenuate the phase separation of N protein and restrict its regulatory functions in NF-κB activation. These results suggest that LLPS of N protein provides a platform to induce NF-κB hyper-activation, which could be a potential therapeutic target against COVID-19 severe pneumonia.


Subject(s)
COVID-19/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , NF-kappa B/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Signal Transduction , A549 Cells , Acrylates/pharmacology , Animals , COVID-19/drug therapy , COVID-19/pathology , Chlorocebus aethiops , HEK293 Cells , HeLa Cells , Humans , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/pathology , Phosphoproteins/metabolism , Vero Cells
4.
Sci Rep ; 11(1): 23928, 2021 12 14.
Article in English | MEDLINE | ID: covidwho-1585797

ABSTRACT

Identification of transcriptional regulatory mechanisms and signaling networks involved in the response of host cells to infection by SARS-CoV-2 is a powerful approach that provides a systems biology view of gene expression programs involved in COVID-19 and may enable the identification of novel therapeutic targets and strategies to mitigate the impact of this disease. In this study, our goal was to identify a transcriptional regulatory network that is associated with gene expression changes between samples infected by SARS-CoV-2 and those that are infected by other respiratory viruses to narrow the results on those enriched or specific to SARS-CoV-2. We combined a series of recently developed computational tools to identify transcriptional regulatory mechanisms involved in the response of epithelial cells to infection by SARS-CoV-2, and particularly regulatory mechanisms that are specific to this virus when compared to other viruses. In addition, using network-guided analyses, we identified kinases associated with this network. The results identified pathways associated with regulation of inflammation (MAPK14) and immunity (BTK, MBX) that may contribute to exacerbate organ damage linked with complications of COVID-19. The regulatory network identified herein reflects a combination of known hits and novel candidate pathways supporting the novel computational pipeline presented herein to quickly narrow down promising avenues of investigation when facing an emerging and novel disease such as COVID-19.


Subject(s)
COVID-19/genetics , Gene Expression Profiling/methods , SARS-CoV-2/pathogenicity , Sequence Analysis, RNA/methods , A549 Cells , Cell Line , Epithelial Cells/chemistry , Epithelial Cells/cytology , Epithelial Cells/virology , Gene Expression Regulation , Humans , Models, Biological , Systems Biology
5.
Sci Rep ; 11(1): 24442, 2021 12 24.
Article in English | MEDLINE | ID: covidwho-1577650

ABSTRACT

Therapeutic interventions targeting viral infections remain a significant challenge for both the medical and scientific communities. While specific antiviral agents have shown success as therapeutics, viral resistance inevitably develops, making many of these approaches ineffective. This inescapable obstacle warrants alternative approaches, such as the targeting of host cellular factors. Respiratory syncytial virus (RSV), the major respiratory pathogen of infants and children worldwide, causes respiratory tract infection ranging from mild upper respiratory tract symptoms to severe life-threatening lower respiratory tract disease. Despite the fact that the molecular biology of the virus, which was originally discovered in 1956, is well described, there is no vaccine or effective antiviral treatment against RSV infection. Here, we demonstrate that targeting host factors, specifically, mTOR signaling, reduces RSV protein production and generation of infectious progeny virus. Further, we show that this approach can be generalizable as inhibition of mTOR kinases reduces coronavirus gene expression, mRNA transcription and protein production. Overall, defining virus replication-dependent host functions may be an effective means to combat viral infections, particularly in the absence of antiviral drugs.


Subject(s)
Coronavirus/metabolism , Respiratory Syncytial Virus, Human/metabolism , TOR Serine-Threonine Kinases/metabolism , Viral Proteins/metabolism , A549 Cells , Coronavirus/drug effects , Coronavirus/genetics , Gene Expression Regulation, Viral/drug effects , Humans , Protein Biosynthesis/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , RNA Interference , RNA, Small Interfering/metabolism , Rapamycin-Insensitive Companion of mTOR Protein/antagonists & inhibitors , Rapamycin-Insensitive Companion of mTOR Protein/genetics , Rapamycin-Insensitive Companion of mTOR Protein/metabolism , Regulatory-Associated Protein of mTOR/antagonists & inhibitors , Regulatory-Associated Protein of mTOR/genetics , Regulatory-Associated Protein of mTOR/metabolism , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus Infections/pathology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/drug effects , Respiratory Syncytial Virus, Human/isolation & purification , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics , Viral Proteins/genetics
6.
Life Sci Alliance ; 5(3)2022 03.
Article in English | MEDLINE | ID: covidwho-1552086

ABSTRACT

Murine neural stem cells (NSCs) were recently shown to release piRNA-containing exosomes/microvesicles (Ex/Mv) for exerting antiviral immunity, but it remains unknown if these Ex/Mv could target SARS-CoV-2 and whether the PIWI-piRNA system is important for these antiviral actions. Here, using in vitro infection models, we show that hypothalamic NSCs (htNSCs) Ex/Mv provided an innate immunity protection against SARS-CoV-2. Importantly, enhanced antiviral actions were achieved by using induced Ex/Mv that were derived from induced htNSCs through twice being exposed to several RNA fragments of SARS-CoV-2 genome, a process that was designed not to involve protein translation of these RNA fragments. The increased antiviral effects of these induced Ex/Mv were associated with increased expression of piRNA species some of which could predictably target SARS-CoV-2 genome. Knockout of piRNA-interacting protein PIWIL2 in htNSCs led to reductions in both innate and induced antiviral effects of Ex/Mv in targeting SARS-CoV-2. Taken together, this study demonstrates a case suggesting Ex/Mv from certain cell types have innate and adaptive immunity against SARS-CoV-2, and the PIWI-piRNA system is important for these antiviral actions.


Subject(s)
Argonaute Proteins/metabolism , COVID-19/immunology , COVID-19/metabolism , Cell-Derived Microparticles/metabolism , Exosomes , RNA, Small Interfering/metabolism , RNA/metabolism , SARS-CoV-2 , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Animals , Genome, Viral , Humans , Hypothalamus/metabolism , Immune System , Immunity, Innate , In Vitro Techniques , Mice
7.
PLoS One ; 16(11): e0260283, 2021.
Article in English | MEDLINE | ID: covidwho-1523456

ABSTRACT

SARS-CoV-2 viral attachment and entry into host cells is mediated by a direct interaction between viral spike glycoproteins and membrane bound angiotensin-converting enzyme 2 (ACE2). The receptor binding motif (RBM), located within the S1 subunit of the spike protein, incorporates the majority of known ACE2 contact residues responsible for high affinity binding and associated virulence. Observation of existing crystal structures of the SARS-CoV-2 receptor binding domain (SRBD)-ACE2 interface, combined with peptide array screening, allowed us to define a series of linear native RBM-derived peptides that were selected as potential antiviral decoy sequences with the aim of directly binding ACE2 and attenuating viral cell entry. RBM1 (16mer): S443KVGGNYNYLYRLFRK458, RBM2A (25mer): E484GFNCYFPLQSYGFQPTNGVGYQPY508, RBM2B (20mer): F456NCYFPLQSYGFQPTNGVGY505 and RBM2A-Sc (25mer): NYGLQGSPFGYQETPYPFCNFVQYG. Data from fluorescence polarisation experiments suggested direct binding between RBM peptides and ACE2, with binding affinities ranging from the high nM to low µM range (Kd = 0.207-1.206 µM). However, the RBM peptides demonstrated only modest effects in preventing SRBD internalisation and showed no antiviral activity in a spike protein trimer neutralisation assay. The RBM peptides also failed to suppress S1-protein mediated inflammation in an endogenously expressing ACE2 human cell line. We conclude that linear native RBM-derived peptides are unable to outcompete viral spike protein for binding to ACE2 and therefore represent a suboptimal approach to inhibiting SARS-CoV-2 viral cell entry. These findings reinforce the notion that larger biologics (such as soluble ACE2, 'miniproteins', nanobodies and antibodies) are likely better suited as SARS-CoV-2 cell-entry inhibitors than short-sequence linear peptides.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antiviral Agents/pharmacology , Peptides/pharmacology , Protein Binding/drug effects , Spike Glycoprotein, Coronavirus/immunology , Virus Internalization , A549 Cells , Humans , Protein Interaction Domains and Motifs
8.
Sci Rep ; 11(1): 22195, 2021 11 12.
Article in English | MEDLINE | ID: covidwho-1514424

ABSTRACT

To initiate SARS-CoV-2 infection, the Receptor Binding Domain (RBD) on the viral spike protein must first bind to the host receptor ACE2 protein on pulmonary and other ACE2-expressing cells. We hypothesized that cardiac glycoside drugs might block the binding reaction between ACE2 and the Spike (S) protein, and thus block viral penetration into target cells. To test this hypothesis we developed a biochemical assay for ACE2:Spike binding, and tested cardiac glycosides as inhibitors of binding. Here we report that ouabain, digitoxin, and digoxin, as well as sugar-free derivatives digitoxigenin and digoxigenin, are high-affinity competitive inhibitors of ACE2 binding to the Original [D614] S1 and the α/ß/γ [D614G] S1 proteins. These drugs also inhibit ACE2 binding to the Original RBD, as well as to RBD proteins containing the ß [E484K], Mink [Y453F] and α/ß/γ [N501Y] mutations. As hypothesized, we also found that ouabain, digitoxin and digoxin blocked penetration by SARS-CoV-2 Spike-pseudotyped virus into human lung cells, and infectivity by native SARS-CoV-2. These data indicate that cardiac glycosides may block viral penetration into the target cell by first inhibiting ACE2:RBD binding. Clinical concentrations of ouabain and digitoxin are relatively safe for short term use for subjects with normal hearts. It has therefore not escaped our attention that these common cardiac medications could be deployed worldwide as inexpensive repurposed drugs for anti-COVID-19 therapy.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/drug therapy , Cardiotonic Agents/pharmacology , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects , A549 Cells , Animals , COVID-19/metabolism , Chlorocebus aethiops , Digitoxin/pharmacology , Digoxin/pharmacology , Humans , Lung/drug effects , Lung/metabolism , Ouabain/pharmacology , Protein Binding/drug effects , SARS-CoV-2/physiology , Vero Cells
9.
Viruses ; 13(10)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470998

ABSTRACT

Nuclear transport and vesicle trafficking are key cellular functions involved in the pathogenesis of RNA viruses. Among other pleiotropic effects on virus-infected host cells, ivermectin (IVM) inhibits nuclear transport mechanisms mediated by importins and atorvastatin (ATV) affects actin cytoskeleton-dependent trafficking controlled by Rho GTPases signaling. In this work, we first analyzed the response to infection in nasopharyngeal swabs from SARS-CoV-2-positive and -negative patients by assessing the gene expression of the respective host cell drug targets importins and Rho GTPases. COVID-19 patients showed alterations in KPNA3, KPNA5, KPNA7, KPNB1, RHOA, and CDC42 expression compared with non-COVID-19 patients. An in vitro model of infection with Poly(I:C), a synthetic analog of viral double-stranded RNA, triggered NF-κB activation, an effect that was halted by IVM and ATV treatment. Importin and Rho GTPases gene expression was also impaired by these drugs. Furthermore, through confocal microscopy, we analyzed the effects of IVM and ATV on nuclear to cytoplasmic importin α distribution, alone or in combination. Results showed a significant inhibition of importin α nuclear accumulation under IVM and ATV treatments. These findings confirm transcriptional alterations in importins and Rho GTPases upon SARS-CoV-2 infection and point to IVM and ATV as valid drugs to impair nuclear localization of importin α when used at clinically-relevant concentrations.


Subject(s)
Active Transport, Cell Nucleus/drug effects , Atorvastatin/pharmacology , COVID-19/drug therapy , Ivermectin/pharmacology , SARS-CoV-2/drug effects , alpha Karyopherins/metabolism , A549 Cells , Actin Cytoskeleton/drug effects , Animals , Antiviral Agents/pharmacology , Cell Line, Tumor , Chlorocebus aethiops , Drug Repositioning , HeLa Cells , Humans , NF-kappa B/metabolism , Vero Cells , rho GTP-Binding Proteins/metabolism
10.
Viruses ; 13(10)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470997

ABSTRACT

We report the in vitro efficacy of ion-channel inhibitors amantadine, memantine and rimantadine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In VeroE6 cells, rimantadine was most potent followed by memantine and amantadine (50% effective concentrations: 36, 80 and 116 µM, respectively). Rimantadine also showed the highest selectivity index, followed by amantadine and memantine (17.3, 12.2 and 7.6, respectively). Similar results were observed in human hepatoma Huh7.5 and lung carcinoma A549-hACE2 cells. Inhibitors interacted in a similar antagonistic manner with remdesivir and had a similar barrier to viral escape. Rimantadine acted mainly at the viral post-entry level and partially at the viral entry level. Based on these results, rimantadine showed the most promise for treatment of SARS-CoV-2.


Subject(s)
Amantadine/pharmacology , Antiviral Agents/pharmacology , COVID-19/drug therapy , Memantine/pharmacology , Rimantadine/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Cell Line, Tumor , Chlorocebus aethiops , Denmark , Drug Repositioning , Humans , Ion Channels/antagonists & inhibitors , Vero Cells
11.
J Immunol ; 207(10): 2521-2533, 2021 11 15.
Article in English | MEDLINE | ID: covidwho-1468558

ABSTRACT

Many patients with coronavirus disease 2019 in intensive care units suffer from cytokine storm. Although anti-inflammatory therapies are available to treat the problem, very often, these treatments cause immunosuppression. Because angiotensin-converting enzyme 2 (ACE2) on host cells serves as the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to delineate a SARS-CoV-2-specific anti-inflammatory molecule, we designed a hexapeptide corresponding to the spike S1-interacting domain of ACE2 receptor (SPIDAR) that inhibited the expression of proinflammatory molecules in human A549 lung cells induced by pseudotyped SARS-CoV-2, but not vesicular stomatitis virus. Accordingly, wild-type (wt), but not mutated (m), SPIDAR inhibited SARS-CoV-2 spike S1-induced activation of NF-κB and expression of IL-6 and IL-1ß in human lung cells. However, wtSPIDAR remained unable to reduce activation of NF-κB and expression of proinflammatory molecules in lungs cells induced by TNF-α, HIV-1 Tat, and viral dsRNA mimic polyinosinic-polycytidylic acid, indicating the specificity of the effect. The wtSPIDAR, but not mutated SPIDAR, also hindered the association between ACE2 and spike S1 of SARS-CoV-2 and inhibited the entry of pseudotyped SARS-CoV-2, but not vesicular stomatitis virus, into human ACE2-expressing human embryonic kidney 293 cells. Moreover, intranasal treatment with wtSPIDAR, but not mutated SPIDAR, inhibited lung activation of NF-κB, protected lungs, reduced fever, improved heart function, and enhanced locomotor activities in SARS-CoV-2 spike S1-intoxicated mice. Therefore, selective targeting of SARS-CoV-2 spike S1-to-ACE2 interaction by wtSPIDAR may be beneficial for coronavirus disease 2019.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Anti-Inflammatory Agents/therapeutic use , COVID-19/therapy , Lung/immunology , Peptides/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/immunology , Cytokines/metabolism , Female , HEK293 Cells , Humans , Inflammation Mediators/metabolism , Locomotion , Male , Mice , Molecular Targeted Therapy , NF-kappa B/metabolism , Peptides/genetics , Peptides/therapeutic use , Signal Transduction , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
12.
mBio ; 12(5): e0137221, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1462899

ABSTRACT

Interleukin6 (IL-6) is a key driver of hyperinflammation in COVID-19, and its level strongly correlates with disease progression. To investigate whether variability in COVID-19 severity partially results from differential IL-6 expression, functional single-nucleotide polymorphisms (SNPs) of IL-6 were determined in Chinese COVID-19 patients with mild or severe illness. An Asian-common IL-6 haplotype defined by promoter SNP rs1800796 and intronic SNPs rs1524107 and rs2066992 correlated with COVID-19 severity. Homozygote carriers of C-T-T variant haplotype were at lower risk of developing severe symptoms (odds ratio, 0.256; 95% confidence interval, 0.088 to 0.739; P = 0.007). This protective haplotype was associated with lower levels of IL-6 and its antisense long noncoding RNA IL-6-AS1 by cis-expression quantitative trait loci analysis. The differences in expression resulted from the disturbance of stimulus-dependent bidirectional transcription of the IL-6/IL-6-AS1 locus by the polymorphisms. The protective rs2066992-T allele disrupted a conserved CTCF-binding locus at the enhancer elements of IL-6-AS1, which transcribed antisense to IL-6 and induces IL-6 expression in inflammatory responses. As a result, carriers of the protective allele had significantly reduced IL-6-AS1 expression and attenuated IL-6 induction in response to acute inflammatory stimuli and viral infection. Intriguingly, this low-producing variant that is endemic to present-day Asia was found in early humans who had inhabited mainland Asia since ∼40,000 years ago but not in other ancient humans, such as Neanderthals and Denisovans. The present study suggests that an individual's IL-6 genotype underlies COVID-19 outcome and may be used to guide IL-6 blockade therapy in Asian patients. IMPORTANCE Overproduction of cytokine interleukin-6 (IL-6) is a hallmark of severe COVID-19 and is believed to play a critical role in exacerbating the excessive inflammatory response. Polymorphisms in IL-6 account for the variability of IL-6 expression and disparities in infectious diseases, but its contribution to the clinical presentation of COVID-19 has not been reported. Here, we investigated IL-6 polymorphisms in severe and mild cases of COVID-19 in a Chinese population. The variant haplotype C-T-T, represented by rs1800796, rs1524107, and rs2066992 at the IL-6 locus, was reduced in patients with severe illness; in contrast, carriers of the wild-type haplotype G-C-G had higher risk of severe illness. Mechanistically, the protective variant haplotype lost CTCF binding at the IL-6 intron and responded poorly to inflammatory stimuli, which may protect the carriers from hyperinflammation in response to acute SARS-CoV-2 infection. These results point out the possibility that IL-6 genotypes underlie the differential viral virulence during the outbreak of COVID-19. The risk loci we identified may serve as a genetic marker to screen high-risk COVID-19 patients.


Subject(s)
COVID-19/metabolism , COVID-19/prevention & control , Interleukin-6/metabolism , A549 Cells , Genotype , Haplotypes/genetics , HeLa Cells , Humans , Polymorphism, Single Nucleotide/genetics , Real-Time Polymerase Chain Reaction , Software
13.
Viruses ; 13(10)2021 10 04.
Article in English | MEDLINE | ID: covidwho-1463827

ABSTRACT

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.


Subject(s)
Coronavirus Infections/veterinary , Deltacoronavirus/genetics , Deltacoronavirus/metabolism , Genes, Reporter/genetics , Luciferases/genetics , A549 Cells , Animals , Cell Line , Chlorocebus aethiops , Chromosomes, Artificial, Bacterial/genetics , Coronavirus Infections/pathology , Deltacoronavirus/growth & development , Dogs , Genome, Viral/genetics , Humans , Luciferases/biosynthesis , Madin Darby Canine Kidney Cells , Nanostructures , Swine , Swine Diseases/virology , Vero Cells , Virus Replication/genetics
14.
Molecules ; 26(19)2021 Oct 07.
Article in English | MEDLINE | ID: covidwho-1463770

ABSTRACT

The COVID-19 pandemic outbreak prompts an urgent need for efficient therapeutics, and repurposing of known drugs has been extensively used in an attempt to get to anti-SARS-CoV-2 agents in the shortest possible time. The glycoside rutin shows manifold pharmacological activities and, despite its use being limited by its poor solubility in water, it is the active principle of many pharmaceutical preparations. We herein report our in silico and experimental investigations of rutin as a SARS-CoV-2 Mpro inhibitor and of its water solubility improvement obtained by mixing it with l-arginine. Tests of the rutin/l-arginine mixture in a cellular model of SARS-CoV-2 infection highlighted that the mixture still suffers from unfavorable pharmacokinetic properties, but nonetheless, the results of this study suggest that rutin might be a good starting point for hit optimization.


Subject(s)
Antiviral Agents/pharmacology , Arginine/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Rutin/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Coronavirus 3C Proteases/metabolism , Humans , Molecular Docking Simulation , Protease Inhibitors/pharmacology , SARS-CoV-2/metabolism , Solubility
15.
Front Immunol ; 12: 725240, 2021.
Article in English | MEDLINE | ID: covidwho-1463472

ABSTRACT

Ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus strains is posing new COVID-19 diagnosis and treatment challenges. To help efforts to meet these challenges we examined data acquired from proteomic analyses of human SARS-CoV-2-infected cell lines and samples from COVID-19 patients. Initially, 129 unique peptides were identified, which were rigorously evaluated for repeats, disorders, polymorphisms, antigenicity, immunogenicity, toxicity, allergens, sequence similarity to human proteins, and contributions from other potential cross-reacting pathogenic species or the human saliva microbiome. We also screened SARS-CoV-2-infected NBHE and A549 cell lines for presence of antigenic peptides, and identified paratope peptides from crystal structures of SARS-CoV-2 antigen-antibody complexes. We then selected four antigen peptides for docking with known viral unbound T-cell receptor (TCR), class I and II peptide major histocompatibility complex (pMHC), and identified paratope sequences. We also tested the paratope binding affinity of SARS-CoV T- and B-cell peptides that had been previously experimentally validated. The resultant antigenic peptides have high potential for generating SARS-CoV-2-specific antibodies, and the paratope peptides can be directly used to develop a COVID-19 diagnostics assay. The presented genomics and proteomics-based in-silico approaches have apparent utility for identifying new diagnostic peptides that could be used to fight SARS-CoV-2.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/metabolism , Epitopes, B-Lymphocyte/metabolism , Epitopes, T-Lymphocyte/metabolism , Peptides/metabolism , Pulmonary Alveoli/pathology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , COVID-19/immunology , Cell Line , Coronavirus Nucleocapsid Proteins/genetics , Epitope Mapping , Epitopes, B-Lymphocyte/genetics , Epitopes, T-Lymphocyte/genetics , HLA Antigens/metabolism , Humans , Molecular Docking Simulation , Peptides/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Proteomics , Receptors, Antigen/metabolism , Spike Glycoprotein, Coronavirus/genetics
16.
Sci Rep ; 11(1): 20012, 2021 10 08.
Article in English | MEDLINE | ID: covidwho-1462029

ABSTRACT

There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of - 3 to - 4 at a concentration of 10-100 µg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 µg/ mL (p < 0.05 compared to untreated controls). In vivo studies using transgenic mice expressing the ACE-2 receptor, dosed nasally with SARS-COV-2 (426,000 TCID50/mL) showed a trend for nasal GCPQ (20 mg/kg) to inhibit viral load in the respiratory tract and brain, although the study was not powered to detect statistical significance. GCPQ's electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 h after nasal dosing. With a no observed adverse effect level of 18 mg/kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Nasal Sprays , SARS-CoV-2/drug effects , A549 Cells , Animals , Antiviral Agents/administration & dosage , Chlorocebus aethiops , Humans , Male , Methylation , Mice, Inbred BALB C , Mice, Transgenic , SARS-CoV-2/physiology , Surface-Active Agents/administration & dosage , Surface-Active Agents/therapeutic use , Vero Cells , Viral Load/drug effects
17.
Dis Markers ; 2021: 6803510, 2021.
Article in English | MEDLINE | ID: covidwho-1443673

ABSTRACT

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most significant public health threat worldwide. Patients with severe COVID-19 usually have pneumonia concomitant with local inflammation and sometimes a cytokine storm. Specific components of the SARS-CoV-2 virus trigger lung inflammation, and recruitment of immune cells to the lungs exacerbates this process, although much remains unknown about the pathogenesis of COVID-19. Our study of lung type II pneumocyte cells (A549) demonstrated that ORF7, an open reading frame (ORF) in the genome of SARS-CoV-2, induced the production of CCL2, a chemokine that promotes the chemotaxis of monocytes, and decreased the expression of IL-8, a chemokine that recruits neutrophils. A549 cells also had an increased level of IL-6. The results of our chemotaxis Transwell assay suggested that ORF7 augmented monocyte infiltration and reduced the number of neutrophils. We conclude that the ORF7 of SARS-CoV-2 may have specific effects on the immunological changes in tissues after infection. These results suggest that the functions of other ORFs of SARS-CoV-2 should also be comprehensively examined.


Subject(s)
COVID-19/metabolism , Chemotaxis , Monocytes/pathology , Neutrophils/pathology , Open Reading Frames/physiology , Pneumonia/pathology , Viral Proteins/metabolism , A549 Cells , Chemokine CCL2/metabolism , Humans , In Vitro Techniques , Monocytes/immunology , Monocytes/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Pneumonia/immunology , Pneumonia/metabolism , SARS-CoV-2/metabolism , Viral Proteins/genetics
18.
Science ; 374(6567): eabj3624, 2021 10 29.
Article in English | MEDLINE | ID: covidwho-1440797
19.
Sci Rep ; 11(1): 19161, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440480

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with fatal pulmonary fibrosis. Small interfering RNAs (siRNAs) can be developed to induce RNA interference against SARS-CoV-2, and their susceptible target sites can be inferred by Argonaute crosslinking immunoprecipitation sequencing (AGO CLIP). Here, by reanalysing AGO CLIP data in RNA viruses, we delineated putative AGO binding in the conserved non-structural protein 12 (nsp12) region encoding RNA-dependent RNA polymerase (RdRP) in SARS-CoV-2. We utilised the inferred AGO binding to optimise the local RNA folding parameter to calculate target accessibility and predict all potent siRNA target sites in the SARS-CoV-2 genome, avoiding sequence variants. siRNAs loaded onto AGO also repressed seed (positions 2-8)-matched transcripts by acting as microRNAs (miRNAs). To utilise this, we further screened 13 potential siRNAs whose seed sequences were matched to known antifibrotic miRNAs and confirmed their miRNA-like activity. A miR-27-mimicking siRNA designed to target the nsp12 region (27/RdRP) was validated to silence a synthesised nsp12 RNA mimic in lung cell lines and function as an antifibrotic miR-27 in regulating target transcriptomes related to TGF-ß signalling. siRNA sequences with an antifibrotic miRNA-like activity that could synergistically treat COVID-19 are available online ( http://clip.korea.ac.kr/covid19 ).


Subject(s)
Argonaute Proteins/genetics , COVID-19/prevention & control , MicroRNAs/genetics , RNA, Small Interfering/genetics , SARS-CoV-2/genetics , A549 Cells , Argonaute Proteins/metabolism , Base Sequence , Binding Sites/genetics , COVID-19/virology , Cell Line , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Gene Expression Profiling/methods , HeLa Cells , Humans , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/metabolism , RNA Interference , RNA-Seq/methods , SARS-CoV-2/physiology , Sequence Homology, Nucleic Acid
20.
Viruses ; 13(8)2021 08 12.
Article in English | MEDLINE | ID: covidwho-1436097

ABSTRACT

Type III interferons (lambda IFNs) are a quite new, small family of three closely related cytokines with interferon-like activity. Attention to IFN-λ is mainly focused on direct antiviral activity in which, as with IFN-α, viral genome replication is inhibited without the participation of immune system cells. The heterodimeric receptor for lambda interferons is exposed mainly on epithelial cells, which limits its possible action on other cells, thus reducing the likelihood of developing undesirable side effects compared to type I IFN. In this study, we examined the antiviral potential of exogenous human IFN-λ1 in cellular models of viral infection. To study the protective effects of IFN-λ1, three administration schemes were used: 'preventive' (pretreatment); 'preventive/therapeutic' (pre/post); and 'therapeutic' (post). Three IFN-λ1 concentrations (from 10 to 500 ng/mL) were used. We have shown that human IFN-λ1 restricts SARS-CoV-2 replication in Vero cells with all three treatment schemes. In addition, we have shown a decrease in the viral loads of CHIKV and IVA with the 'preventive' and 'preventive/therapeutic' regimes. No significant antiviral effect of IFN-λ1 against AdV was detected. Our study highlights the potential for using IFN-λ as a broad-spectrum therapeutic agent against respiratory RNA viruses.


Subject(s)
Adenoviruses, Human/drug effects , Chikungunya virus/drug effects , Influenza A virus/drug effects , Interferons/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Adenoviruses, Human/physiology , Animals , Chikungunya virus/physiology , Chlorocebus aethiops , Dose-Response Relationship, Drug , Gene Expression Regulation , Humans , Influenza A virus/physiology , Interferons/therapeutic use , Interleukins , RNA Virus Infections/drug therapy , RNA Virus Infections/prevention & control , Recombinant Proteins/pharmacology , SARS-CoV-2/physiology , Vero Cells , Viral Load/drug effects , Virus Replication/drug effects
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