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1.
ACS Appl Bio Mater ; 5(2): 483-491, 2022 02 21.
Article in English | MEDLINE | ID: covidwho-1805546

ABSTRACT

Interleukin-mediated deep cytokine storm, an aggressive inflammatory response to SARS-CoV-2 virus infection in COVID-19 patients, is correlated directly with lung injury, multi-organ failure, and poor prognosis of severe COVID-19 patients. Curcumin (CUR), a phenolic antioxidant compound obtained from turmeric (Curcuma longa L.), is well-known for its strong anti-inflammatory activity. However, its in vivo efficacy is constrained due to poor bioavailability. Herein, we report that CUR-encapsulated polysaccharide nanoparticles (CUR-PS-NPs) potently inhibit the release of cytokines, chemokines, and growth factors associated with damage of SARS-CoV-2 spike protein (CoV2-SP)-stimulated liver Huh7.5 and lung A549 epithelial cells. Treatment with CUR-PS-NPs effectively attenuated the interaction of ACE2 and CoV2-SP. The effects of CUR-PS-NPs were linked to reduced NF-κB/MAPK signaling which in turn decreased CoV2-SP-mediated phosphorylation of p38 MAPK, p42/44 MAPK, and p65/NF-κB as well as nuclear p65/NF-κB expression. The findings of the study strongly indicate that organic NPs of CUR can be used to control hyper-inflammatory responses and prevent lung and liver injuries associated with CoV2-SP-mediated cytokine storm.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Curcumin/pharmacology , Cytokine Release Syndrome/prevention & control , MAP Kinase Signaling System/drug effects , NF-kappa B/metabolism , Nanoparticles/chemistry , Signal Transduction/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/metabolism , Anti-Inflammatory Agents/pharmacokinetics , Cell Survival/drug effects , Chemokines/biosynthesis , Curcumin/chemistry , Curcumin/pharmacokinetics , Cytokines/biosynthesis , Humans , Intercellular Signaling Peptides and Proteins/biosynthesis , Phosphorylation , Spike Glycoprotein, Coronavirus/physiology
2.
Mol Neurobiol ; 59(3): 1850-1861, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1712348

ABSTRACT

SARS-CoV-2 infects cells via its spike protein binding to its surface receptor on target cells and results in acute symptoms involving especially the lungs known as COVID-19. However, increasing evidence indicates that many patients develop a chronic condition characterized by fatigue and neuropsychiatric symptoms, termed long-COVID. Most of the vaccines produced so far for COVID-19 direct mammalian cells via either mRNA or an adenovirus vector to express the spike protein, or administer recombinant spike protein, which is recognized by the immune system leading to the production of neutralizing antibodies. Recent publications provide new findings that may help decipher the pathogenesis of long-COVID. One paper reported perivascular inflammation in brains of deceased patients with COVID-19, while others showed that the spike protein could damage the endothelium in an animal model, that it could disrupt an in vitro model of the blood-brain barrier (BBB), and that it can cross the BBB resulting in perivascular inflammation. Moreover, the spike protein appears to share antigenic epitopes with human molecular chaperons resulting in autoimmunity and can activate toll-like receptors (TLRs), leading to release of inflammatory cytokines. Moreover, some antibodies produced against the spike protein may not be neutralizing, but may change its conformation rendering it more likely to bind to its receptor. As a result, one wonders whether the spike protein entering the brain or being expressed by brain cells could activate microglia, alone or together with inflammatory cytokines, since protective antibodies could not cross the BBB, leading to neuro-inflammation and contributing to long-COVID. Hence, there is urgent need to better understand the neurotoxic effects of the spike protein and to consider possible interventions to mitigate spike protein-related detrimental effects to the brain, possibly via use of small natural molecules, especially the flavonoids luteolin and quercetin.


Subject(s)
COVID-19/complications , Spike Glycoprotein, Coronavirus/physiology , COVID-19/etiology , COVID-19/virology , Humans , Prospective Studies
3.
J Clin Invest ; 132(4)2022 02 15.
Article in English | MEDLINE | ID: covidwho-1685790

ABSTRACT

Infection with SARS-CoV-2, the causative agent of COVID-19, causes mild to moderate disease in most patients but carries a risk of morbidity and mortality. Seriously affected individuals manifest disorders of hemostasis and a cytokine storm, but it is not understood how these manifestations of severe COVID-19 are linked. Here, we showed that the SARS-CoV-2 spike protein engaged the CD42b receptor to activate platelets via 2 distinct signaling pathways and promoted platelet-monocyte communication through the engagement of P selectin/PGSL-1 and CD40L/CD40, which led to proinflammatory cytokine production by monocytes. These results explain why hypercoagulation, monocyte activation, and a cytokine storm are correlated in patients severely affected by COVID-19 and suggest a potential target for therapeutic intervention.


Subject(s)
Blood Platelets/physiology , COVID-19/blood , Inflammation/blood , Monocytes/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/physiology , Blood Platelets/metabolism , CD40 Antigens/blood , CD40 Ligand/blood , Cell Communication , Cytokine Release Syndrome , Cytokines , HEK293 Cells , Humans , P-Selectin/blood
4.
Molecules ; 27(3)2022 Jan 26.
Article in English | MEDLINE | ID: covidwho-1650788

ABSTRACT

The entry of the SARS-CoV-2, a causative agent of COVID-19, into human host cells is mediated by the SARS-CoV-2 spike (S) glycoprotein, which critically depends on the formation of complexes involving the spike protein receptor-binding domain (RBD) and the human cellular membrane receptor angiotensin-converting enzyme 2 (hACE2). Using classical site density functional theory (SDFT) and structural bioinformatics methods, we investigate binding and conformational properties of these complexes and study the overlooked role of water-mediated interactions. Analysis of the three-dimensional reference interaction site model (3DRISM) of SDFT indicates that water mediated interactions in the form of additional water bridges strongly increases the binding between SARS-CoV-2 spike protein and hACE2 compared to SARS-CoV-1-hACE2 complex. By analyzing structures of SARS-CoV-2 and SARS-CoV-1, we find that the homotrimer SARS-CoV-2 S receptor-binding domain (RBD) has expanded in size, indicating large conformational change relative to SARS-CoV-1 S protein. Protomer with the up-conformational form of RBD, which binds with hACE2, exhibits stronger intermolecular interactions at the RBD-ACE2 interface, with differential distributions and the inclusion of specific H-bonds in the CoV-2 complex. Further interface analysis has shown that interfacial water promotes and stabilizes the formation of CoV-2/hACE2 complex. This interaction causes a significant structural rigidification of the spike protein, favoring proteolytic processing of the S protein for the fusion of the viral and cellular membrane. Moreover, conformational dynamics simulations of RBD motions in SARS-CoV-2 and SARS-CoV-1 point to the role in modification of the RBD dynamics and their impact on infectivity.


Subject(s)
Angiotensin-Converting Enzyme 2/ultrastructure , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , COVID-19/physiopathology , Computational Biology/methods , Density Functional Theory , Humans , Models, Theoretical , Protein Binding , Protein Domains , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/physiology , Structure-Activity Relationship
5.
Viral Immunol ; 34(3): 165-173, 2021 04.
Article in English | MEDLINE | ID: covidwho-1569564

ABSTRACT

The current pandemic is caused by the coronavirus disease 2019 (COVID-19), which is, in turn, induced by a novel coronavirus (SARS-CoV-2) that triggers an acute respiratory disease. In recent years, the emergence of SARS-CoV-2 is the third highly pathogenic event and large-scale epidemic affecting the human population. It follows the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 and the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012. This novel SARS-CoV-2 employs the angiotensin-converting enzyme 2 (ACE2) receptor, like SARS-CoV, and spreads principally in the respiratory tract. The viral spike (S) protein of coronaviruses facilities the attachment to the cellular receptor, entrance, and membrane fusion. The S protein is a glycoprotein and is critical to elicit an immune response. Glycosylation is a biologically significant post-translational modification in virus surface proteins. These glycans play important roles in the viral life cycle, structure, immune evasion, and cell infection. However, it is necessary to search for new information about viral behavior and immunological host's response after SARS-CoV-2 infection. The present review discusses the implications of the CoV-2 S protein glycosylation in the SARS-CoV-2/ACE2 interaction and the immunological response. Elucidation of the glycan repertoire on the spike protein can propel research for the development of an appropriate vaccine.


Subject(s)
Angiotensin-Converting Enzyme 2/physiology , COVID-19/immunology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/physiology , Glycosylation , Humans , SARS-CoV-2/chemistry , SARS-CoV-2/genetics
6.
Nat Methods ; 18(12): 1477-1488, 2021 12.
Article in English | MEDLINE | ID: covidwho-1541247

ABSTRACT

Emergence of new viral agents is driven by evolution of interactions between viral proteins and host targets. For instance, increased infectivity of SARS-CoV-2 compared to SARS-CoV-1 arose in part through rapid evolution along the interface between the spike protein and its human receptor ACE2, leading to increased binding affinity. To facilitate broader exploration of how pathogen-host interactions might impact transmission and virulence in the ongoing COVID-19 pandemic, we performed state-of-the-art interface prediction followed by molecular docking to construct a three-dimensional structural interactome between SARS-CoV-2 and human. We additionally carried out downstream meta-analyses to investigate enrichment of sequence divergence between SARS-CoV-1 and SARS-CoV-2 or human population variants along viral-human protein-interaction interfaces, predict changes in binding affinity by these mutations/variants and further prioritize drug repurposing candidates predicted to competitively bind human targets. We believe this resource ( http://3D-SARS2.yulab.org ) will aid in development and testing of informed hypotheses for SARS-CoV-2 etiology and treatments.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Virus Attachment , Biological Evolution , COVID-19/immunology , Genetic Variation , Humans , Models, Molecular , Molecular Structure , Protein Conformation , Spike Glycoprotein, Coronavirus/physiology
7.
Sci Rep ; 11(1): 22860, 2021 11 24.
Article in English | MEDLINE | ID: covidwho-1532106

ABSTRACT

The worse outcome of COVID-19 in people with diabetes mellitus could be related to the non-enzymatic glycation of human ACE2, leading to a more susceptible interaction with virus Spike protein. We aimed to evaluate, through a computational approach, the interaction between human ACE2 receptor and SARS-CoV-2 Spike protein under different conditions of hyperglycemic environment. A computational analysis was performed, based on the X-ray crystallographic structure of the Spike Receptor-Binding Domain (RBD)-ACE2 system. The possible scenarios of lysine aminoacid residues on surface transformed by glycation were considered: (1) on ACE2 receptor; (2) on Spike protein; (3) on both ACE2 receptor and Spike protein. In comparison to the native condition, the number of polar bonds (comprising both hydrogen bonds and salt bridges) in the poses considered are 10, 6, 6, and 4 for the states ACE2/Spike both native, ACE2 native/Spike glycated, ACE2 glycated/Spike native, ACE2/Spike both glycated, respectively. The analysis highlighted also how the number of non-polar contacts (in this case, van der Waals and aromatic interactions) significantly decreases when the lysine aminoacid residues undergo glycation. Following non-enzymatic glycation, the number of interactions between human ACE2 receptor and SARS-CoV-2 Spike protein is decreased in comparison to the unmodified model. The reduced affinity of the Spike protein for ACE2 receptor in case of non-enzymatic glycation may shift the virus to multiple alternative entry routes.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Hyperglycemia/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/physiology , COVID-19/metabolism , COVID-19/pathology , Computational Biology/methods , Computer Simulation , Humans , Hyperglycemia/immunology , Molecular Dynamics Simulation , Protein Binding , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/physiology
8.
PLoS Comput Biol ; 17(11): e1009560, 2021 11.
Article in English | MEDLINE | ID: covidwho-1523396

ABSTRACT

Severe acute respiratory coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is of zoonotic origin. Evolutionary analyses assessing whether coronaviruses similar to SARS-CoV-2 infected ancestral species of modern-day animal hosts could be useful in identifying additional reservoirs of potentially dangerous coronaviruses. We reasoned that if a clade of species has been repeatedly exposed to a virus, then their proteins relevant for viral entry may exhibit adaptations that affect host susceptibility or response. We perform comparative analyses across the mammalian phylogeny of angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2, in order to uncover evidence for selection acting at its binding interface with the SARS-CoV-2 spike protein. We uncover that in rodents there is evidence for adaptive amino acid substitutions at positions comprising the ACE2-spike interaction interface, whereas the variation within ACE2 proteins in primates and some other mammalian clades is not consistent with evolutionary adaptations. We also analyze aminopeptidase N (APN), the receptor for the human coronavirus 229E, a virus that causes the common cold, and find evidence for adaptation in primates. Altogether, our results suggest that the rodent and primate lineages may have had ancient exposures to viruses similar to SARS-CoV-2 and HCoV-229E, respectively.


Subject(s)
COVID-19/genetics , COVID-19/virology , Coronavirus Infections/genetics , Coronavirus Infections/virology , SARS-CoV-2/genetics , Adaptation, Physiological/genetics , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/physiology , Animals , CD13 Antigens/genetics , CD13 Antigens/physiology , Common Cold/genetics , Common Cold/virology , Computational Biology , Coronavirus 229E, Human/genetics , Coronavirus 229E, Human/physiology , Evolution, Molecular , Genomics , Host Microbial Interactions/genetics , Host Microbial Interactions/physiology , Host Specificity/genetics , Host Specificity/physiology , Humans , Mammals/genetics , Mammals/virology , Phylogeny , Protein Interaction Domains and Motifs/genetics , Receptors, Virus/genetics , Receptors, Virus/physiology , SARS-CoV-2/physiology , Selection, Genetic , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/physiology , Virus Internalization
9.
Viruses ; 13(11)2021 11 03.
Article in English | MEDLINE | ID: covidwho-1502528

ABSTRACT

Men are disproportionately affected by the coronavirus disease-2019 (COVID-19), and face higher odds of severe illness and death compared to women. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial injury are not defined. We determined the effects of SARS-CoV-2 spike protein-mediated endothelial injury under conditions of exposure to androgen dihydrotestosterone (DHT) and tumor necrosis factor-a (TNF-α) and tested potentially therapeutic effects of mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in men and women diagnosed with COVID-19. Exposure of endothelial cells (ECs) in vitro to DHT exacerbated spike protein S1-mediated endothelial injury transcripts for the cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 and anti-fibrinolytic PAI-1 (p < 0.05), and increased THP-1 monocyte adhesion to ECs (p = 0.032). Spironolactone dramatically reduced DHT+S1-induced endothelial activation. TNF-α exacerbated S1-induced EC activation, which was abrogated by pretreatment with spironolactone. Analysis from patients hospitalized with COVID-19 showed concordant higher circulating VCAM-1 and E-Selectin levels in men, compared to women. A beneficial effect of the FDA-approved drug spironolactone was observed on endothelial cells in vitro, supporting a rationale for further evaluation of mineralocorticoid antagonism as an adjunct treatment in COVID-19.


Subject(s)
COVID-19/pathology , Dihydrotestosterone/pharmacology , Endothelium, Vascular/pathology , Inflammation , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/physiology , Spironolactone/pharmacology , Angiotensin Receptor Antagonists/pharmacology , COVID-19/physiopathology , COVID-19/virology , Cell Adhesion Molecules/blood , Cells, Cultured , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Female , Humans , Male , Sex Characteristics , Tumor Necrosis Factor-alpha/pharmacology , Tumor Necrosis Factor-alpha/physiology , Valsartan/pharmacology
10.
Science ; 374(6573): 1353-1360, 2021 Dec 10.
Article in English | MEDLINE | ID: covidwho-1483980

ABSTRACT

The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report the structure, function, and antigenicity of its full-length spike (S) trimer as well as those of the Gamma and Kappa variants, and compare their characteristics with the G614, Alpha, and Beta variants. Delta S can fuse membranes more efficiently at low levels of cellular receptor angiotensin converting enzyme 2 (ACE2), and its pseudotyped viruses infect target cells substantially faster than the other five variants, possibly accounting for its heightened transmissibility. Each variant shows different rearrangement of the antigenic surface of the amino-terminal domain of the S protein but only makes produces changes in the receptor binding domain (RBD), making the RBD a better target for therapeutic antibodies.


Subject(s)
Immune Evasion , Membrane Fusion , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Antibody Affinity , Antigens, Viral/immunology , Cell Line , Epitopes/immunology , Humans , Models, Molecular , Mutation , Protein Conformation , Protein Domains , Protein Multimerization , Receptors, Coronavirus/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/physiology
11.
Viruses ; 13(10)2021 09 30.
Article in English | MEDLINE | ID: covidwho-1444331

ABSTRACT

Both the SARS-CoV-2 pandemic and emergence of variants of concern have highlighted the need for functional antibody assays to monitor the humoral response over time. Antibodies directed against the spike (S) protein of SARS-CoV-2 are an important component of the neutralizing antibody response. In this work, we report that in a subset of patients-despite a decline in total S-specific antibodies-neutralizing antibody titers remain at a similar level for an average of 98 days in longitudinal sampling of a cohort of 59 Hispanic/Latino patients exposed to SARS-CoV-2. Our data suggest that 100% of seroconverting patients make detectable neutralizing antibody responses which can be quantified by a surrogate viral neutralization test. Examination of sera from ten out of the 59 subjects which received mRNA-based vaccination revealed that both IgG titers and neutralizing activity of sera were higher after vaccination compared to a cohort of 21 SARS-CoV-2 naïve subjects. One dose was sufficient for the induction of a neutralizing antibody, but two doses were necessary to reach 100% surrogate virus neutralization in subjects irrespective of previous SARS-CoV-2 natural infection status. Like the pattern observed after natural infection, the total anti-S antibodies titers declined after the second vaccine dose; however, neutralizing activity remained relatively constant for more than 80 days after the first vaccine dose. Furthermore, our data indicates that-compared with mRNA vaccination-natural infection induces a more robust humoral immune response in unexposed subjects. This work is an important contribution to understanding the natural immune response to the novel coronavirus in a population severely impacted by SARS-CoV-2. Furthermore, by comparing the dynamics of the immune response after the natural infection vs. the vaccination, these findings suggest that functional neutralizing antibody tests are more relevant indicators than the presence or absence of binding antibodies.


Subject(s)
Immunity, Humoral/physiology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/physiology , Adult , Aged , Antibodies, Neutralizing/analysis , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , COVID-19/immunology , COVID-19/physiopathology , COVID-19 Vaccines/immunology , Female , Follow-Up Studies , Humans , Immunity, Humoral/genetics , Immunity, Humoral/immunology , Male , Middle Aged , Protein Binding/genetics , Protein Domains/genetics , Puerto Rico/epidemiology , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/immunology , Vaccination
12.
Curr Opin Virol ; 50: 173-182, 2021 10.
Article in English | MEDLINE | ID: covidwho-1415336

ABSTRACT

The COVID-19 (coronavirus disease 2019) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to loss of human life in millions and devastating socio-economic consequences worldwide. The disease has created urgent needs for intervention strategies to control the crisis and meeting these needs requires a deep understanding of the structure-function relationships of viral proteins and relevant host factors. The trimeric spike (S) protein of the virus decorates the viral surface and is an important target for development of diagnostics, therapeutics and vaccines. Rapid progress in the structural biology of SARS-CoV-2 S protein has been made since the early stage of the pandemic, advancing our knowledge on the viral entry process considerably. In this review, we summarize our latest understanding of the structure of the SARS-CoV-2 S protein and discuss the implications for vaccines and therapeutics.


Subject(s)
Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/chemistry , Binding Sites , COVID-19 Vaccines/immunology , Protein Domains , Spike Glycoprotein, Coronavirus/physiology
14.
Int J Biol Sci ; 17(13): 3305-3319, 2021.
Article in English | MEDLINE | ID: covidwho-1372133

ABSTRACT

An inflammatory cytokine storm is considered an important cause of death in severely and critically ill COVID-19 patients, however, the relationship between the SARS-CoV-2 spike (S) protein and the host's inflammatory cytokine storm is not clear. Here, the qPCR results indicated that S protein induced a significantly elevated expression of multiple inflammatory factor mRNAs in peripheral blood mononuclear cells (PBMCs), whereas RS-5645 ((4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5-trimethoxyphenyl)methanone) attenuated the expression of the most inflammatory factor mRNAs. RS-5645 also significantly reduced the cellular ratios of CD45+/IFNγ+, CD3+/IFNγ+, CD11b+/IFNγ+, and CD56+/IFNγ+ in human PBMCs. In addition, RS-5645 effectively inhibited the activation of inflammatory cells and reduced inflammatory damage to lung tissue in mice. Sequencing results of 16S rRNA v3+v4 in mouse alveolar lavage fluid showed that there were 494 OTUs overlapping between the alveolar lavage fluid of mice that underwent S protein+ LPS-combined intervention (M) and RS-5645-treated mice (R), while R manifested 64 unique OTUs and M exhibited 610 unique OTUs. In the alveoli of group R mice, the relative abundances of microorganisms belonging to Porphyromonas, Rothia, Streptococcus, and Neisseria increased significantly, while the relative abundances of microorganisms belonging to Psychrobacter, Shimia, and Sporosarcina were significantly diminished. The results of KEGG analysis indicated that the alveolar microbiota of mice in the R group can increase translation and reduce the activity of amino acid metabolism pathways. COG analysis results indicated that the abundance of proteins involved in ribosomal structure and biogenesis related to metabolism was augmented in the alveolar microbiota of the mice in the R group, while the abundance of proteins involved in secondary metabolite biosynthesis was significantly reduced. Therefore, our research results showed that RS-5645 attenuated pulmonary inflammatory cell infiltration and the inflammatory storm induced by the S protein and LPS by modulating the pulmonary microbiota.


Subject(s)
Anti-Inflammatory Agents/pharmacology , COVID-19/immunology , Cytokine Release Syndrome/prevention & control , Lipopolysaccharides/pharmacology , Lung/microbiology , Microbiota/drug effects , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/physiology , Animals , Antigens, CD/immunology , COVID-19/virology , Cytokine Release Syndrome/immunology , Disease Models, Animal , Humans , Interferon-gamma/immunology , Male , Mice , Mice, Inbred BALB C
15.
Arch Pharm (Weinheim) ; 354(11): e2100160, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1370365

ABSTRACT

Boswellic acids (BAs) have been shown to possess antiviral activity. Using bioinformatic methods, it was tested whether or not acetyl-11-keto-ß-boswellic acid (AKBA), 11-keto-ß-boswellic acid (KBA), ß-boswellic acid (BBA), and the phosphorylated active metabolite of Remdesivir® (RGS-P3) bind to functional proteins of SARS-CoV-2, that is, the replicase polyprotein P0DTD1, the spike glycoprotein P0DTC2, and the nucleoprotein P0DTC9. Using P0DTD1, AKBA and KBA showed micromolar binding affinity to the RNA-dependent RNA polymerase (RdRp) and to the main proteinase complex Mpro . Phosphorylated BAs even bond in the nanomolar range. Due to their positive and negative charges, BAs and RGS-P3 bond to corresponding negative and positive areas of the protein. BAs and RGS-P3 docked in the tunnel-like cavity of RdRp. BAs also docked into the elongated surface rim of viral Mpro . In both cases, binding occurred with active site amino acids in the lower micromolecular to upper nanomolar range. KBA, BBA, and RGS-P3 also bond to P0DTC2 and P0DTC9. The binding energies for BAs were in the range of -5.8 to -6.3 kcal/mol. RGS-P3 and BAs occluded the centrally located pore of the donut-like protein structure of P0DTC9 and, in the case of P0DTC2, RGS-P3 and BAs impacted the double-wing-like protein structure. The data of this bioinformatics study clearly show that BAs bind to three functional proteins of the SARS-CoV-2 virus responsible for adhesion and replication, as does RGS-P3, a drug on the market to treat this disease. The binding effectiveness of BAs can be increased through phosphate esterification. Whether or not BAs are druggable against the SARS-CoV-2 disease remains to be established.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , COVID-19 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/physiology , Triterpenes/pharmacology , Viral Proteins/physiology , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antiviral Agents/pharmacology , Binding Sites/physiology , Boswellia , COVID-19/drug therapy , COVID-19/virology , Computational Biology/methods , Humans , Molecular Docking Simulation , Nucleoproteins/metabolism , Polyproteins/metabolism , Prodrugs/pharmacology , Protein Binding/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Structure-Activity Relationship
16.
J Med Virol ; 93(9): 5638-5643, 2021 09.
Article in English | MEDLINE | ID: covidwho-1363682

ABSTRACT

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have emerged, posing a renewed threat to coronavirus disease 2019 containment and to vaccine and drug efficacy. In this study, we analyzed more than 1,000,000 SARS-CoV-2 genomic sequences deposited up to April 27, 2021, on the GISAID public repository, and identified a novel T478K mutation located on the SARS-CoV-2 Spike protein. The mutation is structurally located in the region of interaction with human receptor ACE2 and was detected in 11,435 distinct cases. We show that T478K has appeared and risen in frequency since January 2021, predominantly in Mexico and the United States, but we could also detect it in several European countries.


Subject(s)
COVID-19/virology , Genome, Viral , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/physiology , Europe , Humans , Mexico , Mutation , Phylogeny , Spike Glycoprotein, Coronavirus/genetics , United States
17.
Virology ; 562: 142-148, 2021 10.
Article in English | MEDLINE | ID: covidwho-1331288

ABSTRACT

SARS-CoV, MERS-CoV, and potentially SARS-CoV-2 emerged as novel human coronaviruses following cross-species transmission from animal hosts. Although the receptor binding characteristics of human coronaviruses are well documented, the role of carbohydrate binding in addition to recognition of proteinaceous receptors has not been fully explored. Using natural glycan microarray technology, we identified N-glycans in the human lung that are recognized by various human and animal coronaviruses. All viruses tested, including SARS-CoV-2, bound strongly to a range of phosphorylated, high mannose N-glycans and to a very specific set of sialylated structures. Examination of two linked strains, human CoV OC43 and bovine CoV Mebus, reveals shared binding to the sialic acid form Neu5Gc (not found in humans), supporting the evidence for cross-species transmission of the bovine strain. Our findings, revealing robust recognition of lung glycans, suggest that these receptors could play a role in the initial stages of coronavirus attachment and entry.


Subject(s)
COVID-19/virology , Host Microbial Interactions/physiology , Middle East Respiratory Syndrome Coronavirus/metabolism , Polysaccharides/metabolism , SARS-CoV-2/metabolism , Animals , Cattle , Humans , Lung/metabolism , Mannose/chemistry , Middle East Respiratory Syndrome Coronavirus/physiology , N-Acetylneuraminic Acid/chemistry , Phosphorylation , Protein Array Analysis , Protein Binding , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/physiology
18.
PLoS Pathog ; 17(7): e1009715, 2021 07.
Article in English | MEDLINE | ID: covidwho-1315897

ABSTRACT

SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/etiology , SARS Virus/physiology , SARS-CoV-2/physiology , Severe Acute Respiratory Syndrome/etiology , Spike Glycoprotein, Coronavirus/physiology , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/genetics , COVID-19/virology , HEK293 Cells , Humans , Mutation, Missense , Severe Acute Respiratory Syndrome/genetics , Severe Acute Respiratory Syndrome/virology
19.
PLoS Pathog ; 17(7): e1009706, 2021 07.
Article in English | MEDLINE | ID: covidwho-1305581

ABSTRACT

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.


Subject(s)
COVID-19/drug therapy , COVID-19/virology , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Virus Internalization/drug effects , Ammonium Chloride/pharmacology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/physiology , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , Chloroquine/pharmacology , Clathrin/metabolism , Drug Synergism , Endocytosis/drug effects , Endocytosis/physiology , Endosomes/drug effects , Endosomes/metabolism , Humans , Hydrogen-Ion Concentration/drug effects , Hydroxychloroquine/administration & dosage , Macrolides/pharmacology , Niclosamide/administration & dosage , Niclosamide/pharmacology , Protein Binding/drug effects , Protein Domains , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/physiology , Vero Cells
20.
J Med Virol ; 93(9): 5638-5643, 2021 09.
Article in English | MEDLINE | ID: covidwho-1293241

ABSTRACT

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have emerged, posing a renewed threat to coronavirus disease 2019 containment and to vaccine and drug efficacy. In this study, we analyzed more than 1,000,000 SARS-CoV-2 genomic sequences deposited up to April 27, 2021, on the GISAID public repository, and identified a novel T478K mutation located on the SARS-CoV-2 Spike protein. The mutation is structurally located in the region of interaction with human receptor ACE2 and was detected in 11,435 distinct cases. We show that T478K has appeared and risen in frequency since January 2021, predominantly in Mexico and the United States, but we could also detect it in several European countries.


Subject(s)
COVID-19/virology , Genome, Viral , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/physiology , Europe , Humans , Mexico , Mutation , Phylogeny , Spike Glycoprotein, Coronavirus/genetics , United States
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