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1.
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
2.
Commun Biol ; 4(1): 1389, 2021 12 16.
Article in English | MEDLINE | ID: covidwho-1585764

ABSTRACT

In light of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants potentially undermining humoral immunity, it is important to understand the fine specificity of the antiviral antibodies. We screened 20 COVID-19 patients for antibodies against 9 different SARS-CoV-2 proteins observing responses against the spike (S) proteins, the receptor-binding domain (RBD), and the nucleocapsid (N) protein which were of the IgG1 and IgG3 subtypes. Importantly, mutations which typically occur in the B.1.351 "South African" variant, significantly reduced the binding of anti-RBD antibodies. Nine of 20 patients were critically ill and were considered high-risk (HR). These patients showed significantly higher levels of transforming growth factor beta (TGF-ß) and myeloid-derived suppressor cells (MDSC), and lower levels of CD4+ T cells expressing LAG-3 compared to standard-risk (SR) patients. HR patients evidenced significantly higher anti-S1/RBD IgG antibody levels and an increased neutralizing activity. Importantly, a large proportion of S protein-specific antibodies were glycosylation-dependent and we identified a number of immunodominant linear epitopes within the S1 and N proteins. Findings derived from this study will not only help us to identify the most relevant component of the anti-SARS-CoV-2 humoral immune response but will also enable us to design more meaningful immunomonitoring methods for anti-COVID-19 vaccines.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Viral Proteins/immunology , Adaptive Immunity/immunology , Adult , Aged , COVID-19/virology , COVID-19 Vaccines/immunology , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/metabolism , Female , Humans , Immunity, Humoral/immunology , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Male , Middle Aged , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism
3.
Front Immunol ; 12: 736529, 2021.
Article in English | MEDLINE | ID: covidwho-1515533

ABSTRACT

Various authors have hypothesized carotid body (CB) involvement in Coronavirus Disease 2019 (COVID-19), through direct invasion or indirect effects by systemic stimuli ('cytokine storm', angiotensin-converting enzyme [ACE]1/ACE2 imbalance). However, empirical evidence is limited or partial. Here, we present an integrated histopathological and virological analysis of CBs sampled at autopsy from four subjects (2 males and 2 females; age: >70 years old) who died of COVID-19. Histopathological, immunohistochemical and molecular investigation techniques were employed to characterize Severe Acute Respiratory Syndrome - Coronavirus 2 (SARS-CoV2) viral invasion and inflammatory reaction. SARS-CoV2 RNA was detected in the CBs of three cases through Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR). In these cases, positive immunostaining for Nucleocapsid and Spike protein were also demonstrated, mainly at the level of large roundish cells consistent with type I cells, confirming direct CB invasion. In these cases, T lymphocytes showed focal aggregations in the CBs, suggestive of local inflammatory reaction. Blood congestion and microthrombosis were also found in one of the positive cases. Intriguingly, microthrombosis, blood congestion and microhaemorrages were also bilaterally detected in the CBs of the negative case, supporting the possibility of COVID-19 effects on the CB even in the absence of its direct invasion. SARS-CoV-2 direct invasion of the CB is confirmed through both immunohistochemistry and RT-PCR, with likely involvement of different cell types. We also reported histopathological findings which could be ascribed to local and/or systemic actions of SARS-CoV-2 and which could potentially affect chemoreception.


Subject(s)
COVID-19 , Carotid Body , SARS-CoV-2 , Aged , Autopsy , COVID-19/pathology , COVID-19/virology , Carotid Body/pathology , Carotid Body/virology , Coronavirus Nucleocapsid Proteins/metabolism , Female , Humans , Male , Phosphoproteins/metabolism , RNA, Viral/analysis , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism
4.
Eur J Med Chem ; 227: 113966, 2022 Jan 05.
Article in English | MEDLINE | ID: covidwho-1487705

ABSTRACT

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented in human history. As a major structural protein, nucleocapsid protein (NPro) is critical to the replication of SARS-CoV-2. In this work, 17 NPro-targeting phenanthridine derivatives were rationally designed and synthesized, based on the crystal structure of NPro. Most of these compounds can interact with SARS-CoV-2 NPro tightly and inhibit the replication of SARS-CoV-2 in vitro. Compounds 12 and 16 exhibited the most potent anti-viral activities with 50% effective concentration values of 3.69 and 2.18 µM, respectively. Furthermore, site-directed mutagenesis of NPro and Surface Plasmon Resonance (SPR) assays revealed that 12 and 16 target N-terminal domain (NTD) of NPro by binding to Tyr109. This work found two potent anti-SARS-CoV-2 bioactive compounds and also indicated that SARS-CoV-2 NPro-NTD can be a target for new anti-virus agents.


Subject(s)
Antiviral Agents/chemistry , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Phenanthridines/chemistry , SARS-CoV-2/metabolism , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Cell Survival/drug effects , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/metabolism , Drug Design , Humans , Kinetics , Molecular Docking Simulation , Phenanthridines/metabolism , Phenanthridines/pharmacology , Phenanthridines/therapeutic use , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , Protein Binding , Protein Structure, Tertiary , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Vero Cells
5.
J Virol ; 95(16): e0018721, 2021 07 26.
Article in English | MEDLINE | ID: covidwho-1486048

ABSTRACT

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P < 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S71NWHFYYLGTGPHADLRYRT90 and the interaction between N protein and p53. In the nucleus, the binding of N protein to p53 maintained consistently high-level expression of p53, which activated the p53-DREAM pathway. The key domain of the N protein interacting with p53 was revealed to be S171RGNSQNRGNNQGRGASQNRGGNN194 (NS171-N194), in which G183RG185 are core residues. NS171-N194 and G183RG185 were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the NS171-N194 domain of the PEDV N protein were screened through molecular docking. Hyperoside could antagonize N protein-induced S-phase arrest by interfering with interaction between N protein and p53 and inhibit viral replication (P < 0.05). The above-described experiments were also validated in porcine intestinal cells, and data were in line with results in Vero E6 cells. Therefore, these results reveal the PEDV N protein interacts with p53 to activate the p53-DREAM pathway, and subsequently induces S-phase arrest to create a favorable environment for virus replication. These findings provide new insight into the PEDV-host interaction and the design of novel antiviral strategies against PEDV. IMPORTANCE Many viruses subvert the host cell cycle to create a cellular environment that promotes viral growth. PEDV, an emerging and reemerging coronavirus, has led to substantial economic loss in the global swine industry. Our study is the first to demonstrate that PEDV N-induced cell cycle arrest during the S-phase promotes viral replication. We identified a novel mechanism of PEDV N-induced S-phase arrest, where the binding of PEDV N protein to p53 maintains consistently high levels of p53 expression in the nucleus to mediate S-phase arrest by activating the p53-DREAM pathway. Furthermore, a small molecular compound, hyperoside, targeted the PEDV N protein, interfering with the interaction between the N protein and p53 and, importantly, inhibited PEDV replication by antagonizing cell cycle arrest. This study reveals a new mechanism of PEDV-host interaction and also provides a novel antiviral strategy for PEDV. These data provide a foundation for further research into coronavirus-host interactions.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus Nucleocapsid Proteins/chemistry , Host-Pathogen Interactions/drug effects , Porcine epidemic diarrhea virus/drug effects , Quercetin/analogs & derivatives , Tumor Suppressor Protein p53/chemistry , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , Binding Sites , Cell Line , Chlorocebus aethiops , Coronavirus Infections/drug therapy , Coronavirus Infections/genetics , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Epithelial Cells/drug effects , Epithelial Cells/virology , Gene Expression Regulation , High-Throughput Screening Assays , Host-Pathogen Interactions/genetics , Molecular Docking Simulation , Nuclear Localization Signals , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/metabolism , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Quercetin/chemistry , Quercetin/pharmacology , S Phase Cell Cycle Checkpoints/drug effects , S Phase Cell Cycle Checkpoints/genetics , Signal Transduction , Swine , Swine Diseases/drug therapy , Swine Diseases/genetics , Swine Diseases/metabolism , Swine Diseases/virology , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Vero Cells , Virus Replication/drug effects
6.
Int J Mol Sci ; 22(20)2021 Oct 14.
Article in English | MEDLINE | ID: covidwho-1470888

ABSTRACT

The ongoing COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a globally leading public health concern over the past two years. Despite the development and administration of multiple vaccines, the mutation of newer strains and challenges to universal immunity has shifted the focus to the lack of efficacious drugs for therapeutic intervention for the disease. As with SARS-CoV, MERS-CoV, and other non-respiratory viruses, flavonoids present themselves as a promising therapeutic intervention given their success in silico, in vitro, in vivo, and more recently, in clinical studies. This review focuses on data from in vitro studies analyzing the effects of flavonoids on various key SARS-CoV-2 targets and presents an analysis of the structure-activity relationships for the same. From 27 primary papers, over 69 flavonoids were investigated for their activities against various SARS-CoV-2 targets, ranging from the promising 3C-like protease (3CLpro) to the less explored nucleocapsid (N) protein; the most promising were quercetin and myricetin derivatives, baicalein, baicalin, EGCG, and tannic acid. We further review promising in silico studies featuring activities of flavonoids against SARS-CoV-2 and list ongoing clinical studies involving the therapeutic potential of flavonoid-rich extracts in combination with synthetic drugs or other polyphenols and suggest prospects for the future of flavonoids against SARS-CoV-2.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Flavonoids/therapeutic use , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Coronavirus Nucleocapsid Proteins/metabolism , Flavonoids/chemistry , Flavonoids/pharmacology , Humans , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/physiology , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , Rhinovirus/drug effects , Rhinovirus/physiology , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Virus Internalization/drug effects
7.
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
8.
PLoS Biol ; 19(10): e3001425, 2021 10.
Article in English | MEDLINE | ID: covidwho-1463301

ABSTRACT

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that drives interactions between N proteins in condensates, and deletion of this region disrupts phase separation. We also identified small molecules that alter the size and shape of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , SARS-CoV-2/metabolism , Viral Genome Packaging/physiology , Animals , COVID-19/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Genome, Viral , Genomics , HEK293 Cells , Humans , Nucleocapsid Proteins/genetics , Phosphoproteins/metabolism , Protein Domains , RNA, Viral/genetics , SARS-CoV-2/genetics , Vero Cells
9.
Microbiol Spectr ; 9(2): e0090821, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1452921

ABSTRACT

Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, and, as of yet, none of the currently available broad-spectrum drugs or vaccines can effectively control these diseases. Host antiviral proteins play an important role in inhibiting viral proliferation. One of the isoforms of cytoplasmic poly(A)-binding protein (PABP), PABPC4, is an RNA-processing protein, which plays an important role in promoting gene expression by enhancing translation and mRNA stability. However, its function in viruses remains poorly understood. Here, we report that the host protein, PABPC4, could be regulated by transcription factor SP1 and broadly inhibits the replication of CoVs, covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. PABPC4 recruited the E3 ubiquitin ligase MARCH8/MARCHF8 to the N protein for ubiquitination. Ubiquitinated N protein was recognized by the cargo receptor NDP52/CALCOCO2, which delivered it to the autolysosomes for degradation, resulting in impaired viral proliferation. In addition to regulating gene expression, these data demonstrate a novel antiviral function of PABPC4, which broadly suppresses CoVs by degrading the N protein via the selective autophagy pathway. This study will shed light on the development of broad anticoronaviral therapies. IMPORTANCE Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, but none of the currently available drugs or vaccines can effectively control these diseases. During viral infection, the host will activate the interferon (IFN) signaling pathways and host restriction factors in maintaining the innate antiviral responses and suppressing viral replication. This study demonstrated that the host protein, PABPC4, interacts with the nucleocapsid (N) proteins from eight CoVs covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family. PABPC4 could be regulated by SP1 and broadly inhibits the replication of CoVs by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. This study significantly increases our understanding of the novel host restriction factor PABPC4 against CoV replication and will help develop novel antiviral strategies.


Subject(s)
Autophagy/physiology , Blood Proteins/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus/growth & development , Poly(A)-Binding Proteins/metabolism , Virus Replication/physiology , Animals , Cell Line , Chlorocebus aethiops , HEK293 Cells , Humans , Infectious bronchitis virus/growth & development , Murine hepatitis virus/growth & development , Nuclear Proteins/metabolism , Porcine epidemic diarrhea virus/growth & development , Proteolysis , Sp1 Transcription Factor/metabolism , Swine , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Vero Cells
10.
Proc Natl Acad Sci U S A ; 118(42)2021 10 19.
Article in English | MEDLINE | ID: covidwho-1447424

ABSTRACT

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35-0.74], P = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type-dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.


Subject(s)
COVID-19/prevention & control , Coronavirus Nucleocapsid Proteins/metabolism , Glycogen Synthase Kinase 3/antagonists & inhibitors , Lithium Compounds/therapeutic use , Adult , Aged , Female , Glycogen Synthase Kinase 3/metabolism , HEK293 Cells , Humans , Lithium Compounds/pharmacology , Male , Middle Aged , Molecular Targeted Therapy , Phosphoproteins/metabolism , Phosphorylation/drug effects , Retrospective Studies
11.
Int J Biol Sci ; 17(14): 3889-3897, 2021.
Article in English | MEDLINE | ID: covidwho-1438863

ABSTRACT

Intraviral protein-protein interactions (PPIs) of SARS-CoV-2 in host cells may provide useful information for deep understanding of virology of SARS-CoV-2. In this study, 22 of 55 interactions of the structural and accessory proteins of SARS-CoV-2 were identified by biomolecular fluorescence complementation (BiFC) assay. The nucleocapsid (N) protein was found to have the most interactions among the structural and accessory proteins of SARS-CoV-2, and also specifically interacted with the putative packaging signal (PS) of SARS-CoV-2. We also demonstrated that the PS core containing PS576 RNA bears a functional PS, important for the assembly of the viral RNA into virus like particles (VLPs), and the packaging of SARS-CoV-2 RNA was N dependent.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , SARS-CoV-2/metabolism , Virus Assembly , HEK293 Cells , Humans , Phosphoproteins/metabolism , Protein Interaction Maps
12.
J Virol ; 95(23): e0139621, 2021 11 09.
Article in English | MEDLINE | ID: covidwho-1434896

ABSTRACT

Emerging evidence suggests that endothelial activation plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) and multiorgan failure in patients with coronavirus disease 2019 (COVID-19). However, the molecular mechanisms underlying endothelial activation in COVID-19 patients remain unclear. In this study, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins that potently activate human endothelial cells were screened to elucidate the molecular mechanisms involved in endothelial activation. It was found that nucleocapsid protein (NP) of SARS-CoV-2 significantly activated human endothelial cells through Toll-like receptor 2 (TLR2)/NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Remarkably, though the protein sequences of N proteins from coronaviruses are highly conserved, only NP from SARS-CoV-2 induced endothelial activation. The NPs from other coronaviruses such as SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HUB1-CoV, and influenza virus H1N1 did not activate endothelial cells. These findings are consistent with the results from clinical investigations showing broad endotheliitis and organ injury in severe COVID-19 patients. In conclusion, the study provides insights on SARS-CoV-2-induced vasculopathy and coagulopathy and suggests that simvastatin, an FDA-approved lipid-lowering drug, may help prevent the pathogenesis and improve the outcome of COVID-19 patients. IMPORTANCE Coronavirus disease 2019 (COVID-19), caused by the betacoronavirus SARS-CoV-2, is a worldwide challenge for health care systems. The leading cause of mortality in patients with COVID-19 is hypoxic respiratory failure from acute respiratory distress syndrome (ARDS). To date, pulmonary endothelial cells (ECs) have been largely overlooked as a therapeutic target in COVID-19, yet emerging evidence suggests that these cells contribute to the initiation and propagation of ARDS by altering vessel barrier integrity, promoting a procoagulative state, inducing vascular inflammation and mediating inflammatory cell infiltration. Therefore, a better mechanistic understanding of the vasculature is of utmost importance. In this study, we screened the SARS-CoV-2 viral proteins that potently activate human endothelial cells and found that nucleocapsid protein (NP) significantly activated human endothelial cells through TLR2/NF-κB and MAPK signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Our results provide insights on SARS-CoV-2-induced vasculopathy and coagulopathy, and suggests that simvastatin, an FDA-approved lipid-lowering drug, may benefit to prevent the pathogenesis and improve the outcome of COVID-19 patients.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/virology , SARS-CoV-2 , Signal Transduction , Simvastatin/pharmacology , COVID-19/virology , Cell Line , Human Umbilical Vein Endothelial Cells , Humans , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Toll-Like Receptor 2/metabolism
13.
Glycobiology ; 31(9): 1080-1092, 2021 09 20.
Article in English | MEDLINE | ID: covidwho-1434394

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , Protein Processing, Post-Translational/physiology , SARS-CoV-2/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Coronavirus Nucleocapsid Proteins/chemistry , Glycosylation , HEK293 Cells , Humans , Phosphorylation , SARS-CoV-2/chemistry
14.
Int J Mol Sci ; 22(18)2021 Sep 13.
Article in English | MEDLINE | ID: covidwho-1409702

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic with high infectivity and mortality has caused severe social and economic impacts worldwide. Growing reports of COVID-19 patients with multi-organ damage indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) may also disturb the cardiovascular system. Herein, we used human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) as the in vitro platform to examine the consequence of SARS-CoV2 infection on iCMs. Differentiated iCMs expressed the primary SARS-CoV2 receptor angiotensin-converting enzyme-II (ACE2) and the transmembrane protease serine type 2 (TMPRSS2) receptor suggesting the susceptibility of iCMs to SARS-CoV2. Following the infection of iCMs with SARS-CoV2, the viral nucleocapsid (N) protein was detected in the host cells, demonstrating the successful infection. Bioinformatics analysis revealed that the SARS-CoV2 infection upregulates several inflammation-related genes, including the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The pretreatment of iCMs with TNF-α for 24 h, significantly increased the expression of ACE2 and TMPRSS2, SASR-CoV2 entry receptors. The TNF-α pretreatment enhanced the entry of GFP-expressing SARS-CoV2 pseudovirus into iCMs, and the neutralization of TNF-α ameliorated the TNF-α-enhanced viral entry. Collectively, SARS-CoV2 elevated TNF-α expression, which in turn enhanced the SARS-CoV2 viral entry. Our findings suggest that, TNF-α may participate in the cytokine storm and aggravate the myocardial damage in COVID-19 patients.


Subject(s)
COVID-19/complications , Cardiovascular Diseases/immunology , Cytokine Release Syndrome/immunology , SARS-CoV-2/immunology , Tumor Necrosis Factor-alpha/metabolism , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , Cardiovascular Diseases/virology , Cell Differentiation , Cell Line , Computational Biology , Coronavirus Nucleocapsid Proteins/metabolism , Cytokine Release Syndrome/pathology , Cytokine Release Syndrome/virology , Humans , Induced Pluripotent Stem Cells , Myocardium/cytology , Myocardium/immunology , Myocardium/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/virology , Phosphoproteins/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Up-Regulation/immunology , Virus Internalization/drug effects
15.
mBio ; 12(5): e0131621, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1406604

ABSTRACT

Coronaviruses (CoVs) are emergent pathogens that may cause life-threatening respiratory diseases in humans. Understanding of CoV-host interactions may help to identify novel therapeutic targets. MOV10 is an RNA helicase involved in different steps of cellular RNA metabolism. Both MOV10 antiviral and proviral activities have been described in a limited number of viruses, but this protein has not been previously associated with CoVs. We found that during Middle East respiratory syndrome coronavirus (MERS-CoV) infection, MOV10 aggregated in cytoplasmic structures colocalizing with viral nucleocapsid (N) protein. MOV10-N interaction was confirmed by endogenous MOV10 coimmunoprecipitation, and the presence of other cellular proteins was also detected in MOV10 complexes. MOV10 silencing significantly increased both N protein accumulation and virus titer, with no changes in the accumulation of viral RNAs. Moreover, MOV10 overexpression caused a 10-fold decrease in viral titers. These data indicated that MOV10 has antiviral activity during MERS-CoV infection. We postulated that this activity could be mediated by viral RNA sequestration, and in fact, RNA immunoprecipitation data showed the presence of viral RNAs in the MOV10 cytoplasmic complexes. Expression of wild-type MOV10 or of a MOV10 mutant without helicase activity in MOV10 knockout cell lines, developed by CRISPR-Cas technology, indicated that the helicase activity of MOV10 was required for its antiviral effect. Interestingly MOV10-N interaction was conserved in other mildly or highly pathogenic human CoVs, including the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), although MOV10 antiviral activity was found only in highly pathogenic CoVs, suggesting a potential role of MOV10 in the modulation of human CoVs pathogenesis. IMPORTANCE Coronaviruses (CoVs) are emerging pathogens causing life-threatening diseases in humans. Knowledge of virus-host interactions and viral subversion mechanisms of host pathways is required for the development of effective countermeasures against CoVs. The interaction between cellular RNA helicase MOV10 and nucleocapsid (N) protein from several human CoVs is shown. Using MERS-CoV as a model, we demonstrate that MOV10 has antiviral function, requiring its helicase activity, most likely mediated by viral RNA sequestration in cytoplasmic ribonucleoprotein structures. Furthermore, we found that MOV10 antiviral activity may act only in highly pathogenic human CoVs, suggesting a role for MOV10 in modulating CoVs pathogenesis. The present study uncovers a complex network of viral and cellular RNAs and proteins interaction modulating the antiviral response against CoVs.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus Nucleocapsid Proteins/metabolism , RNA Helicases/metabolism , RNA Helicases/pharmacology , Animals , Blotting, Western , Cell Line, Tumor , Chlorocebus aethiops , Fluorescent Antibody Technique , Humans , Immunoprecipitation , RNA, Viral/metabolism , Vero Cells , Virus Replication/drug effects
16.
Mol Syst Biol ; 17(9): e10079, 2021 09.
Article in English | MEDLINE | ID: covidwho-1406892

ABSTRACT

We modeled 3D structures of all SARS-CoV-2 proteins, generating 2,060 models that span 69% of the viral proteome and provide details not available elsewhere. We found that ˜6% of the proteome mimicked human proteins, while ˜7% was implicated in hijacking mechanisms that reverse post-translational modifications, block host translation, and disable host defenses; a further ˜29% self-assembled into heteromeric states that provided insight into how the viral replication and translation complex forms. To make these 3D models more accessible, we devised a structural coverage map, a novel visualization method to show what is-and is not-known about the 3D structure of the viral proteome. We integrated the coverage map into an accompanying online resource (https://aquaria.ws/covid) that can be used to find and explore models corresponding to the 79 structural states identified in this work. The resulting Aquaria-COVID resource helps scientists use emerging structural data to understand the mechanisms underlying coronavirus infection and draws attention to the 31% of the viral proteome that remains structurally unknown or dark.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Host-Pathogen Interactions/genetics , Protein Processing, Post-Translational , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Transport Systems, Neutral/chemistry , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Binding Sites , COVID-19/genetics , COVID-19/metabolism , COVID-19/virology , Computational Biology/methods , Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Models, Molecular , Molecular Mimicry , Neuropilin-1/chemistry , Neuropilin-1/genetics , Neuropilin-1/metabolism , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Interaction Mapping/methods , Protein Multimerization , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viroporin Proteins/chemistry , Viroporin Proteins/genetics , Viroporin Proteins/metabolism , Virus Replication
17.
Int J Biol Macromol ; 190: 636-648, 2021 Nov 01.
Article in English | MEDLINE | ID: covidwho-1401500

ABSTRACT

SARS-CoV-2 nucleocapsid (N) protein undergoes RNA-induced phase separation (LLPS) and sequesters the host key stress granule (SG) proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and 2 (G3BP1 and G3BP2) to inhibit SG formation. This will allow viral packaging and propagation in host cells. Based on a genomic-guided meta-analysis, here we identify upstream regulatory elements modulating the expression of G3BP1 and G3BP2 (collectively called G3BP1/2). Using this strategy, we have identified FOXA1, YY1, SYK, E2F-1, and TGFBR2 as activators and SIN3A, SRF, and AKT-1 as repressors of G3BP1/2 genes. Panels of the activators and repressors were then used to identify drugs that change their gene expression signatures. Two drugs, imatinib, and decitabine have been identified as putative modulators of G3BP1/2 genes and their regulators, suggesting their role as COVID-19 mitigation agents. Molecular docking analysis suggests that both drugs bind to G3BP1/2 with a much higher affinity than the SARS-CoV-2 N protein. This study reports imatinib and decitabine as candidate drugs against N protein and G3BP1/2 protein.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , COVID-19/drug therapy , Coronavirus Nucleocapsid Proteins/chemistry , DNA Helicases/chemistry , Decitabine/chemistry , Imatinib Mesylate/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Poly-ADP-Ribose Binding Proteins/chemistry , RNA Helicases/chemistry , RNA Recognition Motif Proteins/chemistry , RNA-Binding Proteins/chemistry , SARS-CoV-2/chemistry , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/metabolism , COVID-19/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , DNA Helicases/antagonists & inhibitors , DNA Helicases/metabolism , Decitabine/pharmacology , Drug Delivery Systems , Genomics , Imatinib Mesylate/pharmacology , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Poly-ADP-Ribose Binding Proteins/antagonists & inhibitors , Poly-ADP-Ribose Binding Proteins/metabolism , RNA Helicases/antagonists & inhibitors , RNA Helicases/metabolism , RNA Recognition Motif Proteins/antagonists & inhibitors , RNA Recognition Motif Proteins/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism
18.
Nat Commun ; 12(1): 1936, 2021 03 29.
Article in English | MEDLINE | ID: covidwho-1387331

ABSTRACT

The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.


Subject(s)
Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Binding Sites , COVID-19/virology , Dimerization , Molecular Dynamics Simulation , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Protein Conformation , Protein Domains
19.
Nat Commun ; 12(1): 502, 2021 01 21.
Article in English | MEDLINE | ID: covidwho-1387327

ABSTRACT

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein's central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Viral Matrix Proteins/metabolism , COVID-19/genetics , COVID-19/metabolism , Cell Membrane/virology , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , DNA Helicases/genetics , DNA Helicases/metabolism , Humans , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Protein Binding , Protein Domains , RNA Helicases/genetics , RNA Helicases/metabolism , RNA Recognition Motif Proteins/genetics , RNA Recognition Motif Proteins/metabolism , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics
20.
Mol Cell ; 80(6): 1078-1091.e6, 2020 12 17.
Article in English | MEDLINE | ID: covidwho-1386333

ABSTRACT

We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33°C and 37°C) and reduced at room temperature (22°C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.


Subject(s)
COVID-19/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , Genome, Viral , Nucleocapsid/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/genetics , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/genetics , Drug Evaluation, Preclinical , HEK293 Cells , Humans , Nucleocapsid/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , SARS-CoV-2/genetics , Vero Cells
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