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1.
J Phys Chem Lett ; 12(51): 12249-12255, 2021 Dec 30.
Article in English | MEDLINE | ID: covidwho-1586057

ABSTRACT

SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratory's refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics that inhibit coronavirus budding.


Subject(s)
Coronavirus Envelope Proteins/metabolism , Molecular Dynamics Simulation , SARS-CoV-2/physiology , Viral Matrix Proteins/metabolism , COVID-19/pathology , COVID-19/virology , Coronavirus Envelope Proteins/chemistry , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Humans , Protein Multimerization , Protein Transport , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/chemistry
2.
Curr Res Transl Med ; 69(4): 103309, 2021 10.
Article in English | MEDLINE | ID: covidwho-1459004

ABSTRACT

PURPOSE OF THE STUDY: Currently no treatment has been proven to be efficacious for patients with early symptoms of COVID-19. Although most patients present mild or moderate symptoms, up to 5-10% may have a poor disease progression, so there is an urgent need for effective drugs, which can be administered even before the onset of severe symptoms, i.e. when the course of the disease is modifiable. Recently, promising results of several studies on oral ivermectin have been published, which has prompted us to conduct the present review of the scientific literature. METHODS: A narrative review has been carried out, focusing on the following four main topics: a) short-term efficacy in the treatment of the disease, b) long-term efficacy in the treatment of patients with post-acute symptoms of COVID-19, c) efficacy in the prophylaxis of the disease, and c) safety of ivermectin. RESULTS: The reviewed literature suggests that there seems to be sufficient evidence about the safety of oral ivermectin, as well as the efficacy of the drug in the early-treatment and the prophylaxis of COVID-19. CONCLUSIONS: In the view of the available evidence, the Frontline COVID-19 Critical Care Alliance (FLCCC) recommends the use of oral ivermectin for both prophylaxis and early-treatment of COVID-19. Further well-designed studies should be conducted in order to explore the efficacy and safety of invermectin at low and high doses, following different dosing schedules, in both, the short and long-term treatment.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Drug Repositioning , Ivermectin/therapeutic use , SARS-CoV-2/drug effects , Antiviral Agents/adverse effects , COVID-19/prevention & control , Case-Control Studies , Dose-Response Relationship, Drug , Humans , Ivermectin/administration & dosage , Ivermectin/adverse effects , Ivermectin/pharmacology , Meta-Analysis as Topic , Multicenter Studies as Topic , Practice Guidelines as Topic , Protein Transport/drug effects , RNA Viruses/drug effects , Randomized Controlled Trials as Topic , Time Factors , Treatment Outcome
4.
Int J Mol Sci ; 22(10)2021 May 17.
Article in English | MEDLINE | ID: covidwho-1383880

ABSTRACT

Numerous viruses hijack cellular protein trafficking pathways to mediate cell entry or to rearrange membrane structures thereby promoting viral replication and antagonizing the immune response. Adaptor protein complexes (AP), which mediate protein sorting in endocytic and secretory transport pathways, are one of the conserved viral targets with many viruses possessing AP-interacting motifs. We present here different mechanisms of viral interference with AP complexes and the functional consequences that allow for efficient viral propagation and evasion of host immune defense. The ubiquity of this phenomenon is evidenced by the fact that there are representatives for AP interference in all major viral families, covered in this review. The best described examples are interactions of human immunodeficiency virus and human herpesviruses with AP complexes. Several other viruses, like Ebola, Nipah, and SARS-CoV-2, are pointed out as high priority disease-causative agents supporting the need for deeper understanding of virus-AP interplay which can be exploited in the design of novel antiviral therapies.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , HIV-1/metabolism , Herpesviridae/metabolism , SARS-CoV-2/metabolism , Ebolavirus/metabolism , Endocytosis , Humans , Nipah Virus/metabolism , Protein Transport , Virus Release , Virus Replication
5.
J Biol Chem ; 295(36): 12686-12696, 2020 09 04.
Article in English | MEDLINE | ID: covidwho-1387615

ABSTRACT

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.


Subject(s)
Epithelial Cells/metabolism , Membrane Proteins/metabolism , Proteolysis , Serine Proteases/metabolism , A549 Cells , Cells, Cultured , HEK293 Cells , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutation , Protein Domains , Protein Transport , Respiratory Mucosa/cytology , Serine Proteases/chemistry , Serine Proteases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Trypsin/metabolism
6.
Int J Mol Sci ; 21(19)2020 Oct 01.
Article in English | MEDLINE | ID: covidwho-1389385

ABSTRACT

Tom70 is a versatile adaptor protein of 70 kDa anchored in the outer membrane of mitochondria in metazoa, fungi and amoeba. The tertiary structure was resolved for the Tom70 of yeast, showing 26 α-helices, most of them participating in the formation of 11 tetratricopeptide repeat (TPR) motifs. Tom70 serves as a docking site for cytosolic chaperone proteins and co-chaperones and is thereby involved in the uptake of newly synthesized chaperone-bound proteins in mitochondrial biogenesis. In yeast, Tom70 additionally mediates ER-mitochondria contacts via binding to sterol transporter Lam6/Ltc1. In mammalian cells, TOM70 promotes endoplasmic reticulum (ER) to mitochondria Ca2+ transfer by association with the inositol-1,4,5-triphosphate receptor type 3 (IP3R3). TOM70 is specifically targeted by the Bcl-2-related protein MCL-1 that acts as an anti-apoptotic protein in macrophages infected by intracellular pathogens, but also in many cancer cells. By participating in the recruitment of PINK1 and the E3 ubiquitin ligase Parkin, TOM70 can be implicated in the development of Parkinson's disease. TOM70 acts as receptor of the mitochondrial antiviral-signaling protein (MAVS) and thereby participates in the corresponding system of innate immunity against viral infections. The protein encoded by Orf9b in the genome of SARS-CoV-2 binds to TOM70, probably compromising the synthesis of type I interferons.


Subject(s)
Immunity, Innate , Mitochondrial Membrane Transport Proteins/chemistry , Animals , Betacoronavirus/genetics , Binding Sites , Humans , Mitochondrial Membrane Transport Proteins/metabolism , Open Reading Frames , Protein Binding , Protein Transport , SARS-CoV-2
7.
J Gen Virol ; 102(8)2021 08.
Article in English | MEDLINE | ID: covidwho-1368372

ABSTRACT

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein's surface. Here we used IBV propagated in embryonated hens' eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.


Subject(s)
Coronavirus/physiology , Polysaccharides/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Alkaloids/chemistry , Alkaloids/pharmacology , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Chromatography, Liquid , Computational Biology/methods , Coronavirus/drug effects , Coronavirus Infections/veterinary , Gene Expression Regulation, Viral , Glycosylation/drug effects , Infectious bronchitis virus/physiology , Models, Molecular , Molecular Conformation , Molecular Weight , Neutralization Tests , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Polysaccharides/chemistry , Poultry Diseases/virology , Protein Transport , Spectrometry, Mass, Electrospray Ionization , Spike Glycoprotein, Coronavirus/genetics , Structure-Activity Relationship , Virus Replication/drug effects
8.
Med Hypotheses ; 155: 110662, 2021 10.
Article in English | MEDLINE | ID: covidwho-1347760
9.
Viruses ; 13(8)2021 07 29.
Article in English | MEDLINE | ID: covidwho-1335227

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.


Subject(s)
COVID-19/virology , Methyltransferases/metabolism , RNA Caps/genetics , RNA, Viral/genetics , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Antibodies, Monoclonal/analysis , Humans , Methyltransferases/analysis , Methyltransferases/genetics , Protein Transport , RNA Caps/metabolism , RNA, Viral/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Viral Nonstructural Proteins/analysis , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/analysis , Viral Regulatory and Accessory Proteins/genetics
10.
Nat Cell Biol ; 23(8): 846-858, 2021 08.
Article in English | MEDLINE | ID: covidwho-1309445

ABSTRACT

The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity.


Subject(s)
Autophagy-Related Proteins/metabolism , Cell Membrane/metabolism , Membrane Proteins/metabolism , Vesicular Transport Proteins/metabolism , Autophagosomes/metabolism , Autophagy-Related Proteins/genetics , HEK293 Cells , HeLa Cells , Humans , Immunoblotting , Immunoprecipitation , Membrane Proteins/genetics , Microscopy, Confocal , Protein Transport/physiology , Vesicular Transport Proteins/genetics
11.
J Biol Chem ; 297(2): 100940, 2021 08.
Article in English | MEDLINE | ID: covidwho-1293905

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 envelope protein (S2-E) is a conserved membrane protein that is important for coronavirus (CoV) assembly and budding. Here, we describe the recombinant expression and purification of S2-E in amphipol-class amphipathic polymer solutions, which solubilize and stabilize membrane proteins, but do not disrupt membranes. We found that amphipol delivery of S2-E to preformed planar bilayers results in spontaneous membrane integration and formation of viroporin cation channels. Amphipol delivery of the S2-E protein to human cells results in plasma membrane integration, followed by retrograde trafficking to the trans-Golgi network and accumulation in swollen perinuclear lysosomal-associated membrane protein 1-positive vesicles, likely lysosomes. CoV envelope proteins have previously been proposed to manipulate the luminal pH of the trans-Golgi network, which serves as an accumulation station for progeny CoV particles prior to cellular egress via lysosomes. Delivery of S2-E to cells will enable chemical biological approaches for future studies of severe acute respiratory syndrome coronavirus 2 pathogenesis and possibly even development of "Trojan horse" antiviral therapies. Finally, this work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.


Subject(s)
Cell Membrane/drug effects , Coronavirus Envelope Proteins/metabolism , Polymers/pharmacology , Propylamines/pharmacology , Surface-Active Agents/pharmacology , trans-Golgi Network/metabolism , Cell Membrane/metabolism , Coronavirus Envelope Proteins/genetics , HeLa Cells , Humans , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Lysosomes/metabolism , Polymers/chemistry , Propylamines/chemistry , Protein Transport , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Surface-Active Agents/chemistry
12.
Pharmacol Res Perspect ; 9(4): e00798, 2021 08.
Article in English | MEDLINE | ID: covidwho-1269136

ABSTRACT

Therapeutic regimens for the COVID-19 pandemics remain unmet. In this line, repurposing of existing drugs against known or predicted SARS-CoV-2 protein actions have been advanced, while natural products have also been tested. Here, we propose that p-cymene, a natural monoterpene, can act as a potential novel agent for the treatment of SARS-CoV-2-induced COVID-19 and other RNA-virus-induced diseases (influenza, rabies, Ebola). We show by extensive molecular simulations that SARS-CoV-2 C-terminal structured domain contains a nuclear localization signal (NLS), like SARS-CoV, on which p-cymene binds with low micromolar affinity, impairing nuclear translocation of this protein and inhibiting viral replication, as verified by preliminary in vitro experiments. A similar mechanism may occur in other RNA-viruses (influenza, rabies and Ebola), also verified in vitro for influenza, by interaction of p-cymene with viral nucleoproteins, and structural modification of their NLS site, weakening its interaction with importin A. This common mechanism of action renders therefore p-cymene as a possible antiviral, alone, or in combination with other agents, in a broad spectrum of RNA viruses, from SARS-CoV-2 to influenza A infections.


Subject(s)
Antiviral Agents/pharmacology , Cymenes/pharmacology , Influenza A Virus, H1N1 Subtype/physiology , Nucleocapsid Proteins/metabolism , SARS-CoV-2/physiology , Animals , Antiviral Agents/chemistry , Cell Nucleus/metabolism , Cell Nucleus/virology , Chlorocebus aethiops , Cymenes/chemistry , Dogs , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Madin Darby Canine Kidney Cells , Models, Molecular , Molecular Dynamics Simulation , Nuclear Localization Signals , Nucleocapsid Proteins/chemistry , Protein Conformation , Protein Domains , Protein Transport , SARS-CoV-2/drug effects , Vero Cells , Virus Replication/drug effects
13.
Blood ; 138(4): 344-349, 2021 07 29.
Article in English | MEDLINE | ID: covidwho-1255893

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with the hypercoagulable state. Tissue factor (TF) is the primary cellular initiator of coagulation. Most of the TF expressed on cell surfaces remains cryptic. Sphingomyelin (SM) is responsible for maintaining TF in the encrypted state, and hydrolysis of SM by acid sphingomyelinase (ASMase) increases TF activity. ASMase was shown to play a role in virus infection biology. In the present study, we investigated the role of ASMase in SARS-CoV-2 infection-induced TF procoagulant activity. Infection of human monocyte-derived macrophages (MDMs) with SARS-CoV-2 spike protein pseudovirus (SARS-CoV-2-SP-PV) markedly increased TF procoagulant activity at the cell surface and released TF+ extracellular vesicles. The pseudovirus infection did not increase either TF protein expression or phosphatidylserine externalization. SARS-CoV-2-SP-PV infection induced the translocation of ASMase to the outer leaflet of the plasma membrane, which led to the hydrolysis of SM in the membrane. Pharmacologic inhibitors or genetic silencing of ASMase attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Inhibition of the SARS-CoV-2 receptor, angiotensin-converting enzyme-2, attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Overall, our data suggest that SARS-CoV-2 infection activates the coagulation by decrypting TF through activation of ASMase. Our data suggest that the US Food and Drug Administration-approved functional inhibitors of ASMase may help treat hypercoagulability in patients with COVID-19.


Subject(s)
COVID-19/blood , Macrophages/virology , Membrane Proteins/physiology , SARS-CoV-2 , Sphingomyelin Phosphodiesterase/physiology , Spike Glycoprotein, Coronavirus/physiology , Thrombophilia/etiology , Thromboplastin/physiology , Angiotensin-Converting Enzyme 2/physiology , COVID-19/complications , Cell-Derived Microparticles , Enzyme Activation , Humans , Hydrolysis , Macrophages/enzymology , Molecular Targeted Therapy , Plasmids , Protein Transport , RNA Interference , RNA, Small Interfering/genetics , Receptors, Virus/physiology , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Sphingomyelins/physiology , Thrombophilia/blood , Thrombophilia/drug therapy , Thrombophilia/enzymology
15.
Biochem Biophys Res Commun ; 529(2): 251-256, 2020 08 20.
Article in English | MEDLINE | ID: covidwho-1220683

ABSTRACT

The nucleocapsid protein is significant in the formation of viral RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), accounting for the largest proportion of viral structural proteins. Here, we report for the first time that the 11S proteasomal activator PA28γ regulates the intracellular abundance of the SARS-CoV-2 N protein (nCoV N). Furthermore, we have identified proteasome activator PA28γ as a nCoV N binding protein by co-immunoprecipitation assay. As a result of their interaction, nCoV N could be degraded by PA28γ-20S in vitro degradation assay. This was also demonstrated by blocking de novo protein synthesis with cycloheximide. The stability of nCoV N in PA28γ-knockout cells was greater than in PA28γ-wildtype cells. Notably, immunofluorescence staining revealed that knockout of the PA28γ gene in cells led to the transport of nCoV N from the nucleus to the cytoplasm. Overexpression of PA28γ enhanced proteolysis of nCoV N compared to that in PA28γ-N151Y cells containing a dominant-negative PA28γ mutation, which reduced this process. These results suggest that PA28γ binding is important in regulating 20S proteasome activity, which in turn regulates levels of the critical nCoV N nucleocapsid protein of SARS-CoV-2, furthering our understanding of the pathogenesis of COVID-19.


Subject(s)
Autoantigens/metabolism , Betacoronavirus/metabolism , Coronavirus Infections/metabolism , Nucleocapsid Proteins/metabolism , Pneumonia, Viral/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , COVID-19 , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , HEK293 Cells , Humans , In Vitro Techniques , Pandemics , Phosphoproteins , Pneumonia, Viral/virology , Protein Binding , Protein Stability , Protein Transport , SARS-CoV-2
16.
IUBMB Life ; 73(6): 843-854, 2021 06.
Article in English | MEDLINE | ID: covidwho-1219298

ABSTRACT

The 78 kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum (ER)-resident molecular chaperone. GRP78 is a member of the 70 kDa heat shock family of proteins involved in correcting and clearing misfolded proteins in the ER. In response to cellular stress, GRP78 escapes from the ER and moves to the plasma membrane where it (a) functions as a receptor for many ligands, and (b) behaves as an autoantigen for autoantibodies that contribute to human disease and cancer. Cell surface GRP78 (csGRP78) associates with the major histocompatibility complex class I (MHC-I), and is the port of entry for several viruses, including the predictive binding of the novel SARS-CoV-2. Furthermore, csGRP78 is found in association with partners as diverse as the teratocarcinoma-derived growth factor 1 (Cripto), the melanocortin-4 receptor (MC4R) and the DnaJ-like protein MTJ-1. CsGRP78 also serves as a receptor for a large variety of ligands including activated α2 -macroglobulin (α2 M*), plasminogen kringle 5 (K5), microplasminogen, the voltage-dependent anion channel (VDAC), tissue factor (TF), and the prostate apoptosis response-4 protein (Par-4). In this review, we discuss the mechanisms involved in the translocation of GRP78 from the ER to the cell surface, and the role of secreted GRP78 and its autoantibodies in cancer and neurological disorders.


Subject(s)
Autoimmune Diseases of the Nervous System/immunology , COVID-19/transmission , Heat-Shock Proteins/physiology , Neoplasm Proteins/physiology , Nerve Tissue Proteins/physiology , Receptors, Cell Surface/physiology , Receptors, Virus/physiology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Autoantibodies/immunology , Autoantigens/immunology , Autoimmune Diseases of the Nervous System/metabolism , Cell Survival , Endoplasmic Reticulum Stress/physiology , Exosomes , GPI-Linked Proteins/metabolism , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/immunology , Humans , Ligands , Neoplasm Invasiveness , Neoplasm Proteins/immunology , Nerve Tissue Proteins/immunology , Protein Domains , Protein Transport , Signal Transduction , Tumor Microenvironment , Unfolded Protein Response/physiology , Virus Internalization
17.
Int J Biol Sci ; 17(6): 1547-1554, 2021.
Article in English | MEDLINE | ID: covidwho-1206441

ABSTRACT

Suppression of type I interferon (IFN) response is one pathological outcome of the infection of highly pathogenic human coronaviruses. To effect this, severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 encode multiple IFN antagonists. In this study, we reported on the IFN antagonism of SARS-CoV-2 main protease NSP5. NSP5 proteins of both SARS-CoV and SARS-CoV-2 counteracted Sendai virus-induced IFN production. NSP5 variants G15S and K90R commonly seen in circulating strains of SARS-CoV-2 retained the IFN-antagonizing property. The suppressive effect of NSP5 on IFN-ß gene transcription induced by RIG-I, MAVS, TBK1 and IKKϵ suggested that NSP5 likely acts at a step downstream of IRF3 phosphorylation in the cytoplasm. NSP5 did not influence steady-state expression or phosphorylation of IRF3, suggesting that IRF3, regardless of its phosphorylation state, might not be the substrate of NSP5 protease. However, nuclear translocation of phosphorylated IRF3 was severely compromised in NSP5-expressing cells. Taken together, our work revealed a new mechanism by which NSP5 proteins encoded by SARS-CoV and SARS-CoV-2 antagonize IFN production by retaining phosphorylated IRF3 in the cytoplasm. Our findings have implications in rational design and development of antiviral agents against SARS-CoV-2.


Subject(s)
Cell Nucleus/metabolism , Coronavirus 3C Proteases/metabolism , Interferon Regulatory Factor-3/metabolism , Interferon Type I/biosynthesis , SARS-CoV-2/enzymology , Animals , COVID-19/virology , Chlorocebus aethiops , Humans , Phosphorylation , Protein Transport , Vero Cells
18.
Biochim Biophys Acta Biomembr ; 1863(6): 183590, 2021 06 01.
Article in English | MEDLINE | ID: covidwho-1188312

ABSTRACT

The envelope protein E of the SARS-CoV coronavirus is an archetype of viroporin. It is a small hydrophobic protein displaying ion channel activity that has proven highly relevant in virus-host interaction and virulence. Ion transport through E channel was shown to alter Ca2+ homeostasis in the cell and trigger inflammation processes. Here, we study transport properties of the E viroporin in mixed solutions of potassium and calcium chloride that contain a fixed total concentration (mole fraction experiments). The channel is reconstituted in planar membranes of different lipid compositions, including a lipid mixture that mimics the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane where the virus localizes within the cell. We find that the E ion conductance changes non-monotonically with the total ionic concentration displaying an Anomalous Mole Fraction Effect (AMFE) only when charged lipids are present in the membrane. We also observe that E channel insertion in ERGIC-mimic membranes - including lipid with intrinsic negative curvature - enhances ion permeation at physiological concentrations of pure CaCl2 or KCl solutions, with a preferential transport of Ca2+ in mixed KCl-CaCl2 solutions. Altogether, our findings demonstrate that the presence of calcium modulates the transport properties of the E channel by interacting preferentially with charged lipids through different mechanisms including direct Coulombic interactions and possibly inducing changes in membrane morphology.


Subject(s)
Calcium/metabolism , SARS Virus/metabolism , Viroporin Proteins/metabolism , Amino Acid Sequence , Calcium Channels/metabolism , Ion Transport , Membrane Lipids/metabolism , Protein Binding , Protein Transport , Solutions , Viroporin Proteins/chemistry
19.
Front Immunol ; 12: 663586, 2021.
Article in English | MEDLINE | ID: covidwho-1190318

ABSTRACT

As of January 2021, SARS-CoV-2 has killed over 2 million individuals across the world. As such, there is an urgent need for vaccines and therapeutics to reduce the burden of COVID-19. Several vaccines, including mRNA, vector-based vaccines, and inactivated vaccines, have been approved for emergency use in various countries. However, the slow roll-out of vaccines and insufficient global supply remains a challenge to turn the tide of the pandemic. Moreover, vaccines are important tools for preventing the disease but therapeutic tools to treat patients are also needed. As such, since the beginning of the pandemic, repurposed FDA-approved drugs have been sought as potential therapeutic options for COVID-19 due to their known safety profiles and potential anti-viral effects. One of these drugs is ivermectin (IVM), an antiparasitic drug created in the 1970s. IVM later exerted antiviral activity against various viruses including SARS-CoV-2. In this review, we delineate the story of how this antiparasitic drug was eventually identified as a potential treatment option for COVID-19. We review SARS-CoV-2 lifecycle, the role of the nucleocapsid protein, the turning points in past research that provided initial 'hints' for IVM's antiviral activity and its molecular mechanism of action- and finally, we culminate with the current clinical findings.


Subject(s)
Active Transport, Cell Nucleus/drug effects , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Ivermectin/therapeutic use , SARS-CoV-2/drug effects , Animals , Cell Line , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Coronavirus Nucleocapsid Proteins/metabolism , Drug Repositioning , Humans , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , Protein Transport/drug effects , SARS-CoV-2/growth & development , Vero Cells , Virus Replication/drug effects , alpha Karyopherins/antagonists & inhibitors , beta Karyopherins/antagonists & inhibitors
20.
Cells ; 10(4)2021 03 28.
Article in English | MEDLINE | ID: covidwho-1154291

ABSTRACT

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by progressive loss of dopaminergic neurons in the substantia nigra, intraneuronal deposition of misfolded proteins known as Lewy bodies, and chronic neuroinflammation. PD can arise from monogenic mutations, but in most cases, the etiology is unclear. Viral infection is gaining increasing attentions as a trigger of PD. In this study, we investigated whether the PD-causative 620 aspartate (D) to asparagine (N) mutation in the vacuolar protein sorting 35 ortholog (Vps35) precipitated herpes simplex virus (HSV) infection. We observed that ectopic expression of Vps35 significantly reduced the proliferation and release of HSV-1 virions; the D620N mutation rendered Vps35 a partial loss of such inhibitory effects. Tetherin is a host cell protein capable of restricting the spread of encapsulated viruses including HSV-1 and SARS-Cov-2, both of which are implicated in the development of parkinsonism. Compared with cells overexpressing wildtype Vps35, cells expressing mutant Vps35 with D620N had less Tetherin on cell surfaces. Real-time and static cell imaging revealed that Tetherin recycled through Vps35-positive endosomes. Expression of Vps35 with D620N reduced endosomal dynamics and frequency of motile Tetherin-containing vesicles, a sign of defective production of recycling carriers. Our study suggests that the D620N mutation in Vps35 hinders Tetherin trafficking to cell surfaces and facilitates virus spread.


Subject(s)
Bone Marrow Stromal Antigen 2/metabolism , Parkinson Disease/metabolism , Parkinson Disease/virology , Simplexvirus/metabolism , Vesicular Transport Proteins/metabolism , COVID-19/virology , Cell Line, Tumor , Endosomes/metabolism , Humans , Mutation , Parkinson Disease/genetics , Protein Transport/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Simplexvirus/pathogenicity , Transfection , Vesicular Transport Proteins/genetics , Virus Replication/genetics
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