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1.
J Virol ; 97(6): e0054923, 2023 Jun 29.
Article in English | MEDLINE | ID: covidwho-20245375

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) has caused huge economic losses to the global pig industry. The swine enteric coronavirus spike (S) protein recognizes various cell surface molecules to regulate viral infection. In this study, we identified 211 host membrane proteins related to the S1 protein by pulldown combined with liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis. Among these, heat shock protein family A member 5 (HSPA5) was identified through screening as having a specific interaction with the PEDV S protein, and positive regulation of PEDV infection was validated by knockdown and overexpression tests. Further studies verified the role of HSPA5 in viral attachment and internalization. In addition, we found that HSPA5 interacts with S proteins through its nucleotide-binding structural domain (NBD) and that polyclonal antibodies can block viral infection. In detail, HSPA5 was found to be involved in viral trafficking via the endo-/lysosomal pathway. Inhibition of HSPA5 activity during internalization would reduce the subcellular colocalization of PEDV with lysosomes in the endo-/lysosomal pathway. Together, these findings show that HSPA5 is a novel PEDV potential target for the creation of therapeutic drugs. IMPORTANCE PEDV infection causes severe piglet mortality and threatens the global pig industry. However, the complex invasion mechanism of PEDV makes its prevention and control difficult. Here, we determined that HSPA5 is a novel target for PEDV which interacts with its S protein and is involved in viral attachment and internalization, influencing its transport via the endo-/lysosomal pathway. Our work extends knowledge about the relationship between the PEDV S and host proteins and provides a new therapeutic target against PEDV infection.


Subject(s)
Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Animals , Swine , Chlorocebus aethiops , Porcine epidemic diarrhea virus/physiology , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization , Chromatography, Liquid , Tandem Mass Spectrometry , Lysosomes/metabolism , Vero Cells
2.
ACS Sens ; 8(6): 2159-2168, 2023 Jun 23.
Article in English | MEDLINE | ID: covidwho-20245129

ABSTRACT

In addition to efficacious vaccines and antiviral therapeutics, reliable and flexible in-home personal use diagnostics for the detection of viral antigens are needed for effective control of the COVID-19 pandemic. Despite the approval of several PCR-based and affinity-based in-home COVID-19 testing kits, many of them suffer from problems such as a high false-negative rate, long waiting time, and short storage period. Using the enabling one-bead-one-compound (OBOC) combinatorial technology, several peptidic ligands with a nanomolar binding affinity toward the SARS-CoV-2 spike protein (S-protein) were successfully discovered. Taking advantage of the high surface area of porous nanofibers, immobilization of these ligands on nanofibrous membranes allows the development of personal use sensors that can achieve low nanomolar sensitivity in the detection of the S-protein in saliva. This simple biosensor employing naked-eye reading exhibits detection sensitivity comparable to some of the current FDA-approved home detection kits. Furthermore, the ligand used in the biosensor was found to detect the S-protein derived from both the original strain and the Delta variant. The workflow reported here may enable us to rapidly respond to the development of home-based biosensors against future viral outbreaks.


Subject(s)
Biosensing Techniques , COVID-19 , Humans , COVID-19/diagnosis , Spike Glycoprotein, Coronavirus/chemistry , SARS-CoV-2 , Ligands , COVID-19 Testing , Colorimetry , Pandemics , Peptides
3.
Nature ; 619(7969): 403-409, 2023 Jul.
Article in English | MEDLINE | ID: covidwho-20242865

ABSTRACT

The entry of SARS-CoV-2 into host cells depends on the refolding of the virus-encoded spike protein from a prefusion conformation, which is metastable after cleavage, to a lower-energy stable postfusion conformation1,2. This transition overcomes kinetic barriers for fusion of viral and target cell membranes3,4. Here we report a cryogenic electron microscopy (cryo-EM) structure of the intact postfusion spike in a lipid bilayer that represents the single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membrane-interacting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Cryoelectron Microscopy , Lipid Bilayers , Virus Internalization , Membrane Fusion , Protein Conformation
4.
Int J Biol Macromol ; 242(Pt 4): 125190, 2023 Jul 01.
Article in English | MEDLINE | ID: covidwho-20230951

ABSTRACT

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that, because of its broad host range, poses a potential threat to public health. Here, to identify the neutralizing B-cell epitopes within the S1-CTD protein, we generated three anti-PDCoV monoclonal antibodies (mAbs). Of these, the antibody designated 4E-3 effectively neutralized PDCoV with an IC50 of 3.155 µg/mL. mAb 4E-3 and one other, mAb 2A-12, recognized different linear B-cell epitopes. The minimal fragment recognized by mAb 4E-3 was mapped to 280FYSDPKSAV288 and designated S280-288, the minimal fragment recognized by mAb 2A-12 was mapped to 506TENNRFTT513, and designated S506-513. Subsequently, alanine (A)-scanning mutagenesis indicated that Asp283, Lys285, and Val288 were the critical residues recognized by mAb 4E-3. The S280-288 epitope induces PDCoV specific neutralizing antibodies in mice, demonstrating that it is a neutralizing epitope. Of note, the S280-288 coupled to Keyhole Limpet Hemocyanin (KLH) produces PDCoV neutralizing antibodies in vitro and in vivo, in challenged piglets it potentiates interferon-γ responses and provides partial protection against disease. This is the first report about the PDCoV S protein neutralizing epitope, which will contribute to research of PDCoV-related pathogenic mechanism, vaccine design and antiviral drug development.


Subject(s)
Epitopes, B-Lymphocyte , Immunodominant Epitopes , Animals , Swine , Mice , Spike Glycoprotein, Coronavirus/chemistry , Antibodies, Neutralizing
5.
Int J Biol Macromol ; 244: 125182, 2023 Jul 31.
Article in English | MEDLINE | ID: covidwho-20230950

ABSTRACT

The COVID-19 pandemic, caused by SARS-CoV-2, has become a global public health crisis. The entry of SARS-CoV-2 into host cells is facilitated by the binding of its spike protein (S1-RBD) to the host receptor hACE2. Small molecule compounds targeting S1-RBD-hACE2 interaction could provide an alternative therapeutic strategy sensitive to viral mutations. In this study, we identified G7a as a hit compound that targets the S1-RBD-hACE2 interaction, using high-throughput screening in the SARS2-S pseudovirus model. To enhance the antiviral activity of G7a, we designed and synthesized a series of novel 7-azaindole derivatives that bind to the S1-RBD-hACE2 interface. Surprisingly, ASM-7 showed excellent antiviral activity and low cytotoxicity, as confirmed by pseudovirus and native virus assays. Molecular docking and molecular dynamics simulations revealed that ASM-7 could stably bind to the binding interface of S1-RBD-hACE2, forming strong non-covalent interactions with key residues. Furthermore, the binding of ASM-7 caused alterations in the structural dynamics of both S1-RBD and hACE2, resulting in a decrease in their binding affinity and ultimately impeding the viral invasion of host cells. Our findings demonstrate that ASM-7 is a promising lead compound for developing novel therapeutics against SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Molecular Docking Simulation , Spike Glycoprotein, Coronavirus/chemistry , Pandemics , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Protein Binding
6.
Int J Biol Macromol ; 242(Pt 4): 125153, 2023 Jul 01.
Article in English | MEDLINE | ID: covidwho-20230938

ABSTRACT

The SARS-CoV-2 spike protein (S) represents an important viral component that is required for successful viral infection in humans owing to its essential role in recognition of and entry to host cells. The spike is also an appealing target for drug designers who develop vaccines and antivirals. This article is important as it summarizes how molecular simulations successfully shaped our understanding of spike conformational behavior and its role in viral infection. MD simulations found that the higher affinity of SARS-CoV-2-S to ACE2 is linked to its unique residues that add extra electrostatic and van der Waal interactions in comparison to the SARS-CoV S. This illustrates the spread potential of the pandemic SARS-CoV-2 relative to the epidemic SARS-CoV. Different mutations at the S-ACE2 interface, which is believed to increase the transmission of the new variants, affected the behavior and binding interactions in different simulations. The contributions of glycans to the opening of S were revealed via simulations. The immune evasion of S was linked to the spatial distribution of glycans. This help the virus to escape the immune system recognition. This article is important as it summarizes how molecular simulations successfully shaped our understanding of spike conformational behavior and its role in viral infection. This will pave the way to us preparing for the next pandemic as the computational tools are tailored to help fight new challenges.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Molecular Dynamics Simulation , Protein Binding , Angiotensin-Converting Enzyme 2/chemistry , Polysaccharides
7.
J Phys Chem B ; 127(20): 4396-4405, 2023 05 25.
Article in English | MEDLINE | ID: covidwho-2324522

ABSTRACT

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is considered as a key target for the design and development of COVID-19 drugs and inhibitors. Due to their unique structure and properties, ionic liquids (ILs) have many special interactions with proteins, showing great potential in biomedicine. Nevertheless, few research studies have been carried out on ILs and the spike RBD protein. Here, we explore the interaction of ILs and the RBD protein through large-scale molecular dynamics simulations (4 µs in total). It was found that IL cations with long alkyl chain lengths (nchain) could spontaneously bind to the cavity region of the RBD protein. The longer the alkyl chain is, the stabler the cations bind to the protein. The binding free energy (ΔG) had the same trend, peaking at nchain = 12 with -101.19 kJ/mol. The cationic chain lengths and their fit to the pocket are decisive factors that influence the binding strength of cations and proteins. The cationic imidazole ring has a high contact frequency with phenylalanine and tryptophan, and the hydrophobic residues phenylalanine, valine, leucine, and isoleucine are the most interacting residues with side chains of cations. Meanwhile, through analysis of the interaction energy, the hydrophobic and π-π interactions are the main contributors to the high affinity between cations and the RBD protein. In addition, the long-chain ILs would also act on the protein through clustering. These studies not only provide insights into the molecular interaction between ILs and the RBD of SARS-CoV-2 but also contribute to the rational design of IL-based drugs, drug carriers, and selective inhibitors as a therapeutic for SARS-CoV-2.


Subject(s)
COVID-19 , Ionic Liquids , Humans , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Ionic Liquids/chemistry , Molecular Dynamics Simulation , Protein Binding , Cations , Phenylalanine/metabolism
8.
Vet Q ; 40(1): 243-249, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-2315258

ABSTRACT

Several cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection transmitted from human owners to their dogs have recently been reported. The first ever case of SARS-CoV-2 transmission from a human owner to a domestic cat was confirmed on March 27, 2020. A tiger from a zoo in New York, USA, was also reportedly infected with SARS-CoV-2. It is believed that SARS-CoV-2 was transmitted to tigers from their caretakers, who were previously infected with this virus. On May 25, 2020, the Dutch Minister of Agriculture, Nature and Food Quality reported that two employees were infected with SARS-CoV-2 transmitted from minks. These reports have influenced us to perform a comparative analysis among angiotensin-converting enzyme 2 (ACE2) homologous proteins for verifying the conservation of specific protein regions. One of the most conserved peptides is represented by the peptide "353-KGDFR-357 (H. sapiens ACE2 residue numbering), which is located on the surface of the ACE2 molecule and participates in the binding of SARS-CoV-2 spike receptor binding domain (RBD). Multiple sequence alignments of the ACE2 proteins by ClustalW, whereas the three-dimensional structure of its binding region for the spike glycoprotein of SARS-CoV-2 was assessed by means of Spanner, a structural homology modeling pipeline method. In addition, evolutionary phylogenetic tree analysis by ETE3 was used. ACE2 works as a receptor for the SARS-CoV-2 spike glycoprotein between humans, dogs, cats, tigers, minks, and other animals, except for snakes. The three-dimensional structure of the KGDFR hosting protein region involved in direct interactions with SARS-CoV-2 spike RBD of the mink ACE2 appears to form a loop structurally related to the human ACE2 corresponding protein loop, despite of the reduced available protein length (401 residues of the mink ACE2 available sequence vs 805 residues of the human ACE2). The multiple sequence alignments of the ACE2 proteins shows high homology and complete conservation of the five amino acid residues: 353-KGDFR-357 with humans, dogs, cats, tigers, minks, and other animals, except for snakes. Where the information revealed from our examinations can support precision vaccine design and the discovery of antiviral therapeutics, which will accelerate the development of medical countermeasures, the World Health Organization recently reported on the possible risks of reciprocal infections regarding SARS-CoV-2 transmission from animals to humans.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/transmission , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/transmission , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/genetics , COVID-19 , Cats , Coronavirus Infections/prevention & control , Dogs , Humans , Mink , Pandemics/prevention & control , Peptidyl-Dipeptidase A/chemistry , Phylogeny , Pneumonia, Viral/prevention & control , Receptors, Virus/chemistry , Receptors, Virus/genetics , SARS-CoV-2 , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Tigers
9.
Int J Environ Res Public Health ; 19(4)2022 02 18.
Article in English | MEDLINE | ID: covidwho-2318385

ABSTRACT

Several coronaviruses (CoVs) have been identified as human pathogens, including the α-CoVs strains HCoV-229E and HCoV-NL63 and the ß-CoVs strains HCoV-HKU1 and HCoV-OC43. SARS-CoV, MERS-CoV, and SARS-CoV-2 are also classified as ß-coronavirus. New SARS-CoV-2 spike genomic variants are responsible for human-to-human and interspecies transmissibility, consequences of adaptations of strains from animals to humans. The receptor-binding domain (RBD) of SARS-CoV-2 binds to receptor ACE2 in humans and animal species with high affinity, suggesting there have been adaptive genomic variants. New genomic variants including the incorporation, replacement, or deletion of the amino acids at a variety of positions in the S protein have been documented and are associated with the emergence of new strains adapted to different hosts. Interactions between mutated residues and RBD have been demonstrated by structural modelling of variants including D614G, B.1.1.7, B1.351, P.1, P2; other genomic variants allow escape from antibodies generated by vaccines. Epidemiological and molecular tools are being used for real-time tracking of pathogen evolution and particularly new SARS-CoV-2 variants. COVID-19 vaccines obtained from classical and next-generation vaccine production platforms have entered clinicals trials. Biotechnology strategies of the first generation (attenuated and inactivated virus-CoronaVac, CoVaxin; BBIBP-CorV), second generation (replicating-incompetent vector vaccines-ChAdOx-1; Ad5-nCoV; Sputnik V; JNJ-78436735 vaccine-replicating-competent vector, protein subunits, virus-like particles-NVX-CoV2373 vaccine), and third generation (nucleic-acid vaccines-INO-4800 (DNA); mRNA-1273 and BNT 162b (RNA vaccines) have been used. Additionally, dendritic cells (LV-SMENP-DC) and artificial antigen-presenting (aAPC) cells modified with lentiviral vector have also been developed to inhibit viral activity. Recombinant vaccines against COVID-19 are continuously being applied, and new clinical trials have been tested by interchangeability studies of viral vaccines developed by classical and next-generation platforms.


Subject(s)
COVID-19 Vaccines , COVID-19 , Ad26COVS1 , Animals , Biotechnology , COVID-19/prevention & control , Genomics , Humans , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
10.
ACS Infect Dis ; 9(6): 1180-1189, 2023 Jun 09.
Article in English | MEDLINE | ID: covidwho-2313578

ABSTRACT

SARS-CoV and SARS-CoV-2 cell entry begins when spike glycoprotein (S) docks with the human ACE2 (hACE2) receptor. While the two coronaviruses share a common receptor and architecture of S, they exhibit differences in interactions with hACE2 as well as differences in proteolytic processing of S that trigger the fusion machine. Understanding how those differences impact S activation is key to understand its function and viral pathogenesis. Here, we investigate hACE2-induced activation in SARS-CoV and SARS-CoV-2 S using hydrogen/deuterium-exchange mass spectrometry (HDX-MS). HDX-MS revealed differences in dynamics in unbound S, including open/closed conformational switching and D614G-induced S stability. Upon hACE2 binding, notable differences in transduction of allosteric changes were observed extending from the receptor binding domain to regions proximal to proteolytic cleavage sites and the fusion peptide. Furthermore, we report that dimeric hACE2, the native oligomeric form of the receptor, does not lead to any more pronounced structural effect in S compared to saturated monomeric hACE2 binding. These experiments provide mechanistic insights into receptor-induced activation of Sarbecovirus spike proteins.


Subject(s)
COVID-19 , Severe acute respiratory syndrome-related coronavirus , Humans , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Allosteric Regulation , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/chemistry
11.
Proc Natl Acad Sci U S A ; 119(32): e2205690119, 2022 08 09.
Article in English | MEDLINE | ID: covidwho-2311515

ABSTRACT

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.


Subject(s)
COVID-19 , Furin , Proteolysis , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Amino Acid Motifs/genetics , Animals , COVID-19/virology , Chlorocebus aethiops , Furin/chemistry , Humans , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Deletion , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Virus Replication/genetics
12.
J Mol Biol ; 435(13): 168128, 2023 07 01.
Article in English | MEDLINE | ID: covidwho-2306096

ABSTRACT

SARS-CoV-2 virus spike (S) protein is an envelope protein responsible for binding to the ACE2 receptor, driving subsequent entry into host cells. The existence of multiple disulfide bonds in the S protein makes it potentially susceptible to reductive cleavage. Using a tri-part split luciferase-based binding assay, we evaluated the impacts of chemical reduction on S proteins from different virus variants and found that those from the Omicron family are highly vulnerable to reduction. Through manipulation of different Omicron mutations, we found that alterations in the receptor binding module (RBM) are the major determinants of this vulnerability. Specifically we discovered that Omicron mutations facilitate the cleavage of C480-C488 and C379-C432 disulfides, which consequently impairs binding activity and protein stability. The vulnerability of Omicron S proteins suggests a mechanism that can be harnessed to treat specific SARS-CoV-2 strains.


Subject(s)
Spike Glycoprotein, Coronavirus , Humans , Biological Assay , Mutation , Protein Binding , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Oxidation-Reduction , Protein Stability
13.
Biochem Biophys Res Commun ; 660: 43-49, 2023 06 11.
Article in English | MEDLINE | ID: covidwho-2293477

ABSTRACT

The COVID-19 pandemic, caused by SARS-CoV-2, has led to over 750 million infections and 6.8 million deaths worldwide since late 2019. Due to the continuous evolution of SARS-CoV-2, many significant variants have emerged, creating ongoing challenges to the prevention and treatment of the pandemic. Therefore, the study of antibody responses against SARS-CoV-2 is essential for the development of vaccines and therapeutics. Here we perform single particle cryo-electron microscopy (cryo-EM) structure determination of a rabbit monoclonal antibody (RmAb) 9H1 in complex with the SARS-CoV-2 wild-type (WT) spike trimer. Our structural analysis shows that 9H1 interacts with the receptor-binding motif (RBM) region of the receptor-binding domain (RBD) on the spike protein and by directly competing with angiotensin-converting enzyme 2 (ACE2), it blocks the binding of the virus to the receptor and achieves neutralization. Our findings suggest that utilizing rabbit-derived mAbs provides valuable insights into the molecular interactions between neutralizing antibodies and spike proteins and may also facilitate the development of therapeutic antibodies and expand the antibody library.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Antibodies, Monoclonal , Pandemics , Cryoelectron Microscopy , Antibodies, Viral , Receptors, Virus/metabolism , Antibodies, Neutralizing , Protein Binding , Spike Glycoprotein, Coronavirus/chemistry
14.
Ecohealth ; 20(1): 9-17, 2023 Mar.
Article in English | MEDLINE | ID: covidwho-2299096

ABSTRACT

The susceptibility of the white-tailed deer (WTD; Odocoileus virginianus) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted cervids as coronavirus reservoirs. This study aimed to evaluate the angiotensin-converting enzyme 2 (ACE2) residues which bind the spike protein of SARS-CoV-2 from 16 cervids to predict their potential susceptibility to SARS-CoV-2 infection. Eleven out of 16 species presented identical ACE2 key residues to WTD ACE2. Four cervids presented K31N, a variant associated with low SARS-CoV-2 susceptibility. Large herding of cervids with ACE2 key residues identical to that of the WTD can result in extensive reservoirs of SARS-CoV-2. Cervids as potential reservoirs could favor SARS-CoV-2 adaptation and the emergence of new coronavirus strains.


Subject(s)
COVID-19 , Deer , Animals , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Protein Binding
15.
Int J Mol Sci ; 24(8)2023 Apr 21.
Article in English | MEDLINE | ID: covidwho-2295696

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of the Coronavirus Disease 2019 (COVID-19) pandemic, which is still a health issue worldwide mostly due to a high rate of contagiousness conferred by the high-affinity binding between cell viral receptors, Angiotensin-Converting Enzyme 2 (ACE2) and SARS-CoV-2 Spike protein. Therapies have been developed that rely on the use of antibodies or the induction of their production (vaccination), but despite vaccination being still largely protective, the efficacy of antibody-based therapies wanes with the advent of new viral variants. Chimeric Antigen Receptor (CAR) therapy has shown promise for tumors and has also been proposed for COVID-19 treatment, but as recognition of CARs still relies on antibody-derived sequences, they will still be hampered by the high evasion capacity of the virus. In this manuscript, we show the results from CAR-like constructs with a recognition domain based on the ACE2 viral receptor, whose ability to bind the virus will not wane, as Spike/ACE2 interaction is pivotal for viral entry. Moreover, we have developed a CAR construct based on an affinity-optimized ACE2 and showed that both wild-type and affinity-optimized ACE2 CARs drive activation of a T cell line in response to SARS-CoV-2 Spike protein expressed on a pulmonary cell line. Our work sets the stage for the development of CAR-like constructs against infectious agents that would not be affected by viral escape mutations and could be developed as soon as the receptor is identified.


Subject(s)
COVID-19 , Humans , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Protein Binding , COVID-19 Drug Treatment , T-Lymphocytes/metabolism , Carrier Proteins/metabolism
16.
Mol Biol Evol ; 40(4)2023 04 04.
Article in English | MEDLINE | ID: covidwho-2304585

ABSTRACT

Coronaviruses are single-stranded, positive-sense RNA viruses that can infect many mammal and avian species. The Spike (S) protein of coronaviruses binds to a receptor on the host cell surface to promote viral entry. The interactions between the S proteins of coronaviruses and receptors of host cells are extraordinarily complex, with coronaviruses from different genera being able to recognize the same receptor and coronaviruses from the same genus able to bind distinct receptors. As the coronavirus disease 2019 pandemic has developed, many changes in the S protein have been under positive selection by altering the receptor-binding affinity, reducing antibody neutralization activities, or affecting T-cell responses. It is intriguing to determine whether the selection pressure on the S gene differs between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses due to the host shift from nonhuman animals to humans. Here, we show that the S gene, particularly the S1 region, has experienced positive selection in both SARS-CoV-2 and other coronaviruses. Although the S1 N-terminal domain exhibits signals of positive selection in the pairwise comparisons in all four coronavirus genera, positive selection is primarily detected in the S1 C-terminal domain (the receptor-binding domain) in the ongoing evolution of SARS-CoV-2, possibly owing to the change in host settings and the widespread natural infection and SARS-CoV-2 vaccination in humans.


Subject(s)
COVID-19 , Animals , Humans , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , COVID-19 Vaccines , Mammals/metabolism
17.
Sheng Wu Gong Cheng Xue Bao ; 38(9): 3173-3193, 2022 Sep 25.
Article in Chinese | MEDLINE | ID: covidwho-2254670

ABSTRACT

Coronavirus disease (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), with strong contagiousness, high susceptibility and long incubation period. cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain of the viral spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). Here, we briefly reviewed the mechanisms underlying the interaction between SARS-CoV-2 and ACE2, and summarized the latest research progress on SARS-CoV-2 neutralizing monoclonal antibodies and nanobodies, so as to better understand the development process and drug research direction of COVID-19. This review may facilitate understanding the development of neutralizing antibody drugs for emerging infectious diseases, especially for COVID-19.


Subject(s)
COVID-19 , Single-Domain Antibodies , Angiotensin-Converting Enzyme 2 , Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , Humans , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
18.
Antiviral Res ; 213: 105587, 2023 05.
Article in English | MEDLINE | ID: covidwho-2285219

ABSTRACT

Despite the vaccination campaigns for COVID-19, we still cannot control the spread of SARS-CoV-2, as evidenced by the ongoing circulation of the Omicron variants of concern. This highlights the need for broad-spectrum antivirals to further combat COVID-19 and to be prepared for a new pandemic with a (re-)emerging coronavirus. An interesting target for antiviral drug development is the fusion of the viral envelope with host cell membranes, a crucial early step in the replication cycle of coronaviruses. In this study, we explored the use of cellular electrical impedance (CEI) to quantitatively monitor morphological changes in real time, resulting from cell-cell fusion elicited by SARS-CoV-2 spike. The impedance signal in CEI-quantified cell-cell fusion correlated with the expression level of SARS-CoV-2 spike in transfected HEK293T cells. For antiviral assessment, we validated the CEI assay with the fusion inhibitor EK1 and measured a concentration-dependent inhibition of SARS-CoV-2 spike mediated cell-cell fusion (IC50 value of 0.13 µM). In addition, CEI was used to confirm the fusion inhibitory activity of the carbohydrate-binding plant lectin UDA against SARS-CoV-2 (IC50 value of 0.55 µM), which complements prior in-house profiling activities. Finally, we explored the utility of CEI in quantifying the fusogenic potential of mutant spike proteins and in comparing the fusion efficiency of SARS-CoV-2 variants of concern. In summary, we demonstrate that CEI is a powerful and sensitive technology that can be applied to studying the fusion process of SARS-CoV-2 and to screening and characterizing fusion inhibitors in a label-free and non-invasive manner.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Electric Impedance , HEK293 Cells , Spike Glycoprotein, Coronavirus/chemistry , Membrane Fusion , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Anti-Retroviral Agents/pharmacology
20.
J Med Virol ; 95(3): e28625, 2023 03.
Article in English | MEDLINE | ID: covidwho-2280054

ABSTRACT

Recombination is the main contributor to RNA virus evolution, and SARS-CoV-2 during the pandemic produced several recombinants. The most recent SARS-CoV-2 recombinant is the lineage labeled XBB, also known as Gryphon, which arose from BJ.1 and BM.1.1.1. Here we performed a genome-based survey aimed to compare the new recombinant with its parental lineages that never became dominant. Genetic analyses indicated that the recombinant XBB and its first descendant XBB.1 show an evolutionary condition typical of an evolutionary blind background with no further epidemiologically relevant descendant. Genetic variability and expansion capabilities are slightly higher than parental lineages. Bayesian Skyline Plot indicates that XBB reached its plateau around October 6, 2022 and after an initial rapid growth the viral population size did not further expand, and around November 10, 2022 its levels of genetic variability decreased. Simultaneously with the reduction of the XBB population size, an increase of the genetic variability of its first sub-lineage XBB.1 occurred, that in turn reached the plateau around November 9, 2022 showing a kind of vicariance with its direct progenitors. Structure analysis indicates that the affinity for ACE2 surface in XBB/XBB.1 RBDs is weaker than for BA.2 RBD. In conclusion, at present XBB and XBB.1 do not show evidence about a particular danger or high expansion capability. Genome-based monitoring must continue uninterrupted to individuate if further mutations can make XBB more dangerous or generate new subvariants with different expansion capability.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Bayes Theorem , Spike Glycoprotein, Coronavirus/chemistry
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