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1.
PLoS Comput Biol ; 17(12): e1009675, 2021 12.
Article in English | MEDLINE | ID: covidwho-1619980

ABSTRACT

Identifying the epitope of an antibody is a key step in understanding its function and its potential as a therapeutic. Sequence-based clonal clustering can identify antibodies with similar epitope complementarity, however, antibodies from markedly different lineages but with similar structures can engage the same epitope. We describe a novel computational method for epitope profiling based on structural modelling and clustering. Using the method, we demonstrate that sequence dissimilar but functionally similar antibodies can be found across the Coronavirus Antibody Database, with high accuracy (92% of antibodies in multiple-occupancy structural clusters bind to consistent domains). Our approach functionally links antibodies with distinct genetic lineages, species origins, and coronavirus specificities. This indicates greater convergence exists in the immune responses to coronaviruses than is suggested by sequence-based approaches. Our results show that applying structural analytics to large class-specific antibody databases will enable high confidence structure-function relationships to be drawn, yielding new opportunities to identify functional convergence hitherto missed by sequence-only analysis.


Subject(s)
Antigens, Viral/chemistry , COVID-19/immunology , COVID-19/virology , Epitopes, B-Lymphocyte/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Antibodies, Viral/metabolism , Antibody Specificity , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/genetics , Antigen-Antibody Reactions/genetics , Antigen-Antibody Reactions/immunology , Computational Biology , Coronavirus/chemistry , Coronavirus/genetics , Coronavirus/immunology , Databases, Chemical , Epitope Mapping , Epitopes, B-Lymphocyte/genetics , Humans , Mice , Models, Molecular , Pandemics , SARS-CoV-2/genetics , Single-Domain Antibodies/immunology
2.
Biophys J ; 120(14): 2914-2926, 2021 07 20.
Article in English | MEDLINE | ID: covidwho-1605082

ABSTRACT

Infection of human cells by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) relies on its binding to a specific receptor and subsequent fusion of the viral and host cell membranes. The fusion peptide (FP), a short peptide segment in the spike protein, plays a central role in the initial penetration of the virus into the host cell membrane, followed by the fusion of the two membranes. Here, we use an array of molecular dynamics simulations that take advantage of the highly mobile membrane mimetic model to investigate the interaction of the SARS-CoV2 FP with a lipid bilayer representing mammalian cellular membranes at an atomic level and to characterize the membrane-bound form of the peptide. Six independent systems were generated by changing the initial positioning and orientation of the FP with respect to the membrane, and each system was simulated in five independent replicas, each for 300 ns. In 73% of the simulations, the FP reaches a stable, membrane-bound configuration, in which the peptide deeply penetrated into the membrane. Clustering of the results reveals three major membrane-binding modes (binding modes 1-3), in which binding mode 1 populates over half of the data points. Taking into account the sequence conservation among the viral FPs and the results of mutagenesis studies establishing the role of specific residues in the helical portion of the FP in membrane association, the significant depth of penetration of the whole peptide, and the dense population of the respective cluster, we propose that the most deeply inserted membrane-bound form (binding mode 1) represents more closely the biologically relevant form. Analysis of FP-lipid interactions shows the involvement of specific residues, previously described as the "fusion-active core residues," in membrane binding. Taken together, the results shed light on a key step involved in SARS-CoV2 infection, with potential implications in designing novel inhibitors.


Subject(s)
COVID-19 , SARS-CoV-2 , Amino Acid Sequence , Animals , Cell Membrane , Humans , Membrane Fusion , Peptides , RNA, Viral , Spike Glycoprotein, Coronavirus , Virus Internalization
3.
Microbiol Spectr ; 9(3): e0165921, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1598527

ABSTRACT

COVID-19 vaccines are currently being administered worldwide and playing a critical role in controlling the pandemic. They have been designed to elicit neutralizing antibodies against Spike protein of the original SARS-CoV-2, and hence they are less effective against SARS-CoV-2 variants with mutated Spike than the original virus. It is possible that novel variants with abilities of enhanced transmissibility and/or immunoevasion will appear in the near future and perfectly escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Several lines of evidence suggest the contribution of CTLs on the viral control in COVID-19, and CTLs target a wide range of proteins involving comparatively conserved nonstructural proteins. Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2 using computational algorithms, HLA-A*24:02 transgenic mice and the peptide-encapsulated liposomes. We focused on pp1a and HLA-A*24:02 because pp1a is relatively conserved and HLA-A*24:02 is predominant in East Asians such as Japanese. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by a number of mutations in the Sequence Read Archive database of SARS-CoV-2 variants. The information of such conserved epitopes might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any SARS-CoV-2 variants by the induction of both anti-Spike neutralizing antibodies and CTLs specific for conserved epitopes. IMPORTANCE COVID-19 vaccines have been designed to elicit neutralizing antibodies against the Spike protein of the original SARS-CoV-2, and hence they are less effective against variants. It is possible that novel variants will appear and escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2. We focused on pp1a and HLA-A*24:02 because pp1a is conserved and HLA-A*24:02 is predominant in East Asians. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by mutations in the database of SARS-CoV-2 variants. The information might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any variants.


Subject(s)
COVID-19/immunology , Epitopes/immunology , HLA-A24 Antigen/genetics , HLA-A24 Antigen/immunology , Mutation , Polyproteins/genetics , SARS-CoV-2/genetics , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/immunology , Epitopes/genetics , HLA-A24 Antigen/isolation & purification , Humans , Mice , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology
4.
J Virol ; 94(13)2020 06 16.
Article in English | MEDLINE | ID: covidwho-1583223

ABSTRACT

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.


Subject(s)
Calcium/metabolism , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Host-Pathogen Interactions , Ions/metabolism , Membrane Fusion , Middle East Respiratory Syndrome Coronavirus/physiology , Virus Internalization , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Chlorocebus aethiops , Humans , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Models, Molecular , Mutation , Protein Binding , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship , Vero Cells , Virulence , Virus Assembly
5.
PLoS One ; 16(11): e0258645, 2021.
Article in English | MEDLINE | ID: covidwho-1518355

ABSTRACT

All approved coronavirus disease 2019 (COVID-19) vaccines in current use are safe, effective, and reduce the risk of severe illness. Although data on the immunological presentation of patients with COVID-19 is limited, increasing experimental evidence supports the significant contribution of B and T cells towards the resolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Despite the availability of several COVID-19 vaccines with high efficacy, more effective vaccines are still needed to protect against the new variants of SARS-CoV-2. Employing a comprehensive immunoinformatic prediction algorithm and leveraging the genetic closeness with SARS-CoV, we have predicted potential immune epitopes in the structural proteins of SARS-CoV-2. The S and N proteins of SARS-CoV-2 and SARS-CoVs are main targets of antibody detection and have motivated us to design four multi-epitope vaccines which were based on our predicted B- and T-cell epitopes of SARS-CoV-2 structural proteins. The cardinal epitopes selected for the vaccine constructs are predicted to possess antigenic, non-allergenic, and cytokine-inducing properties. Additionally, some of the predicted epitopes have been experimentally validated in published papers. Furthermore, we used the C-ImmSim server to predict effective immune responses induced by the epitope-based vaccines. Taken together, the immune epitopes predicted in this study provide a platform for future experimental validations which may facilitate the development of effective vaccine candidates and epitope-based serological diagnostic assays.


Subject(s)
Computational Biology , Epitope Mapping , SARS-CoV-2/immunology , Viral Structural Proteins/immunology , Amino Acid Sequence , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , Databases as Topic , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Histocompatibility Antigens Class I/metabolism , Humans , Models, Molecular , Protein Conformation , Reproducibility of Results , Viral Structural Proteins/chemistry
6.
Sci Rep ; 11(1): 22042, 2021 11 11.
Article in English | MEDLINE | ID: covidwho-1510622

ABSTRACT

The mutation of SARS-CoV-2 influences viral function as residue replacements affect both physiochemical properties and folding conformations. Although a large amount of data on SARS-CoV-2 is available, the investigation of how viral functions change in response to mutations is hampered by a lack of effective structural analysis. Here, we exploit the advances of protein structure fingerprint technology to study the folding conformational changes induced by mutations. With integration of both protein sequences and folding conformations, the structures are aligned for SARS-CoV to SARS-CoV-2, including Alpha variant (lineage B.1.1.7) and Delta variant (lineage B.1.617.2). The results showed that the virus evolution with change in mutational positions and physicochemical properties increased the affinity between spike protein and ACE2, which plays a critical role in coronavirus entry into human cells. Additionally, these structural variations impact vaccine effectiveness and drug function over the course of SARS-CoV-2 evolution. The analysis of structural variations revealed how the coronavirus has gradually evolved in both structure and function and how the SARS-CoV-2 variants have contributed to more severe acute disease worldwide.


Subject(s)
COVID-19/virology , Evolution, Molecular , Mutation , SARS-CoV-2/genetics , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Humans , Models, Molecular , Protein Conformation , Protein Folding , Protein Interaction Maps , Protein Multimerization , SARS Virus/chemistry , SARS Virus/genetics , SARS Virus/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
7.
Exp Biol Med (Maywood) ; 246(21): 2332-2337, 2021 11.
Article in English | MEDLINE | ID: covidwho-1507096

ABSTRACT

The coronavirus disease COVID-19 has been the cause of millions of deaths worldwide. Among the SARS-CoV-2 proteins, the non-structural protein 1 (NSP1) has great importance during the virus infection process and is present in both alpha and beta-CoVs. Therefore, monitoring of NSP1 polymorphisms is crucial in order to understand their role during infection and virus-induced pathogenicity. Herein, we analyzed how mutations detected in the circulating SARS-CoV-2 in the population of the city of Manaus, Amazonas state, Brazil could modify the tertiary structure of the NSP1 protein. Three mutations were detected in the SARS-CoV-2 NSP1 gene: deletion of the amino acids KSF from positions 141 to 143 (delKSF), SARS-CoV-2, lineage B.1.195; and two substitutions, R29H and R43C, SARS-CoV-2 lineage B.1.1.28 and B.1.1.33, respectively. The delKSF was found in 47 samples, whereas R29H and R43C were found in two samples, one for each mutation. The NSP1 structures carrying the mutations R43C and R29H on the N-terminal portion (e.g. residues 10 to 127) showed minor backbone divergence compared to the Wuhan model. However, the NSP1 C-terminal region (residues 145 to 180) was severely affected in the delKSF and R29H mutants. The intermediate variable region (residues 144 to 148) leads to changes in the C-terminal region, particularly in the delKSF structure. New investigations must be carried out to analyze how these changes affect NSP1 activity during the infection. Our results reinforce the need for continuous genomic surveillance of SARS-CoV-2 to better understand virus evolution and assess the potential impact of the viral mutations on the approved vaccines and future therapies.


Subject(s)
COVID-19/epidemiology , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Amino Acid Sequence/genetics , Amino Acid Substitution/genetics , Brazil/epidemiology , Humans , Polymorphism, Genetic/genetics , Sequence Deletion/genetics
8.
Sci Rep ; 11(1): 13464, 2021 06 29.
Article in English | MEDLINE | ID: covidwho-1500743

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus disease 2019 (COVID-19) that emerged in human populations recently. Severely ill COVID-19 patients exhibit the elevation of proinflammatory cytokines, and such an unbalanced production of proinflammatory cytokines is linked to acute respiratory distress syndrome with high mortality in COVID-19 patients. Our study provides evidence that the ORF3a, M, ORF7a, and N proteins of SARS-CoV-2 were NF-κB activators. The viral sequence from infected zoo lions belonged to clade V, and a single mutation of G251V is found for ORF3a gene compared to all other clades. No significant functional difference was found for clade V ORF3a, indicating the NF-κB activation is conserved among COVID-19 variants. Of the four viral proteins, the ORF7a protein induced the NF-κB dictated proinflammatory cytokines including IL-1α, IL-1ß, IL-6, IL-8, IL-10, TNF-α, and IFNß. The ORF7a protein also induced IL-3, IL-4, IL-7, IL-23. Of 15 different chemokines examined in the study, CCL11, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL26, CCL27, and CXCL9 were significantly upregulated by ORF7. These cytokines and chemokines were frequently elevated in severely ill COVID-19 patients. Our data provide an insight into how SARS-CoV-2 modulates NF-κB signaling and inflammatory cytokine expressions. The ORF7a protein may be a desirable target for strategic developments to minimize uncontrolled inflammation in COVID-19 patients.


Subject(s)
Cytokines/metabolism , NF-kappa B/metabolism , SARS-CoV-2/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , COVID-19/pathology , COVID-19/virology , Chemokines/genetics , Chemokines/metabolism , Cytokines/genetics , HeLa Cells , Humans , Point Mutation , SARS-CoV-2/isolation & purification , Sequence Alignment , Severity of Illness Index , Up-Regulation , Viral Matrix Proteins/genetics , Viral Proteins/genetics , Viroporin Proteins/chemistry , Viroporin Proteins/genetics , Viroporin Proteins/metabolism
9.
Commun Biol ; 4(1): 1240, 2021 10 29.
Article in English | MEDLINE | ID: covidwho-1493232

ABSTRACT

Circular tandem repeat proteins ('cTRPs') are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and structural motifs and form closed circular structures. They can display significant stability and solubility, a wide range of sizes, and are useful as protein display particles for biotechnology applications. However, cTRPs also demonstrate inefficient self-assembly from smaller subunits. In this study, we describe a new generation of cTRPs, with longer repeats and increased interaction surfaces, which enhanced the self-assembly of two significantly different sizes of homotrimeric constructs. Finally, we demonstrated functionalization of these constructs with (1) a hexameric array of peptide-binding SH2 domains, and (2) a trimeric array of anti-SARS CoV-2 VHH domains. The latter proved capable of sub-nanomolar binding affinities towards the viral receptor binding domain and potent viral neutralization function.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Protein Engineering/methods , Proteins/chemistry , Proteins/metabolism , SARS-CoV-2/metabolism , Tandem Repeat Sequences , Amino Acid Sequence , COVID-19/virology , Computer Simulation , Crystallization , HEK293 Cells , Humans , Models, Molecular , Neutralization Tests , Protein Binding , Protein Domains , Protein Folding , Protein Structure, Secondary , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
10.
Int J Mol Sci ; 22(21)2021 Nov 01.
Article in English | MEDLINE | ID: covidwho-1488619

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to global public health and the economy. The enzymatic product of cholesterol 25-hydroxylase (CH25H), 25-Hydroxycholesterol (25-HC), was reported to have potent anti-SARS-CoV-2 activity. Here, we found that the combination of 25-HC with EK1 peptide, a pan-coronavirus (CoV) fusion inhibitor, showed a synergistic antiviral activity. We then used the method of 25-HC modification to design and synthesize a series of 25-HC-modified peptides and found that a 25-HC-modified EK1 peptide (EK1P4HC) was highly effective against infections caused by SARS-CoV-2, its variants of concern (VOCs), and other human CoVs, such as HCoV-OC43 and HCoV-229E. EK1P4HC could protect newborn mice from lethal HCoV-OC43 infection, suggesting that conjugation of 25-HC with a peptide-based viral inhibitor was a feasible and universal strategy to improve its antiviral activity.


Subject(s)
Antiviral Agents/pharmacology , Hydroxycholesterols/chemistry , Lipopeptides/chemistry , SARS-CoV-2/drug effects , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Body Weight/drug effects , COVID-19/drug therapy , COVID-19/virology , Coronavirus 229E, Human/drug effects , Coronavirus 229E, Human/pathogenicity , Coronavirus Infections/drug therapy , Coronavirus Infections/mortality , Coronavirus Infections/virology , Coronavirus OC43, Human/drug effects , Coronavirus OC43, Human/pathogenicity , Disease Models, Animal , Drug Synergism , Humans , Hydroxycholesterols/pharmacology , Hydroxycholesterols/therapeutic use , Lipopeptides/pharmacology , Lipopeptides/therapeutic use , Mice , Mice, Inbred BALB C , Polyethylene Glycols/chemistry , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Survival Rate , Virus Internalization/drug effects
11.
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
12.
Nat Commun ; 12(1): 6103, 2021 10 20.
Article in English | MEDLINE | ID: covidwho-1475296

ABSTRACT

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/isolation & purification , Angiotensin-Converting Enzyme 2/ultrastructure , Animals , Cell Line, Tumor , Crystallography, X-Ray , HEK293 Cells , Humans , Molecular Dynamics Simulation , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , SARS-CoV-2/genetics , Sf9 Cells , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/isolation & purification , Spike Glycoprotein, Coronavirus/ultrastructure , Spodoptera , Surface Plasmon Resonance , Virus Attachment , Virus Internalization
13.
Molecules ; 26(20)2021 Oct 13.
Article in English | MEDLINE | ID: covidwho-1470934

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, the causative agent of coronavirus disease (COVID-19)) has caused relatively high mortality rates in humans throughout the world since its first detection in late December 2019, leading to the most devastating pandemic of the current century. Consequently, SARS-CoV-2 therapeutic interventions have received high priority from public health authorities. Despite increased COVID-19 infections, a vaccine or therapy to cover all the population is not yet available. Herein, immunoinformatics and custommune tools were used to identify B and T-cells epitopes from the available SARS-CoV-2 sequences spike (S) protein. In the in silico predictions, six B cell epitopes QTGKIADYNYK, TEIYQASTPCNGVEG, LQSYGFQPT, IRGDEVRQIAPGQTGKIADYNYKLPD, FSQILPDPSKPSKRS and PFAMQMAYRFNG were cross-reacted with MHC-I and MHC-II T-cells binding epitopes and selected for vaccination in experimental animals for evaluation as candidate vaccine(s) due to their high antigenic matching and conserved score. The selected six peptides were used individually or in combinations to immunize female Balb/c mice. The immunized mice raised reactive antibodies against SARS-CoV-2 in two different short peptides located in receptor binding domain and S2 region. In combination groups, an additive effect was demonstrated in-comparison with single peptide immunized mice. This study provides novel epitope-based peptide vaccine candidates against SARS-CoV-2.


Subject(s)
COVID-19 Vaccines/chemistry , COVID-19/prevention & control , Epitopes, B-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/chemistry , SARS-CoV-2/metabolism , Amino Acid Sequence , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Epitopes, B-Lymphocyte/immunology , Epitopes, B-Lymphocyte/metabolism , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , Female , Humans , Immunization , Mice , Mice, Inbred BALB C , Peptides/chemistry , Peptides/immunology , Peptides/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
14.
Int J Mol Sci ; 22(20)2021 Oct 18.
Article in English | MEDLINE | ID: covidwho-1470893

ABSTRACT

SputnikV is a vaccine against SARS-CoV-2 developed by the Gamaleya National Research Centre for Epidemiology and Microbiology. The vaccine has been shown to induce both humoral and cellular immune responses, yet the mechanisms remain largely unknown. Forty SputnikV vaccinated individuals were included in this study which aimed to demonstrate the location of immunogenic domains of the SARS-CoV-2 S protein using an overlapping peptide library. Additionally, cytokines in the serum of vaccinated and convalescent COVID-19 patients were analyzed. We have found antibodies from both vaccinated and convalescent sera bind to immunogenic regions located in multiple domains of SARS-CoV-2 S protein, including Receptor Binding Domain (RBD), N-terminal Domain (NTD), Fusion Protein (FP) and Heptad Repeats (HRs). Interestingly, many peptides were recognized by immunized and convalescent serum antibodies and correspond to conserved regions in circulating variants of SARS-CoV-2. This breadth of reactivity was still evident 90 days after the first dose of the vaccine, showing that the vaccine has induced a prolonged response. As evidenced by the activation of T cells, cellular immunity strongly suggests the high potency of the SputnikV vaccine against SARS-CoV-2 infection.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Immunity, Cellular , Immunity, Humoral , Adult , Amino Acid Sequence , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/immunology , Cytokines/metabolism , Female , Humans , Male , Peptides/chemistry , Peptides/immunology , Principal Component Analysis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Vaccination
15.
Proc Natl Acad Sci U S A ; 118(44)2021 11 02.
Article in English | MEDLINE | ID: covidwho-1470027

ABSTRACT

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19 Vaccines/pharmacology , COVID-19/immunology , COVID-19/prevention & control , Pandemics , SARS-CoV-2/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Antigen-Presenting Cells/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/epidemiology , COVID-19 Vaccines/administration & dosage , Camelids, New World/immunology , Female , Histocompatibility Antigens Class II/immunology , Humans , Immunity, Cellular , Immunity, Humoral , Immunization, Secondary , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Pandemics/prevention & control , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/genetics , Single-Domain Antibodies/administration & dosage , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
16.
Molecules ; 26(19)2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1463768

ABSTRACT

Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 µM affinity, a value commonly considered the threshold for cellular PDZ-peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.


Subject(s)
Choanoflagellata/chemistry , Choanoflagellata/metabolism , Computational Biology/methods , PDZ Domains , Protein Binding , Amino Acid Sequence , Evolution, Molecular , Humans , Phylogeny , Protein Conformation , Signal Transduction , Software , Substrate Specificity
17.
Viruses ; 13(10)2021 10 11.
Article in English | MEDLINE | ID: covidwho-1460086

ABSTRACT

Coronavirus, an important zoonotic disease, raises concerns of future pandemics. The bat is considered a source of noticeable viruses resulting in human and livestock infections, especially the coronavirus. Therefore, surveillance and genetic analysis of coronaviruses in bats are essential in order to prevent the risk of future diseases. In this study, the genome of HCQD-2020, a novel alphacoronavirus detected in a bat (Eptesicus serotinus), was assembled and described using next-generation sequencing and bioinformatics analysis. The comparison of the whole-genome sequence and the conserved amino acid sequence of replicated proteins revealed that the new strain was distantly related with other known species in the Alphacoronavirus genus. Phylogenetic construction indicated that this strain formed a separated branch with other species, suggesting a new species of Alphacoronavirus. Additionally, in silico prediction also revealed the risk of cross-species infection of this strain, especially in the order Artiodactyla. In summary, this study provided the genetic characteristics of a possible new species belonging to Alphacoronavirus.


Subject(s)
Alphacoronavirus/classification , Alphacoronavirus/genetics , Chiroptera/virology , Coronavirus Infections/veterinary , Genome, Viral/genetics , Alphacoronavirus/isolation & purification , Amino Acid Sequence/genetics , Animals , Artiodactyla/virology , Coronavirus Infections/virology , Phylogeny , Republic of Korea , Sequence Alignment , Whole Genome Sequencing
18.
ACS Chem Biol ; 16(5): 844-856, 2021 05 21.
Article in English | MEDLINE | ID: covidwho-1457790

ABSTRACT

Interferon-induced transmembrane proteins (IFITMs) are S-palmitoylated proteins in vertebrates that restrict a diverse range of viruses. S-palmitoylated IFITM3 in particular engages incoming virus particles, prevents their cytoplasmic entry, and accelerates their lysosomal clearance by host cells. However, how S-palmitoylation modulates the structure and biophysical characteristics of IFITM3 to promote its antiviral activity remains unclear. To investigate how site-specific S-palmitoylation controls IFITM3 antiviral activity, we employed computational, chemical, and biophysical approaches to demonstrate that site-specific lipidation of cysteine 72 enhances the antiviral activity of IFITM3 by modulating its conformation and interaction with lipid membranes. Collectively, our results demonstrate that site-specific S-palmitoylation of IFITM3 directly alters its biophysical properties and activity in cells to prevent virus infection.


Subject(s)
Antiviral Agents/chemistry , Cell Membrane/metabolism , Interferons/chemistry , Lipids/chemistry , Membrane Proteins/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Antiviral Agents/pharmacology , Binding Sites , Cell Membrane/ultrastructure , Computational Biology , Drug Design , Humans , Interferons/pharmacology , Lipoylation , Lysosomes/metabolism , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Signal Transduction
19.
Biomed Res Int ; 2021: 7251119, 2021.
Article in English | MEDLINE | ID: covidwho-1455778

ABSTRACT

Background: B.1.617.1, a variant of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causing respiratory illness is responsible for the second wave of COVID-19 and associated with a high incidence of infectivity and mortality. To mitigate the B.1.617.1 variant of SARS-CoV-2, deciphering the protein structure and immunological responses by employing bioinformatics tools for data mining and analysis is pivotal. Objectives: Here, an in silico approach was employed for deciphering the structure and immune function of the subunit of spike (S) protein of SARS-CoV-2 B.1.617.1 variant. Methods: The partial amino acid sequence of SARS-CoV-2 B.1.617.1 variant S protein was analyzed, and its putative secondary and tertiary structure was predicted. Immunogenic analyses including B- and T-cell epitopes, interferon-gamma (IFN-γ) response, chemokine, and protective antigens for SARS-CoV 2 S proteins were predicted using appropriate tools. Results: B.1.617.1 variant S protein sequence was found to be highly stable and amphipathic. ABCpred and CTLpred analyses led to the identification of two potential antigenic B cell and T cell epitopes with starting amino acid positions at 60 and 82 (for B cell epitopes) and 54 and 98 (for T cell epitopes) having prediction scores > 0.8. Further, RAMPAGE tool was used for determining the allowed and disallowed regions of the three-dimensional predicted structure of SARS-CoV-2 B.1.617.1 variant S protein. Conclusion: Together, the in silico analysis revealed the predicted structure of partial S protein, immunogenic properties, and possible regions for S protein of SARS-CoV-2 and provides a valuable prelude for engineering the targeted vaccine or drug against B.1.617.1 variant of SARS-CoV-2.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/virology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Algorithms , Amino Acid Sequence , COVID-19/immunology , COVID-19/metabolism , Computational Biology/methods , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Humans , Immunogenicity, Vaccine , Protein Binding , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship , Viral Vaccines/immunology
20.
Biochemistry ; 60(40): 2978-2986, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1440443

ABSTRACT

The SARS-CoV-2 spike protein is the primary antigenic determinant of the virus and has been studied extensively, yet the process of membrane fusion remains poorly understood. The fusion domain (FD) of viral glycoproteins is well established as facilitating the initiation of membrane fusion. An improved understanding of the structural plasticity associated with these highly conserved regions aids in our knowledge of the molecular mechanisms that drive viral fusion. Within the spike protein, the FD of SARS-CoV-2 exists immediately following S2' cleavage at the N-terminus of the S2 domain. Here we have shown that following the introduction of a membrane at pH 7.4, the FD undergoes a transition from a random coil to a more structurally well-defined postfusion state. Furthermore, we have classified the domain into two distinct regions, a fusion peptide (FP, S816-G838) and a fusion loop (FL, D839-F855). The FP forms a helix-turn-helix motif upon association with a membrane, and the favorable entropy gained during this transition from a random coil is likely the driving force behind membrane insertion. Membrane depth experiments then revealed the FP is found inserted within the membrane below the lipid headgroups, while the interaction of the FL with the membrane is shallower in nature. Thus, we propose a structural model relevant to fusion at the plasma membrane in which the FP inserts itself just below the phospholipid headgroups and the FL lays upon the lipid membrane surface.


Subject(s)
Cell Membrane/metabolism , Membrane Fusion/physiology , Models, Biological , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , COVID-19/genetics , COVID-19/metabolism , Cell Membrane/genetics , Humans , Protein Binding/physiology , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics
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