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1.
Sci Rep ; 12(1): 3794, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-2004784

ABSTRACT

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.


Subject(s)
Microscopy/methods , SARS-CoV-2/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/transmission , COVID-19/virology , Chlorocebus aethiops , Cytoplasm/chemistry , Cytoplasm/ultrastructure , Cytoplasm/virology , Extracellular Vesicles/chemistry , Extracellular Vesicles/ultrastructure , Giant Cells/chemistry , Giant Cells/physiology , Helium/chemistry , Humans , Ions/chemistry , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
2.
Virology ; 572: 28-43, 2022 07.
Article in English | MEDLINE | ID: covidwho-1991334

ABSTRACT

The newly discovered SARS-CoV-2 Omicron variant B.1.1.529 is a Variant of Concern (VOC) announced by the World Health Organization (WHO). It's becoming increasingly difficult to keep these variants from spreading over the planet. The fifth wave has begun in several countries because of Omicron variant, and it is posing a threat to human civilization. As a result, we need effective vaccination that can tackle Omicron SARS-CoV-2 variants that are bound to emerge. Therefore, the current study is an initiative to design a peptide-based chimeric vaccine that may potentially battle SARS-CoV-2 Omicron variant. As a result, the most relevant epitopes present in the mutagenic areas of Omicron spike protein were identified using a set of computational tools and immunoinformatic techniques to uncover common MHC-1, MHC-II, and B cell epitopes that may have the ability to influence the host immune mechanism. A final of three epitopes from CD8+ T-cell, CD4+ T-cell epitopes, and B-cell were shortlisted from spike protein, and that are highly antigenic, IFN-γ inducer, as well as overlapping for the construction of twelve vaccine models. As a result, the antigenic epitopes were coupled with a flexible and stable peptide linker, and the adjuvant was added at the N-terminal end to create a unique vaccine candidate. The structure of a 3D vaccine candidate was refined, and its quality was assessed by using web servers. However, the applied immunoinformatic study along with the molecular docking and simulation of 12 modeled vaccines constructs against six distinct HLAs, and TLRs (TLR2, and TLR4) complexes revealed that the V1 construct was non-allergenic, non-toxic, highly immunogenic, antigenic, and most stable. The vaccine candidate's stability was confirmed by molecular dynamics investigations. Finally, we studied the expression of the suggested vaccination using codon optimization and in-silico cloning. The current study proposed V1 Multi-Epitope Vaccine (MEV) as a significant vaccine candidate that may help the scientific community to treat SARS-CoV-2 infections.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/prevention & control , COVID-19 Vaccines/genetics , Computational Biology , Epitopes, B-Lymphocyte , Epitopes, T-Lymphocyte/genetics , Humans , Molecular Docking Simulation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Vaccines, Subunit/genetics
3.
Comput Biol Med ; 146: 105598, 2022 07.
Article in English | MEDLINE | ID: covidwho-1982846

ABSTRACT

The critical event in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis is recognition of host cells by the virus, which is facilitated by protein-protein interaction (PPI) of viral Spike-Receptor Binding Domain (S-RBD) and Human Angiotensin Converting Enzyme 2-Receptor (hACE2-R). Thus, disrupting the interaction between S-RBD and hACE2-R is widely accepted as a primary strategy for managing COVID-19. The purpose of this study is to assess the ability of three steroidal lactones (SL) (4-Dehydrowithaferin A, Withaferin A, and Withalongolide A) derived from plants to disrupt the PPI of S-RBD and hACE2-R under two conditions (CON-I and CON-II) using in-silico methods. Under CON-I, 4-Dehydrowithaferin A destabilizing the interactions between S-RBD and hACE2-R, as indicated by an increase in binding energy (BE) from -1028.5 kJ/mol (control) to -896.12 kJ/mol 4-Dehydrowithaferin A exhibited a strong interaction with S-RBD GLY496 with a hydrogen bond occupancy (HBO) of 37.33%. Under CON-II, Withalongolide A was capable of disrupting all types of PPI, as evidenced by an increased BE from -913 kJ/mol (control) to -133.69 kJ/mol and an increased distance (>3.55 nm) between selected AAR combinations of S-RBD and hACE2-R. Withalongolide A formed a hydrogen bond with TYR453 (97%, HBO) of S-RBD, which is required for interaction with hACE2-R's HIS34. Our studies demonstrated that SL molecules have the potential to disrupt the S-RBD and hACE2-R interaction, thereby preventing SARS-CoV-2 from recognizing host cells. The SL molecules can be considered for additional in-vitro and in-vivo studies with this research evidence.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/chemistry , Humans , Lactones/pharmacology , Peptidyl-Dipeptidase A/chemistry , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
4.
Biochem Biophys Res Commun ; 622: 57-63, 2022 09 24.
Article in English | MEDLINE | ID: covidwho-1982609

ABSTRACT

COVID-19 is accompanied by strong inflammatory reaction and is often followed by long-term cognitive disorders. The fragment 674-685 of SARS-Cov-2 spike protein was shown to interact with α7 nicotinic acetylcholine receptor involved in regulating both inflammatory reactions and cognitive functions. Here we show that mice immunized with the peptide corresponding to 674-685 fragment of SARS-Cov-2 spike protein conjugated to hemocyanin (KLH-674-685) demonstrate decreased level of α7 nicotinic acetylcholine receptors, increased levels of IL-1ß and TNFα in the brain and impairment of episodic memory. Choline injections prevented α7 nicotinic receptor decline and memory loss. Mice injected with immunoglobulins obtained from the blood of (KLH-674-685)-immunized mice also demonstrated episodic memory decline. These data allow suggesting that post-COVID memory impairment in humans is related to SARS-Cov-2 spike protein-specific immune reaction. The mechanisms of such effect are being discussed.


Subject(s)
COVID-19 , Memory, Episodic , Animals , Humans , Immunization , Inflammation , Memory Disorders/etiology , Memory Disorders/metabolism , Mice , Neuroinflammatory Diseases , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/adverse effects , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , alpha7 Nicotinic Acetylcholine Receptor/metabolism
5.
Commun Biol ; 5(1): 789, 2022 Aug 05.
Article in English | MEDLINE | ID: covidwho-1978022

ABSTRACT

As new variants of SARS-CoV-2 continue to emerge, it is important to assess the cross-neutralizing capabilities of antibodies naturally elicited during wild type SARS-CoV-2 infection. In the present study, we evaluate the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviruses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which retain their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solve the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 Å and 2.42 Å resolutions, respectively, revealing a similar mode of binding to that between the RBD and ACE2. Furthermore, we provide five additional structures (at resolutions of 4.7 Å, 7.3 Å, 6.4 Å, 3.3 Å, and 6.1 Å) of a second antibody, TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides additional mechanistic understanding about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insights on the likelihood of reinfections.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Monoclonal/chemistry , Antibodies, Viral , Humans , Neutralization Tests , Spike Glycoprotein, Coronavirus/chemistry
6.
Viruses ; 12(1)2020 01 20.
Article in English | MEDLINE | ID: covidwho-1969491

ABSTRACT

Middle East respiratory syndrome (MERS) is an acute, high-mortality-rate, severe infectious disease caused by an emerging MERS coronavirus (MERS-CoV) that causes severe respiratory diseases. The continuous spread and great pandemic potential of MERS-CoV make it necessarily important to develop effective vaccines. We previously demonstrated that the application of Gram-positive enhancer matrix (GEM) particles as a bacterial vector displaying the MERS-CoV receptor-binding domain (RBD) is a very promising MERS vaccine candidate that is capable of producing potential neutralization antibodies. We have also used the rabies virus (RV) as a viral vector to design a recombinant vaccine by expressing the MERS-CoV S1 (spike) protein on the surface of the RV. In this study, we compared the immunological efficacy of the vaccine candidates in BALB/c mice in terms of the levels of humoral and cellular immune responses. The results show that the rabies virus vector-based vaccine can induce remarkably earlier antibody response and higher levels of cellular immunity than the GEM particles vector. However, the GEM particles vector-based vaccine candidate can induce remarkably higher antibody response, even at a very low dose of 1 µg. These results indicate that vaccines constructed using different vaccine vector platforms for the same pathogen have different rates and trends in humoral and cellular immune responses in the same animal model. This discovery not only provides more alternative vaccine development platforms for MERS-CoV vaccine development, but also provides a theoretical basis for our future selection of vaccine vector platforms for other specific pathogens.


Subject(s)
Coronavirus Infections/immunology , Middle East Respiratory Syndrome Coronavirus/immunology , Viral Vaccines/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cell Line , Coronavirus Infections/prevention & control , Genetic Vectors , Humans , Immunization , Immunoglobulin G/blood , Immunoglobulin G/immunology , Lactococcus lactis/genetics , Mice , Mice, Inbred BALB C , Middle East Respiratory Syndrome Coronavirus/genetics , Rabies virus/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , Viral Vaccines/administration & dosage
7.
Cell Mol Biol Lett ; 27(1): 50, 2022 Jun 17.
Article in English | MEDLINE | ID: covidwho-1962733

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), a major international public health concern. Because of very similar amino acid sequences of the seven domain names, SARS-CoV-2 belongs to the Coronavirinae subfamily of the family Coronaviridae, order Nidovirales, and realm Riboviria, placed in exceptional clusters, but categorized as a SARS-like species. As the RNA virus family with the longest genome, the Coronaviridae genome consists of a single strand of positive RNA (25-32 kb in length). Four major structural proteins of this genome include the spike (S), membrane (M), envelope (E), and the nucleocapsid (N) protein, all of which are encoded within the 3' end of the genome. By engaging with its receptor, angiotensin-converting enzyme 2 (ACE2), SARS-CoV-2 infects host cells. According to the most recent epidemiological data, as the illness spread globally, several genetic variations of SARS-CoV-2 appeared quickly, with the World Health Organization (WHO) naming 11 of them. Among these, seven SARS-CoV-2 subtypes have received the most attention. Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.617.2) are now designated as variations of concern (VOC) (B.1.1.529). Lambda (C.37) and Mu are variations of interest (VOI) (B.1.621). The remaining six are either being monitored or are no longer considered a threat. On the basis of studies done so far, antiviral drugs, antibiotics, glucocorticoids, recombinant intravenous immunoglobulin, plasma therapy, and IFN-α2b have been used to treat patients. Moreover, full vaccination is associated with lower infection and helps prevent transmission, but the risk of infection cannot be eliminated completely in vaccinated people.


Subject(s)
COVID-19 , SARS-CoV-2 , Genotype , Humans , Peptidyl-Dipeptidase A , Phenotype , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
8.
J Virol ; 96(15): e0075322, 2022 08 10.
Article in English | MEDLINE | ID: covidwho-1962094

ABSTRACT

Circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the human population leads to further viral evolution. The new variants that arise during this evolution are more infectious. Our data suggest that newer variants have shifted from utilizing both cathepsin/endosome- and TMPRSS2-mediated entry mechanisms to rely on a TMPRSS2-dependent entry pathway. Accordingly, only the early lineages of SARS-CoV-2 are capable of infecting and forming syncytia in Vero/ACE2 cells which lack TMPRSS2 expression. The presence of an intact multibasic furin cleavage site (FCS) in the S protein was a key requirement for cell-to-cell fusion. Deletion of FCS makes SARS-CoV-2 more infectious in vitro but renders it incapable of syncytium formation. Cell-to-cell fusion likely represents an alternative means of virus spread and is resistant to the presence of high levels of neutralizing monoclonal antibodies (MAbs) and immune sera in the media. In this study, we also noted that cells infected with SARS-CoV-2 with an intact FCS or alphavirus replicon expressing S protein (VEErep/S) released high levels of free S1 subunit. The released S1 is capable of activating the TLR4 receptor and inducing a pro-inflammatory response. Thus, S1 activation of TLR4 may be an important contributor to SARS-CoV-2-induced COVID-19 disease and needs to be considered in the design of COVID mRNA vaccines. Lastly, a VEErep/S-replicon was shown to produce large amounts of infectious, syncytium-forming pseudoviruses and thus could represent alternative experimental system for screening inhibitors of virus entry and syncytium formation. IMPORTANCE The results of this study demonstrate that the late lineages of SARS-CoV-2 evolved to more efficient use of the TMPRSS2-mediated entry pathway and gradually lost an ability to employ the cathepsins/endosome-mediated entry. The acquisition of a furin cleavage site (FCS) by SARS-CoV-2-specific S protein made the virus a potent producer of syncytia. Their formation is also determined by expression of ACE2 and TMPRSS2 and is resistant to neutralizing human MAbs and immune sera. Syncytium formation appears to be an alternative means of infection spread following the development of an adaptive immune response. Cells infected with SARS-CoV-2 with an intact FCS secrete high levels of the S1 subunit. The released S1 demonstrates an ability to activate the TLR4 receptor and induce pro-inflammatory cytokines, which represent a hallmark of SARS-CoV-2 pathogenesis. Alphavirus replicons encoding SARS-CoV-2 S protein cause spreading, syncytium-forming infection, and they can be applied as an experimental tool for studying the mechanism of syncytium formation.


Subject(s)
COVID-19 , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/metabolism , Evolution, Molecular , Furin/metabolism , Humans , Immune Sera , SARS-CoV-2/genetics , Signal Transduction , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Toll-Like Receptor 4 , Virus Internalization
9.
Proc Natl Acad Sci U S A ; 119(32): e2205690119, 2022 Aug 09.
Article in English | MEDLINE | ID: covidwho-1960628

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
10.
Int J Mol Sci ; 23(15)2022 Jul 26.
Article in English | MEDLINE | ID: covidwho-1957352

ABSTRACT

Vaccination protects against COVID-19 via the spike protein receptor-binding domain (RBD)-specific antibody formation, but it also affects the innate immunity. The effects of specific antibody induction on neutrophils that can cause severe respiratory inflammation are important, though not completely investigated. In the present study, using a mouse model mimicking SARS-CoV-2 virus particle inhalation, we investigated neutrophil phenotype and activity alterations in the presence of RBD-specific antibodies. Mice were immunized with RBD and a week after a strong antibody response establishment received 100 nm particles in the RBD solution. Control mice received injections of a phosphate buffer instead of RBD. We show that the application of 100 nm particles in the RBD solution elevates neutrophil recruitment to the blood and the airways of RBD-immunized mice rather than in control mice. Analysis of bone marrow cells of mice with induced RBD-specific antibodies revealed the increased population of CXCR2+CD101+ neutrophils. These neutrophils did not demonstrate an enhanced ability of neutrophil extracellular traps (NETs) formation compared to the neutrophils from control mice. Thus, the induction of RBD-specific antibodies stimulates the activation of mature neutrophils that react to RBD-coated particles without triggering excessive inflammation.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Antibodies, Neutralizing , Antibodies, Viral , Antibody Formation , Humans , Inflammation , Neutrophils , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
11.
Int J Mol Sci ; 23(15)2022 Jul 25.
Article in English | MEDLINE | ID: covidwho-1957347

ABSTRACT

Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use.


Subject(s)
Antibodies, Neutralizing , COVID-19 , Animals , Antibodies, Monoclonal , Antibodies, Viral , Humans , Mice , Mutation , Neutralization Tests , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
12.
J Biomed Nanotechnol ; 18(4): 1121-1130, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1950558

ABSTRACT

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused significant death, economic crisis, and the world to almost completely shut down. This present study focused on targeting the novel SARS-CoV-2 envelope protein, which has not been frequently mutating, and the S protein with a much larger peptide capable of inhibiting virus-mammalian cell attraction. In doing so, molecular dynamics software was used here to model six peptides including: NapFFTLUFLTUTE, NapFFSLAFLTATE, NapFFSLUFLSUTE, NapFFTLAFLTATE, NapFFSLUFLSUSE, and NapFFMLUFLMUME. Results showed that two of these completely hydrophobic peptides (NapFFTLUFLTUTE and NapFFMLUFLMUME) had a strong ability to bind to the virus, preventing its binding to a mammalian cell membrane, entering the cell, and replicating by covering many cell attachment sites on SARS-CoV-2. Further cell modeling results demonstrated the low toxicity and suitable pharmacokinetic properties of both peptides making them ideal for additional in vitro and in vivo investigation. In this manner, these two peptides should be further explored for a wide range of present and future COVID-19 therapeutic and prophylactic applications.


Subject(s)
COVID-19 , Nanostructures , Amino Acid Sequence , Animals , Mammals/metabolism , Peptides , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
13.
Virus Res ; 318: 198845, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-1946817

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presents an immense global health problem. Spike (S) protein of coronavirus is the primary determinant of its entry into the host as it consists of both receptor binding and fusion domain. Besides tissue tropism, and host range, coronavirus pathogenesis are primarily controlled by the interaction of S protein with the cell receptor. Moreover, the proteolytic activation of S protein by host cell proteases plays a decisive role. The host-cell proteases have shown to be involved in the proteolysis of S protein and cleaving it into two functional subunits, S1 and S2, during the maturation process. In the present study, the interaction of the S protein of SARS-CoV-2 with different host proteases like furin, cathepsin B, and plasmin has been analyzed using molecular docking and molecular dynamics (MD) simulation. Incorporation of the furin cleavage site (R-R-A-R) in the S protein of SARS-CoV-2 has been studied by mutating the individual amino acid. MD simulation results suggest the polytropic nature of the S protein. Our analysis indicated that a single amino acid substitution in the polybasic cleavage site of S protein perturb the binding of cellular proteases. This mutation study might help to generate an attenuated SARS-CoV-2. Besides, targeting host proteases by inhibitors may result in a practical approach to stop the cellular spread of SARS-CoV-2 and develop its antiviral.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Furin/chemistry , Humans , Molecular Docking Simulation , Peptide Hydrolases/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
14.
Structure ; 30(8): 1062-1074.e4, 2022 Aug 04.
Article in English | MEDLINE | ID: covidwho-1946637

ABSTRACT

The COVID-19 pandemic has prompted a rapid response in vaccine and drug development. Herein, we modeled a complete membrane-embedded SARS-CoV-2 spike glycoprotein and used molecular dynamics simulations with benzene probes designed to enhance discovery of cryptic pockets. This approach recapitulated lipid and host metabolite binding sites previously characterized by cryo-electron microscopy, revealing likely ligand entry routes, and uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop. A full representation of glycan moieties was essential to accurately describe pocket dynamics. A multi-conformational behavior of the 617-628 loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry experiments, supportive of opening and closing dynamics. The pocket is the site of multiple mutations associated with increased transmissibility found in SARS-CoV-2 variants of concern including Omicron. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.


Subject(s)
SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Benzene , Cryoelectron Microscopy , Molecular Dynamics Simulation , Protein Binding , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
15.
Infect Genet Evol ; 102: 105300, 2022 08.
Article in English | MEDLINE | ID: covidwho-1946053

ABSTRACT

Since the beginning of the Coronavirus disease-2019 pandemic, there has been a growing interest in exploring SARS-CoV-2 genetic variation to understand the origin and spread of the pandemic, improve diagnostic methods and develop the appropriate vaccines. The objective of this study was to identify the SARS-CoV-2s lineages circulating in Tunisia and to explore their amino acid signature in order to follow their genome dynamics. Whole genome sequencing and genetic analyses of fifty-eight SARS-CoV-2 samples collected during one-year between March 2020 and March 2021 from the National Influenza Center were performed using three sampling strategies.. Multiple lineage introductions were noted during the initial phase of the pandemic, including B.4, B.1.1, B.1.428.2, B.1.540 and B.1.1.189. Subsequently, lineages B1.160 (24.2%) and B1.177 (22.4%) were dominant throughout the year. The Alpha variant (B.1.1.7 lineage) was identified in February 2021 and firstly observed in the center of our country. In addition, A clear diversity of lineages was observed in the North of the country. A total of 335 mutations including 10 deletions were found. The SARS-CoV-2 proteins ORF1ab, Spike, ORF3a, and Nucleocapsid were observed as mutation hotspots with a mutation frequency exceeding 20%. The 2 most frequent mutations, D614G in S protein and P314L in Nsp12 appeared simultaneously and are often associated with increased viral infectivity. Interestingly, deletions in coding regions causing consequent deletions of amino acids and frame shifts were identified in NSP3, NSP6, S, E, ORF7a, ORF8 and N proteins. These findings contribute to define the COVID-19 outbreak in Tunisia. Despite the country's limited resources, surveillance of SARS-CoV-2 genomic variation should be continued to control the occurrence of new variants.


Subject(s)
COVID-19 , SARS-CoV-2 , Amino Acids/genetics , COVID-19/epidemiology , Genome, Viral , Humans , Mutation , Phylogeny , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Tunisia/epidemiology
16.
Comput Biol Med ; 147: 105709, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1944685

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the contagious coronavirus disease 2019 (COVID-19) which was first identified in Wuhan, China, in December 2019. Around the world, many researchers focused their research on identifying inhibitors against the druggable SARS-CoV-2 targets. The reported genomic mutations have a direct effect on the receptor-binding domain (RBD), which interacts with host angiotensin-converting enzyme 2 (ACE-2) for viral cell entry. These mutations, some of which are variants of concern (VOC), lead to increased morbidity and mortality rates. The newest variants including B.1.617.2 (Delta), AY.1 (Delta plus), and C.37 (Lambda) were considered in this study. Thus, an exhaustive structure-based virtual screening of a ligand library (in which FDA approved drugs are also present) using the drug-likeness screening, molecular docking, ADMET profiling was performed followed by molecular dynamics (MD) simulation, and Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) calculation to identify compounds or drugs can be repurposed for inhibiting the wild type, Delta, Delta plus and Lambda variants of RBD of the spike protein. Based on the virtual screening steps, two FDA approved drugs, Atovaquone (atv) and Praziquantel (prz), were selected and repurposed as the best candidates of SARS-CoV-2 RBD inhibitors. Molecular docking results display that both atv and prz contribute in different interaction with binding site residues (Gln493, Asn501 and Gly502 in the hydrogen bond formation, Phe490 and Tyr505 in the π- π stacking and Tyr449, Ser494, and Phe497 in the vdW interactions) in the wild type, Delta, Delta plus and Lambda variants of RBD of the spike protein. MD simulations revealed that among the eight studied complexes, the wild type-atv and Delta-prz complexes have the most structural stability over the simulation time. Furthermore, MM-PBSA calculation showed that in the atv containing complexes, highest binding affinity is related to the wild type-atv complex and in the prz containing complexes, it is related to the Delta-prz complex. The validation of docking results was done by comparing with experimental data (heparin in complex with wild type and Delta variants). Also, comparison of the obtained results with the result of simulation of the k22 with the studied proteins showed that atv and prz are suitable inhibitors for these proteins, especially wild type t and Delta variant, respectively. Thus, we found that atv and prz are the best candidate for inhibition of wild type and Delta variant of the spike protein. Also, atv can be an appropriate inhibitor for the Lambda variant. Obtained in silico results may help the development of new anti-COVID-19 drugs.


Subject(s)
COVID-19 , SARS-CoV-2 , Adipates , COVID-19/drug therapy , COVID-19/genetics , Drug Repositioning/methods , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation/genetics , Peptidyl-Dipeptidase A/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Succinates
17.
Biophys Chem ; 288: 106824, 2022 09.
Article in English | MEDLINE | ID: covidwho-1944352

ABSTRACT

The novel coronavirus that caused COVID-19 pandemic is SARS-CoV-2. Although various vaccines are currently being used to prevent the disease's severe consequences, there is still a need for medications for those who become infected. The SARS-CoV-2 has a variety of proteins that have been studied extensively since the virus's advent. In this review article, we looked at chemical to molecular aspects of the various structures studied that have pharmaceutical activity and attempted to find a link between drug activity and compound structure. For example, designing of the compounds which bind to the allosteric site and modify hydrogen bonds or the salt bridges can disrupt SARS-CoV2 RBD-ACE2 complex. It seems that quaternary ammonium moiety and quinolin-1-ium structure could act as a negative allosteric modulator to reduce the tendency between spike-ACE2. Pharmaceutical structures with amino heads and hydrophobic tails can block envelope protein to prevent making mature SARS-CoV-2. Also, structures based on naphthalene pharmacophores or isosteres can form a strong bond with the PLpro and form a π-π and the Mpro's active site can be occupied by octapeptide compounds or linear compounds with a similar fitting ability to octapeptide compounds. And for protein RdRp, it is critical to consider pH and pKa so that pKa regulation of compounds to comply with patients is very effective, thus, the presence of tetrazole, phenylpyrazole groups, and analogs of pyrophosphate in the designed drugs increase the likelihood of the RdRp active site inhibition. Finally, it can be deduced that designing hybrid drug molecules along with considering the aforementioned characteristics would be a suitable approach for developing medicines in order to accurate targeting and complete inhibition this virus.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , COVID-19/drug therapy , Humans , Pandemics , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pharmaceutical Preparations , Protein Binding , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase , Spike Glycoprotein, Coronavirus/chemistry
18.
Mol Cell ; 82(11): 2050-2068.e6, 2022 06 02.
Article in English | MEDLINE | ID: covidwho-1937002

ABSTRACT

Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor-binding domain (RBD) and neutralizing antibody epitope presentation, affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Humans , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
19.
PLoS Comput Biol ; 18(7): e1009834, 2022 07.
Article in English | MEDLINE | ID: covidwho-1933194

ABSTRACT

The recent novel coronavirus disease (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is threatening global health. However, an understanding of the interaction of SARS-CoV-2 with human cells, including the physical docking property influenced by the host's genetic diversity, is still lacking. Here, based on germline variants in the UK Biobank covering 502,543 individuals, we revealed the molecular interactions between human angiotensin-converting enzyme 2 (hACE2), which is the representative receptor for SARS-CoV-2 entry, and COVID-19 infection. We identified six nonsense and missense variants of hACE2 from 2585 subjects in the UK Biobank covering 500000 individuals. Using our molecular dynamics simulations, three hACE2 variants from 2585 individuals we selected showed higher levels of binding free energy for docking in the range of 1.44-3.69 kcal/mol. Although there are diverse contributors to SARS-CoV-2 infections, including the mobility of individuals, we analyzed the diagnosis records of individuals with these three variants of hACE2. Our molecular dynamics simulations combined with population-based genomic data provided an atomistic understanding of the interaction between hACE2 and the spike protein of SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Angiotensin-Converting Enzyme 2/genetics , COVID-19/epidemiology , COVID-19/genetics , Humans , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Protein Binding , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
20.
J Am Chem Soc ; 144(29): 13060-13065, 2022 Jul 27.
Article in English | MEDLINE | ID: covidwho-1931308

ABSTRACT

We have used chemical shift perturbation (CSP) and saturation transfer difference (STD) NMR experiments to identify and characterize the binding of selected ligands to the receptor-binding domain (RBD) of the spike glycoprotein (S-protein) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We also subjected full-length S-protein to STD NMR experiments, allowing correlations with RBD-based results. CSPs reveal the binding sites for heparin and fondaparinux, and affinities were measured using CSP titrations. We then show that α-2,3-sialyllactose binds to the S-protein but not to the RBD. Finally, combined CSP and STD NMR experiments show that lifitegrast, a compound used for the treatment of dry eye, binds to the linoleic acid (LA) binding pocket with a dissociation constant in the µM range. This is an interesting finding, as lifitegrast lends itself well as a blueprint for medicinal chemistry, eventually furnishing novel entry inhibitors targeting the highly conserved LA binding site.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2 , Binding Sites , COVID-19/drug therapy , Humans , Ligands , Magnetic Resonance Spectroscopy , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
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