scholarly journals A review of COVID-19: Molecular basis, diagnosis, therapeutics and prevention

2020 ◽  
Vol 4 (3) ◽  
pp. First
Author(s):  
Mai T. N. HUYNH ◽  
Phuc H. T. NGUYEN ◽  
Hieu H. C. PHAN ◽  
Nghia T. H. PHAN ◽  
Kong H. LE ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Vaccine Development ◽  
Transmission Rate ◽  
Cathepsin L ◽  
Virus Transmission ◽  
Open Reading Frames ◽  
Furin Cleavage ◽  
Viral Pathogen ◽  
S Protein ◽  
Nucleoprotein N

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the confirmed viral pathogen of COVID-19, a pandemic originated from Wuhan, China at the end of 2019. Since then, SARS-CoV-2 has rapidly spread across the globe with over 8 million confirmed cases and more than 430.000 deaths worldwide as of mid-June 2020. Similar to other strains of coronavirus, the envelope of SARS-CoV-2 comprises of three structural proteins: S protein (spike), E protein (envelope) and M glycoprotein (membrane). SARS-CoV-2 capsids are spherical or pleomorphic. Each capsid contains a positive-sense single-stranded RNA (+ssRNA-Class IV-Baltimore) associated with nucleoprotein N. The viral RNA genome is approximately 30 kb in length and contains 14 open reading frames (ORFs). The binding affinity of the viral S protein to the ACE2 (angiotensin-converting enzyme 2) receptor facilitates the attachment of SARS-CoV-2 to human epithelial cells. Upon binding, SARS-CoV-2 spike protein is cleaved and activated by TMPRSS2 (transmembrane protease, serine 2) or by cathepsin L at the cleavage site S2', and also by furin at the cleavage site S1/S2. The furin cleavage motif RR_R is a notable feature, firstly found in SARS-CoV-2 S protein, which may increase virus transmission rate. This feature and many others might result from several evolution events in SARS-CoV-2 genome. These events could occur when coronaviruses, including SARS-CoV-2, spread from one host to another. They can be causative to high virulence and transmission rate of future coronavirus strains, which may require the development of newer vaccine generations. To understand of SARS-CoV-2’s structure, infection mechanism, diagnosis, treatment, and vaccine development strategies, a review of current literature is of highly importance to disease control in Vietnam.

scholarly journals Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2

2020 ◽  
Author(s):  
Tianling Ou ◽  
Huihui Mou ◽  
Lizhou Zhang ◽  
Amrita Ojha ◽  
Hyeryun Choe ◽  
...  
Keyword(s):  
Cell Culture ◽  
Clinical Trials ◽  
Viral Infection ◽  
Viral Entry ◽  
Cleavage Site ◽  
Cathepsin L ◽  
Furin Cleavage ◽  
S Protein ◽  
Furin Cleavage Site

AbstractHydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.Author SummaryThe novel pathogenic coronavirus SARS-CoV-2 causes COVID-19 and remains a threat to global public health. Chloroquine and hydroxychloroquine have been shown to prevent viral infection in cell-culture systems, but human clinical trials did not observe a significant improvement in COVID-19 patients treated with these compounds. Here we show that hydroxychloroquine interferes with only one of two somewhat redundant pathways by which the SARS-CoV-2 spike (S) protein is activated to mediate infection. The first pathway is dependent on the endosomal protease cathepsin L and sensitive to hydroxychloroquine, whereas the second pathway is dependent on TMPRSS2, which is unaffected by this compound. We further show that SARS-CoV-2 is more reliant than SARS coronavirus (SARS-CoV-1) on the TMPRSS2 pathway, and that this difference is due to a furin cleavage site present in the SARS-CoV-2 S protein. Finally, we show that combinations of hydroxychloroquine and a clinically tested TMPRSS2 inhibitor work together to effectively inhibit SARS-CoV-2 entry. Thus TMPRSS2 expression on physiologically relevant SARS-CoV-2 target cells may bypass the antiviral activities of hydroxychloroquine, and explain its lack of in vivo efficacy.

scholarly journals Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

2018 ◽  
Vol 92 (19) ◽  
Author(s):  
Shutoku Matsuyama ◽  
Kazuya Shirato ◽  
Miyuki Kawase ◽  
Yutaka Terada ◽  
Kengo Kawachi ◽  
...  
Keyword(s):  
Middle East ◽  
Viral Entry ◽  
Cathepsin L ◽  
Inhibitory Effect ◽  
Middle East Respiratory Syndrome ◽  
Cell Entry ◽  
Target Cells ◽  
Furin Cleavage ◽  
S Protein ◽  
Entry Assay

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin. IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.

scholarly journals The trypsin-enhanced infection of porcine epidemic diarrhea virus is determined by the S2 subunit of the spike glycoprotein

2021 ◽  
Author(s):  
Yubei Tan ◽  
Limeng Sun ◽  
Gang Wang ◽  
Yuejun Shi ◽  
Wanyu Dong ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Cleavage Site ◽  
Vaccine Development ◽  
Syncytium Formation ◽  
Reverse Genetic System ◽  
Site Mutation ◽  
Swine Industry ◽  
S Protein ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) is an enteric pathogen in the swine industry, causing high mortality in neonatal piglets. Efficient PEDV infection usually relies on the presence of trypsin, yet the mechanism of trypsin dependency is ambiguous. Here, we identified two PEDV strains, trypsin-enhanced YN200 and trypsin-independent DR13, in which the spike (S) protein of YN200 exhibits a stronger ability to induce syncytium formation and cleaved by trypsin than that of DR13. Using a full-length infectious YN200 cDNA clone, we confirmed that the S protein is a trypsin dependency determinant by comparison of rYN200 and rYN200-SDR13. To explore the trypsin-associated sites of the YN200 S protein, we then constructed a series of mutations adjacent to the fusion peptide. The results show that the putative S2’ cleavage site (R892G) is not the determinant for virus trypsin dependency. Hence, we generated viruses carrying chimeric S proteins: the S1 subunit, S2 subunit, and S2720∼892 aa domain (NS2’) were individually replaced by the corresponding DR13 sequences. Intriguingly, only the S2 substitution, not the S1 or NS2’ substitutions, provides trypsin-independent growth of YN200. Additionally, the NS2’ recombinant virus significantly abrogated effective infection, indicating a vital role for NS2’ in viral entry. These findings suggest that the trypsin dependency of PEDV is mainly controlled by mutations in the S2 subunit rather than directly trypsin cleavage site. Importance With the emergence of new variants, PEDV remains a major problem in the global swine industry. Efficient PEDV infection usually requires trypsin, while the mechanism of trypsin dependency is complex. Here, we used two PEDV strains, trypsin-enhanced YN200 and trypsin-independent DR13, and results showed that the S protein determined PEDV trypsin dependency by using a reverse genetic system of YN200. The S2 subunit was verified as the main portion of PEDV trypsin dependency, though the putative S2’ site mutation cannot render trypsin-independent growth of YN200. Finally, these results provide some different insight to the PEDV trypsin dependency and might inspire vaccine development.

Identification of a Potent Inhibitor Targeting the Spike Protein of Pandemic Human Coronavirus, SARS-CoV-2 by Computational Methods

2020 ◽  
Author(s):  
SRUTHI UNNI ◽  
Snehal Aouti ◽  
Padmanabhan Balasundaram
Keyword(s):  
Vaccine Development ◽  
Protein S ◽  
Dynamics Simulation ◽  
Spike Protein ◽  
Simulation Studies ◽  
Viral Pathogen ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Chemical Libraries ◽  
Pi Interactions

<p>Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is an emerging new viral pathogen that causes severe respiratory disease. SARS-CoV-2 is responsible for an outbreak of COVID-19 pandemic worldwide. As there are no confirmed antiviral drugs or vaccines currently available for the treatment of COVID-19, discovering potent inhibitors or vaccines are urgently required for the benefit of humanity. The glycosylated Spike protein (S-protein) directly interacts with human angiotensin-converting enzyme 2 (ACE2) receptor through the receptor-binding domain (RBD) of S-protein. As the S-protein is exposed to the surface and is essential for entry into the host, the S-protein can be considered as a first-line therapeutic target for antiviral therapy and vaccine development. In-silico screening, docking and molecular dynamics simulation studies were performed to identify repurposing drugs using DrugBank and PubChem library against the RBD of S-protein. The study identified a laxative drug, Bisoxatin (DB09219), which is used for the treatment of constipation and preparation of the colon for surgical procedures. It binds nicely at the S-protein – ACE2 interface by making substantial pi-pi interactions with Tyr505 in the ‘Site 1’ hook region of RBD and hydrophilic interactions with Glu406, Ser494 and Thr500. Bisoxatin consistently binds to the protein throughout the 100 ns simulation. Taken together, we propose that the discovered molecule, Bisoxatin may be a potent repurpose drug to develop new chemical libraries for inhibiting SARS-CoV-2 entry into the host.</p>

Identification of a Potent Inhibitor Targeting the Spike Protein of Pandemic Human Coronavirus, SARS-CoV-2 by Computational Methods

2020 ◽  
Author(s):  
SRUTHI UNNI ◽  
Snehal Aouti ◽  
Padmanabhan Balasundaram
Keyword(s):  
Vaccine Development ◽  
Protein S ◽  
Dynamics Simulation ◽  
Spike Protein ◽  
Simulation Studies ◽  
Viral Pathogen ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Chemical Libraries ◽  
Pi Interactions

<p>Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is an emerging new viral pathogen that causes severe respiratory disease. SARS-CoV-2 is responsible for an outbreak of COVID-19 pandemic worldwide. As there are no confirmed antiviral drugs or vaccines currently available for the treatment of COVID-19, discovering potent inhibitors or vaccines are urgently required for the benefit of humanity. The glycosylated Spike protein (S-protein) directly interacts with human angiotensin-converting enzyme 2 (ACE2) receptor through the receptor-binding domain (RBD) of S-protein. As the S-protein is exposed to the surface and is essential for entry into the host, the S-protein can be considered as a first-line therapeutic target for antiviral therapy and vaccine development. In-silico screening, docking and molecular dynamics simulation studies were performed to identify repurposing drugs using DrugBank and PubChem library against the RBD of S-protein. The study identified a laxative drug, Bisoxatin (DB09219), which is used for the treatment of constipation and preparation of the colon for surgical procedures. It binds nicely at the S-protein – ACE2 interface by making substantial pi-pi interactions with Tyr505 in the ‘Site 1’ hook region of RBD and hydrophilic interactions with Glu406, Ser494 and Thr500. Bisoxatin consistently binds to the protein throughout the 100 ns simulation. Taken together, we propose that the discovered molecule, Bisoxatin may be a potent repurpose drug to develop new chemical libraries for inhibiting SARS-CoV-2 entry into the host.</p>

scholarly journals Natural isolate and recombinant SARS-CoV-2 rapidly evolve in vitro to higher infectivity through more efficient binding to heparan sulfate and reduced S1/S2 cleavage

2021 ◽  
Author(s):  
Nikita Shiliaev ◽  
Tetyana Lukash ◽  
Oksana Palchevska ◽  
David K Crossman ◽  
Todd J. Green ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Cleavage Site ◽  
Heparin Binding ◽  
Natural Isolate ◽  
Furin Cleavage ◽  
S Protein ◽  
Viral Spread ◽  
Furin Cleavage Site ◽  
Viral Particles

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant, cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increase the positive charge of the surface of this domain, ii) insertions into NTD of heterologous peptides, containing positively charged amino acids, and iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide and makes viruses less capable of syncytia formation. These viral adaptations result in higher affinity of viral particles to heparin sepharose, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers and two orders of magnitude lower GE:PFU ratios. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA+ viruses, evolution to HS binding may result in virus attenuation in vivo.

scholarly journals D614G mutation alters SARS-CoV-2 spike conformational dynamics and protease cleavage susceptibility at the S1/S2 junction

2020 ◽  
Author(s):  
Sophie M-C. Gobeil ◽  
Katarzyna Janowska ◽  
Shana McDowell ◽  
Katayoun Mansouri ◽  
Robert Parks ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Conformational Dynamics ◽  
Vaccine Design ◽  
List Type ◽  
Allosteric Effect ◽  
Furin Cleavage ◽  
Dominant Form ◽  
S Protein ◽  
Protease Cleavage ◽  
Furin Cleavage Site

AbstractThe SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we analyze the D614G mutation in the context of a soluble S ectodomain construct. Cryo-EM structures, antigenicity and proteolysis experiments suggest altered conformational dynamics resulting in enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage altered the conformational dynamics of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on the RBD dynamics triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational dynamics and allostery, and have implications for vaccine design.HighlightsSARS-CoV-2 S ectodomains with or without the K986P, V987P mutations have similar structures, antigenicity and stability.The D614G mutation alters S protein conformational dynamics.D614G enhances protease cleavage susceptibility at the S protein furin cleavage site.Cryo-EM structures reveal allosteric effect of changes at the S1/S2 junction on RBD dynamics.

scholarly journals Emergence of N antigen SARS-CoV-2 genetic variants escaping detection of antigenic tests

2021 ◽  
Author(s):  
Claudia Del Vecchio ◽  
Giuseppina Brancaccio ◽  
Alessandra Rosalba Brazzale ◽  
Enrico Lavezzo ◽  
Francesco Onelia ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Vaccine Development ◽  
Transmission Rate ◽  
Herd Immunity ◽  
Virus Transmission ◽  
Host Cells ◽  
N Gene ◽  
Sequencing Analysis ◽  
Antigen Test ◽  
Rt Pcr

SARS-CoV-2 genetic variants are emerging as a major threat to vaccination efforts worldwide as they may increase virus transmission rate and/or confer the ability to escape vaccine induced immunity with knock on effects on the level of herd immunity and vaccine efficacy respectively. These variants concern the Spike protein, which is encoded by the S gene, involved in virus entry into host cells and the major target of vaccine development. We report here that genetic variants of the N gene can impair our ability to utilize antigenic tests for both diagnosis and mass testing efforts aimed at controlling virus transmission. While conducting a large validation study on the Abbott Panbio COVID-19 Ag test, we noticed that some swab samples failed to generate a positive result in spite of a high viral load in Rt-PCR assays. Sequencing analysis of viruses showing discordant results in the Rt-PCR and antigen assays revealed the presence of multiple disruptive amino-acid substitutions in the N antigen (the viral protein detected in the antigen test) clustered from position 229 to 374 a region known to contain an immunodominant epitope. A relevant fraction of the variants, undetected by the antigen test, contained the mutations A376T coupled to M241I. Intriguingly we found that virus sequences with this mutation were over-represented in the antigen-test-negative and PCR-positive samples and progressively increased in frequency over time in Veneto, a region of Italy that has aggressively scaled up the utilization of antigen tests, which reached nearly 68% of all the SARS-CoV-2 swab assays performed there. We speculate that mass utilization of antigen assays could create a selection pressure on the target that may favor the spread of undetectable virus variants.

scholarly journals A Single Point Mutation Creating a Furin Cleavage Site in the Spike Protein Renders Porcine Epidemic Diarrhea Coronavirus Trypsin Independent for Cell Entry and Fusion

2015 ◽  
Vol 89 (15) ◽  
pp. 8077-8081 ◽  
Author(s):  
Wentao Li ◽  
Oliver Wicht ◽  
Frank J. M. van Kuppeveld ◽  
Qigai He ◽  
Peter J. M. Rottier ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Virus Propagation ◽  
Furin Cleavage ◽  
S Protein ◽  
Diarrhea Virus ◽  
Furin Cleavage Site ◽  
Porcine Epidemic Diarrhea

The emerging porcine epidemic diarrhea virus (PEDV) requires trypsin supplementation to activate its S protein for membrane fusion and virus propagation in cell culture. By substitution of a single amino acid in the S protein, we created a recombinant PEDV with an artificial furin protease cleavage site N terminal of the putative fusion peptide (PEDV-SFCS). PEDV-SFCSexhibited trypsin-independent cell-cell fusion and was able to replicate in culture cells independently of trypsin, though to low titer.

scholarly journals Identification of NPC1 as a novel SARS-CoV-2 intracellular target

2020 ◽  
Author(s):  
Isabel Garcia-Dorival ◽  
Miguel Ángel Cuesta-Geijo ◽  
Lucía Barrado-Gil ◽  
Inmaculada Galindo ◽  
Jesús Urquiza ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Cathepsin L ◽  
Viral Fusion ◽  
S Protein ◽  
Endosomal Membrane ◽  
Common Strategy ◽  
Intracellular Target ◽  
Niemann Pick ◽  
Transmembrane Serine Protease ◽  
Nucleoprotein N

AbstractNiemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol trafficking, which is crucial in the Ebola virus (EBOV) cycle. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cell by binding of the viral spike (S) protein to the ACE2 receptor. This requires S-protein processing either by the surface transmembrane serine protease TMPRSS2 for plasma membrane fusion or cathepsin L for endosomal entry. Additional host factors are required for viral fusion at endosomes. Here, we report a novel interaction of the SARS-CoV-2 nucleoprotein (N) with the cholesterol transporter NPC1. Moreover, small molecules interfering with NPC1 that inhibit EBOV entry, also inhibited human coronavirus. Our findings suggest an important role for NPC1 in SARS-CoV-2 infection, a common strategy shared with EBOV, and a potential therapeutic target to fight against COVID-19.

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