scholarly journals A Furin Cleavage Site Inserted into the Spike Protein of SARS-CoV-2: A Structural Implication?

2020 ◽  
Author(s):  
Wei Li
Keyword(s):  
Cleavage Site ◽  
Structural Data ◽  
Cell Entry ◽  
Spike Protein ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Structural Consequence ◽  
Viral Cell Entry

One notable features of the SARS-CoV-2 genome is that the spike protein of SARS-CoV-2 has a functional polybasic (furin) cleavage site (RRAR) at the S1–S2 boundary through the insertion of 12 nucleotides encoding PRRA. To date, the furin cleavage site (FCS) remains an experimentally uncharted territory both structurally and functionally. For instance, whether or not FCS is actually cleaved, before or after viral cell entry or exit, still remains to be experimentally investigated. With currently available structural data, this article presents a computational structural characterization of the FCS inserted into SARS-CoV-2 spike glycoprotein, and puts forward a set of structural hypothesis against the hypothesis of SARS-CoV-2 from purposeful manipulation: (1), the inserted FCS does not alter, neither stabilize nor de-stabilize, the overall structure of SARS-CoV-2 spike glycoprotein; (2), the net structural consequence of FCS is the insertion of a furin cleavage site into SARS-CoV-2 spike glycoprotein, whose S1 and S2 subunits will still be bonded together even if the FCS is actually cleaved by furin protease.

scholarly journals A novel antibody against the furin cleavage site of SARS-CoV-2 spike protein: effects on proteolytic cleavage and ACE2 binding

2021 ◽  
Author(s):  
Michael G. Spelios ◽  
Jeanne M. Capanelli ◽  
Adam W. Li
Keyword(s):  
Cleavage Site ◽  
High Titer ◽  
High Sensitivity ◽  
Cell Entry ◽  
Spike Protein ◽  
Furin Cleavage ◽  
Proteolytic Activation ◽  
Blocking Antibody ◽  
Furin Cleavage Site ◽  
Site Blocking

AbstractSARS-CoV-2 harbors a unique S1/S2 furin cleavage site within its spike protein, which can be cleaved by furin and other proprotein convertases. Proteolytic activation of SARS-CoV-2 spike protein at the S1/S2 boundary facilitates interaction with host ACE2 receptor for cell entry. To address this, high titer antibody was generated against the SARS-CoV-2-specific furin motif. Using a series of innovative ELISA-based assays, this furin site blocking antibody displayed high sensitivity and specificity for the S1/S2 furin cleavage site, and demonstrated effective blockage of both enzyme-mediated cleavage and spike-ACE2 interaction. The results suggest that immunological blocking of the furin cleavage site may afford a suitable approach to stem proteolytic activation of SARS-CoV-2 spike protein and curtail viral infectivity.

scholarly journals The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

2021 ◽  
Vol 22 (12) ◽  
pp. 6490
Author(s):  
Olga A. Postnikova ◽  
Sheetal Uppal ◽  
Weiliang Huang ◽  
Maureen A. Kane ◽  
Rafael Villasmil ◽  
...  
Keyword(s):  
Amino Acid ◽  
Insertion Sequence ◽  
Experimental Studies ◽  
Spike Protein ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
New Host ◽  
S Protein ◽  
Furin Cleavage Site ◽  
Ribosome Pausing

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

scholarly journals Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects

2020 ◽  
Author(s):  
Hanh T. Nguyen ◽  
Shijian Zhang ◽  
Qian Wang ◽  
Saumya Anang ◽  
Jia Wang ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Cleavage Site ◽  
Virus Entry ◽  
Cytoplasmic Tail ◽  
Syncytium Formation ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
Cytopathic Effects ◽  
Furin Cleavage Site

SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects (syncytium formation). We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced syncytium-forming ability. Notably, the D614G change found in globally predominant SARS-CoV-2 strains increased infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, these two features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both host proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent. IMPORTANCE The development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, a process that contributes to the destruction of the virus-infected cell.

scholarly journals Sequence Analysis Indicates that 2019-nCoV Virus Contains a Putative Furin Cleavage Site at the Boundary of S1 and S2 Domains of Spike Protein

2020 ◽  
Author(s):  
Z. Galen WO
Keyword(s):  
Amino Acid ◽  
Cleavage Site ◽  
Spike Protein ◽  
Sequence Motif ◽  
Furin Cleavage ◽  
Host Cell Membrane ◽  
Virus Family ◽  
Prediction Program ◽  
Furin Cleavage Site ◽  
Novel Coronavirus

The infectious 2019-nCoV virus, which caused the current novel coronavirus pneumonia epidemic outbreak, possesses a unique 4-Amino Acid insert at the boundary of the two subdomains (S1 and S2) of Spike protein based on multiple protein sequence alignment with the large SARS and SARS-related virus family. Using Bat CoV_RaTG13 Spike protein as reference (sharing 97% aa identity) the 4-amino acid insert can be identified as PRRA (AA position 681-684). The effect of the 4-AA insertion is the presence of a furin signature sequence motif (PRRARSV) at the boundary of S1 and S2 domains of spike protein. This sequence motif consists the required Arg residue for P1 and P4 position of Furin site. In addition, it contains Arg at P3 site as well as Ser at P1’ site of furin motif. This sequence motif matches Aerolysin furin site in FurinDB and was predicted to be moderately strong (score 0.62) by ProP, a protease cleavage site prediction program. This finding suggests that the infectious 2019-nCoV virus, unlike SARS viruses, may be processed via cellular furin recognition and cleavage of the spike protein before host cell membrane fusion and entry. This putative furin site in spike protein of 2019-nCoV virus, if proven to be functional, suggests the potential of looking into agents inhibiting furin as therapeutic mean for the treatment of the novel coronavirus pneumonia.

scholarly journals The Polybasic Cleavage Site in SARS-CoV-2 Spike Modulates Viral Sensitivity to Type I Interferon and IFITM2

2021 ◽  
Vol 95 (9) ◽  
Author(s):  
Helena Winstone ◽  
Maria Jose Lista ◽  
Alisha C. Reid ◽  
Clement Bouton ◽  
Suzanne Pickering ◽  
...  
Keyword(s):  
Viral Entry ◽  
Cleavage Site ◽  
Type I Interferons ◽  
Spike Protein ◽  
Type I ◽  
Furin Cleavage ◽  
Independent Manner ◽  
Type I Ifns ◽  
Furin Cleavage Site ◽  
Potent Inhibition

ABSTRACT The cellular entry of severe acute respiratory syndrome-associated coronaviruses types 1 and 2 (SARS-CoV-1 and -2) requires sequential protease processing of the viral spike glycoprotein. The presence of a polybasic cleavage site in SARS-CoV-2 spike at the S1/S2 boundary has been suggested to be a factor in the increased transmissibility of SARS-CoV-2 compared to SARS-CoV-1 by facilitating maturation of the spike precursor by furin-like proteases in the producer cells rather than endosomal cathepsins in the target. We investigate the relevance of the polybasic cleavage site in the route of entry of SARS-CoV-2 and the consequences this has for sensitivity to interferons (IFNs) and, more specifically, the IFN-induced transmembrane (IFITM) protein family that inhibit entry of diverse enveloped viruses. We found that SARS-CoV-2 is restricted predominantly by IFITM2, rather than IFITM3, and the degree of this restriction is governed by route of viral entry. Importantly, removal of the cleavage site in the spike protein renders SARS-CoV-2 entry highly pH and cathepsin dependent in late endosomes, where, like SARS-CoV-1 spike, it is more sensitive to IFITM2 restriction. Furthermore, we found that potent inhibition of SARS-CoV-2 replication by type I but not type II IFNs is alleviated by targeted depletion of IFITM2 expression. We propose that the polybasic cleavage site allows SARS-CoV-2 to mediate viral entry in a pH-independent manner, in part to mitigate against IFITM-mediated restriction and promote replication and transmission. This suggests that therapeutic strategies that target furin-mediated cleavage of SARS-CoV-2 spike may reduce viral replication through the activity of type I IFNs. IMPORTANCE The furin cleavage site in the spike protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals, compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell. Here, we show that SARS-CoV-2 is inhibited by antiviral membrane protein IFITM2 and that the sensitivity is exacerbated by deletion of the furin cleavage site, which restricts viral entry to low pH compartments. Furthermore, we find that IFITM2 is a significant effector of the antiviral activity of type I interferons against SARS-CoV-2 replication. We suggest that one role of the furin cleavage site is to reduce SARS-CoV-2 sensitivity to innate immune restriction, and thus, it may represent a potential therapeutic target for COVID-19 treatment development.

Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects

2020 ◽  
Author(s):  
Hanh T. Nguyen ◽  
Shijian Zhang ◽  
Qian Wang ◽  
Saumya Anang ◽  
Jia Wang ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Cleavage Site ◽  
Virus Entry ◽  
Cytoplasmic Tail ◽  
Syncytium Formation ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
Cytopathic Effects ◽  
Furin Cleavage Site

ABSTRACTSARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects. We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced the ability to form lethal syncytia. Notably, the D614G change found in globally predominant SARS-CoV-2 strains restored infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, two unique features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent.IMPORTANCEThe development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, which contributes to the destruction of the virus-infected cell.

scholarly journals SARS-CoV-2 and MERS-CoV Share the Furin Site CGG-CGG Genetic Footprint

2021 ◽  
Author(s):  
Antonio R. Romeu
Keyword(s):  
Middle East ◽  
Markov Model ◽  
Cleavage Site ◽  
Spike Protein ◽  
Cleavage Sites ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Arginine Residues ◽  
Codon Pair ◽  
Shed Light

The SARS-CoV-2 polybasic furin cleavage site is still a missing link. Remarkably, the two arginine residues of this protease recognition site are encoded by the CGG codon, which is rare in Betacoronavirus. However, the arginine pair is common at viral furin cleavage sites, but are not CGG-CGG encoded. The question is: Is this genetic footprint unique to the SARS-CoV-2? To address the issue, using Perl scripts, here I dissect in detail the NCBI Virus database in order to report the arginine dimers of the Betacoronavirus proteins. The main result reveals that a group of Middle East respiratory syndrome-related coronavirus (MERS-CoV) (isolates: camel/Nigeria/NVx/2016, host: Camelus dromedarius) also have the CGG-CGG arginine pair in the spike protein polybasic furin cleavage region. In addition, CGG-CGG encoded arginine pairs were found in the orf1ab polyprotein from HKU9 and HKU14 Betacoronavirus, as well as, in the nucleocapsid phosphoprotein from few SARS-CoV-2 isolates. To quantify the probability of finding the arginine CGG-CGG codon pair in Betacoronavirus, the likelihood ratio (LR) and a Markov model were defined. In conclusion, it is highly unlikely to find this genetic marker in betacoronaviruses wildlife, but they are there. Collectively, results shed light on recombination as origin of the virus CGG-CGG arginine pair in the S1/S2 cleavage site.

scholarly journals QTQTN motif upstream of the furin-cleavage site plays key role in SARS-CoV-2 infection and pathogenesis.

2021 ◽  
Author(s):  
Michelle N Vu ◽  
Kumari Lokugamage ◽  
Jessica A Plante ◽  
Dionna Scharton ◽  
Bryan A Johnson ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Unusual Feature ◽  
Spike Protein ◽  
Loop Length ◽  
Dependent Manner ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Respiratory Cells

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 1,2. 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 3. 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 glycosylated4, and disruption 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. 

scholarly journals Scalable live-attenuated SARS-CoV-2 vaccine candidate demonstrates preclinical safety and efficacy

2021 ◽  
Vol 118 (29) ◽  
pp. e2102775118
Author(s):  
Ying Wang ◽  
Chen Yang ◽  
Yutong Song ◽  
J. Robert Coleman ◽  
Marcin Stawowczyk ◽  
...  
Keyword(s):  
Weight Loss ◽  
Cleavage Site ◽  
Mass Vaccination ◽  
Amino Acid Sequences ◽  
Spike Protein ◽  
Host Immune System ◽  
Permissive Temperature ◽  
Lung Pathology ◽  
Furin Cleavage ◽  
Furin Cleavage Site

Successfully combating the COVID-19 pandemic depends on mass vaccination with suitable vaccines to achieve herd immunity. Here, we describe COVI-VAC, the only live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine currently in clinical development. COVI-VAC was developed by recoding a segment of the viral spike protein with synonymous suboptimal codon pairs (codon-pair deoptimization), thereby introducing 283 silent (point) mutations. In addition, the furin cleavage site within the spike protein was deleted from the viral genome for added safety of the vaccine strain. Except for the furin cleavage site deletion, the COVI-VAC and parental SARS-CoV-2 amino acid sequences are identical, ensuring that all viral proteins can engage with the host immune system of vaccine recipients. COVI-VAC was temperature sensitive in vitro yet grew robustly (>107 plaque forming units/mL) at the permissive temperature. Tissue viral loads were consistently lower, lung pathology milder, and weight loss reduced in Syrian golden hamsters (Mesocricetus auratus) vaccinated intranasally with COVI-VAC compared to those inoculated with wild-type (WT) virus. COVI-VAC inoculation generated spike IgG antibody levels and plaque reduction neutralization titers similar to those in hamsters inoculated with WT virus. Upon challenge with WT virus, COVI-VAC vaccination reduced lung challenge viral titers, resulted in undetectable virus in the brain, and protected hamsters from almost all SARS-CoV-2–associated weight loss. Highly attenuated COVI-VAC is protective at a single intranasal dose in a relevant in vivo model. This, coupled with its large-scale manufacturing potential, supports its potential use in mass vaccination programs.

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