The S2 Subunit of QX-type Infectious Bronchitis Coronavirus Spike Protein Is an Essential Determinant of Neurotropism

Some coronaviruses (CoVs) have an extra furin cleavage site (RRKR/S, furin-S2′ site) upstream of the fusion peptide in the spike protein, which plays roles in virion adsorption and fusion. Mutation of the S2′ site of QX genotype (QX-type) infectious bronchitis virus (IBV) spike protein (S) in a recombinant virus background results in higher pathogenicity, pronounced neural symptoms and neurotropism when compared with conditions in wild-type IBV (WT-IBV) infected chickens. In this study, we present evidence suggesting that recombinant IBV with a mutant S2′ site (furin-S2′ site) leads to higher mortality. Infection with mutant IBV induces severe encephalitis and breaks the blood–brain barrier. The results of a neutralization test and immunoprotection experiment show that an original serum and vaccine can still provide effective protection in vivo and in vitro. This is the first demonstration of IBV-induced neural symptoms in chickens with encephalitis and the furin-S2′ site as a determinant of neurotropism.

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

10.1101/2021.12.15.472450 ◽

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.

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Inclusion of a Furin Cleavage Site Enhances Antitumor Efficacy against Colorectal Cancer Cells of Ribotoxin α-Sarcin- or RNase T1-Based Immunotoxins

Toxins ◽

10.3390/toxins11100593 ◽

2019 ◽

Vol 11 (10) ◽

pp. 593 ◽

Cited By ~ 5

Author(s):

Javier Ruiz-de-la-Herrán ◽

Jaime Tomé-Amat ◽

Rodrigo Lázaro-Gorines ◽

José G. Gavilanes ◽

Javier Lacadena

Keyword(s):

Cleavage Site ◽

Tumor Antigens ◽

Functional Characterization ◽

Target Cells ◽

Furin Cleavage ◽

Ribonucleolytic Activity ◽

Furin Cleavage Site ◽

Potential Therapeutic Application

Immunotoxins are chimeric molecules that combine the specificity of an antibody to recognize and bind tumor antigens with the potency of the enzymatic activity of a toxin, thus, promoting the death of target cells. Among them, RNases-based immunotoxins have arisen as promising antitumor therapeutic agents. In this work, we describe the production and purification of two new immunoconjugates, based on RNase T1 and the fungal ribotoxin α-sarcin, with optimized properties for tumor treatment due to the inclusion of a furin cleavage site. Circular dichroism spectroscopy, ribonucleolytic activity studies, flow cytometry, fluorescence microscopy, and cell viability assays were carried out for structural and in vitro functional characterization. Our results confirm the enhanced antitumor efficiency showed by these furin-immunotoxin variants as a result of an improved release of their toxic domain to the cytosol, favoring the accessibility of both ribonucleases to their substrates. Overall, these results represent a step forward in the design of immunotoxins with optimized properties for potential therapeutic application in vivo.

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Gene of the month: the 2019-nCoV/SARS-CoV-2 novel coronavirus spike protein

Journal of Clinical Pathology ◽

10.1136/jclinpath-2020-206658 ◽

2020 ◽

Vol 73 (7) ◽

pp. 366-369 ◽

Cited By ~ 23

Author(s):

Tahir S Pillay

Keyword(s):

Receptor Binding ◽

Transmembrane Domain ◽

Vaccine Development ◽

Protein S ◽

Respiratory Illness ◽

Spike Protein ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Cellular Entry ◽

Novel Coronavirus

The year 2020 has seen a major and sustained outbreak of a novel betacoronavirus (severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2) infection that causes fever, severe respiratory illness and pneumonia, a disease called COVID-19. At the time of writing, the death toll was greater than 120 000 worldwide with more than 2 million documented infections. The genome of the CoV encodes a number of structural proteins that facilitate cellular entry and assembly of virions, of which the spike protein S appears to be critical for cellular entry. The spike protein guides the virus to attach to the host cell. The spike protein contains a receptor-binding domain (RBD), a fusion domain and a transmembrane domain. The RBD of spike protein S binds to Angiotensin Converting Enzyme 2 (ACE2) to initiate cellular entry. The spike protein of SARS-CoV-2 shows more than 90% amino acid similarity to the pangolin and bat CoVs and these also use ACE2 as a receptor. Binding of the spike protein to ACE2 exposes the cleavage sites to cellular proteases. Cleavage of the spike protein by transmembrane protease serine 2 and other cellular proteases initiates fusion and endocytosis. The spike protein contains an addition furin cleavage site that may allow it to be ‘preactivated’ and highly infectious after replication. The fundamental role of the spike protein in infectivity suggests that it is an important target for vaccine development, blocking therapy with antibodies and diagnostic antigen-based tests. This review briefly outlines the structure and function of the 2019 novel CoV/SARS-CoV-2 spike protein S.

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A cautionary perspective regarding the isolation and serial propagation of SARS-CoV-2 in Vero cells

npj Vaccines ◽

10.1038/s41541-021-00346-z ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Simon G. P. Funnell ◽

Babak Afrough ◽

John James Baczenas ◽

Neil Berry ◽

Kevin R. Bewley ◽

...

Keyword(s):

Clinical Trials ◽

Genetic Variants ◽

Animal Studies ◽

Vero Cells ◽

In Vitro Assays ◽

Critical Research ◽

Furin Cleavage ◽

Furin Cleavage Site

AbstractAn array of SARS-CoV-2 virus variants have been isolated, propagated and used in in vitro assays, in vivo animal studies and human clinical trials. Observations of working stocks of SARS-CoV-2 suggest that sequential propagation in Vero cells leads to critical changes in the region of the furin cleavage site, which significantly reduce the value of the working stock for critical research studies. Serially propagating SARS-CoV-2 in Vero E6 cells leads to rapid increases in genetic variants while propagation in other cell lines (e.g. Vero/hSLAM) appears to mitigate this risk thereby improving the overall genetic stability of working stocks. From these observations, investigators are urged to monitor genetic variants carefully when propagating SARS-CoV-2 in Vero cells.

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Characterization of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein

Nature Communications ◽

10.1038/s41467-021-23166-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Pui Wang ◽

Siu-Ying Lau ◽

Shaofeng Deng ◽

Pin Chen ◽

Bobo Wing-Yee Mok ◽

...

Keyword(s):

Neutralizing Antibody ◽

T Cell Response ◽

Spike Protein ◽

Wild Type Virus ◽

Wild Type ◽

Cleavage Motif ◽

Sterilizing Immunity

AbstractSARS-CoV-2 is of zoonotic origin and contains a PRRA polybasic cleavage motif which is considered critical for efficient infection and transmission in humans. We previously reported on a panel of attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction of the spike protein. Here, we characterize pathogenicity, immunogenicity, and protective ability of a further cell-adapted SARS-CoV-2 variant, Ca-DelMut, in in vitro and in vivo systems. Ca-DelMut replicates more efficiently than wild type or parental virus in Vero E6 cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut causes no obvious pathological changes and does not induce elevation of proinflammatory cytokines, but still triggers a strong neutralizing antibody and T cell response in hamsters and mice. Ca-DelMut immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, with little sign of virus replication in the upper or lower respiratory tract, demonstrating sterilizing immunity.

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SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients

10.1101/2020.06.19.154930 ◽

2020 ◽

Cited By ~ 3

Author(s):

William B. Klimstra ◽

Natasha L. Tilston-Lunel ◽

Sham Nambulli ◽

James Boslett ◽

Cynthia M. McMillen ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Serum Samples ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Similar Disease ◽

Antibody Titers ◽

Low Passage

AbstractSARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

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Ad26-vector based COVID-19 vaccine encoding a prefusion stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses

10.1101/2020.07.30.227470 ◽

2020 ◽

Cited By ~ 3

Author(s):

Rinke Bos ◽

Lucy Rutten ◽

Joan E.M. van der Lubbe ◽

Mark J.G. Bakkers ◽

Gijs Hardenberg ◽

...

Keyword(s):

Signal Peptide ◽

Neutralizing Antibodies ◽

Etiologic Agent ◽

Wild Type ◽

Furin Cleavage ◽

S Protein ◽

Design Elements ◽

Furin Cleavage Site ◽

Prophylactic Measures

AbstractDevelopment of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g. prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S1) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild type signal peptide was best suited for the correct cleavage needed for a natively-folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-γ. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).

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Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses

npj Vaccines ◽

10.1038/s41541-020-00243-x ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 6

Author(s):

Rinke Bos ◽

Lucy Rutten ◽

Joan E. M. van der Lubbe ◽

Mark J. G. Bakkers ◽

Gijs Hardenberg ◽

...

Keyword(s):

Signal Peptide ◽

Neutralizing Antibodies ◽

Etiologic Agent ◽

Wild Type ◽

Furin Cleavage ◽

S Protein ◽

Design Elements ◽

Furin Cleavage Site ◽

Prophylactic Measures

Abstract Development of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g., prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S2) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild-type signal peptide was best suited for the correct cleavage needed for a natively folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild-type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-γ. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).

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Pathogenicity, immunogenicity, and protective ability of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein

10.21203/rs.3.rs-78635/v1 ◽

2020 ◽

Author(s):

Pui Wang ◽

Siu-Ying Lau ◽

Shaofeng Deng ◽

Pin Chen ◽

Bobo Mok ◽

...

Keyword(s):

Lower Respiratory Tract ◽

Neutralizing Antibody ◽

Spike Protein ◽

Wild Type Virus ◽

Wild Type ◽

Protective Ability ◽

Cleavage Motif ◽

Sterilizing Immunity

Abstract SARS-CoV-2 is zoonotic origin and contains a PRRA polybasic cleavage motif which is considered critical for efficient infection and transmission in humans. We previously reported on a panel of attenuated SARS-CoV-2 variants with deletion at the S1/S2 junction of spike protein. Here we characterize pathogenicity, immunogenicity, and protective ability of a further cell-adapted SARS-CoV-2 variant, Ca-DelMut, in in vitro and in vivo systems. Ca-DelMut replicates more efficiently than wild type or parental virus in cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut causes no apparent pathological changes and does not induce elevated proinflammatory cytokines in hamster infections, but still triggers a strong neutralizing antibody response in hamsters. Ca-DelMut immunized hamsters challenged with wild type SARS-CoV-2 are fully protected with no sign of virus replication in the upper or lower respiratory tract of challenged animals, demonstrating sterilizing immunity.

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Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis

10.1101/2020.08.26.268854 ◽

2020 ◽

Cited By ~ 11

Author(s):

Bryan A. Johnson ◽

Xuping Xie ◽

Birte Kalveram ◽

Kumari G. Lokugamage ◽

Antonio Muruato ◽

...

Keyword(s):

Cleavage Site ◽

Critical Role ◽

Vero Cells ◽

Mutant Virus ◽

Spike Protein ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Wide Range ◽

The World

AbstractSARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site (ΔPRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the ΔPRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the ΔPRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT50) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays.ImportanceAs COVID-19 has impacted the world, understanding how SARS-CoV-2 replicates and causes virulence offers potential pathways to disrupt its disease. By removing the furin cleavage site, we demonstrate the importance of this insertion to SARS-CoV-2 replication and pathogenesis. In addition, the findings with Vero cells indicate the likelihood of cell culture adaptations in virus stocks that can influence reagent generation and interpretation of a wide range of data including neutralization and drug efficacy. Overall, our work highlights the importance of this key motif in SARS-CoV-2 infection and pathogenesis.Article SummaryA deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro.

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