scholarly journals The SARS-CoV-2 Transcriptome and the Dynamics of the S Gene Furin Cleavage Site in Primary Human Airway Epithelia

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Wei Zou ◽  
Min Xiong ◽  
Siyuan Hao ◽  
Elizabeth Yan Zhang ◽  
Nathalie Baumlin ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Vero Cells ◽  
High Rate ◽  
Furin Cleavage ◽  
Airway Epithelia ◽  
Human Airway ◽  
Furin Cleavage Site ◽  
Air Liquid Interface ◽  
Human Airway Epithelia

ABSTRACT The spike (S) polypeptide of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary that contains a furin cleavage site (FCS), 682RRAR↓S686. Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, it acquired deletions surrounding the FCS. We studied the viral transcriptome in Vero cell-derived SARS-CoV-2-infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability of the FCS. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the FCS region: a 12 amino acid deletion (678TNSPRRAR↓SVAS689) that contains the underlined FCS and a 5 amino acid deletion (675QTQTN679) that is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions from 11 infected HAE-ALI cultures of both healthy and lung disease donors revealed that the selective pressure for the FCS maintains the FCS stably in 9 HAE-ALI cultures but with 2 exceptions, in which the FCS deletions are retained at a high rate of >40% after infection of ≥13 days. Our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is likely donor dependent. IMPORTANCE Polarized human airway epithelia at an air-liquid interface (HAE-ALI) are an in vitro model that supports efficient infection of SARS-CoV-2. The spike (S) protein of SARS-CoV-2 contains a furin cleavage site (FCS) at the boundary of the S1 and S2 domains which distinguishes it from SARS-CoV. However, FCS deletion mutants have been identified in patients and in vitro cell cultures, and how the airway epithelial cells maintain the unique FCS remains unknown. We found that HAE-ALI cultures were capable of suppressing two prevalent FCS deletion mutants (Δ678TNSPRRAR↓SVAS689 and Δ675QTQTN679) that were selected during propagation in Vero cells. While such suppression was observed in 9 out of 11 of the tested HAE-ALI cultures derived from independent donors, 2 exceptions that retained a high rate of FCS deletions were also found. Our results present evidence of the donor-dependent properties of human airway epithelia in the evolution of the FCS during infection.

scholarly journals The SARS-CoV-2 transcriptome and the dynamics of the S gene furin cleavage site in primary human airway epithelia

2021 ◽  
Author(s):  
Wei Zou ◽  
Min Xiong ◽  
Siyuan Hao ◽  
Elizabeth Yan Zhang-Chen ◽  
Nathalie Baumlin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Viral Genome ◽  
Cleavage Site ◽  
Vero Cells ◽  
Furin Cleavage ◽  
Airway Epithelia ◽  
Human Airway ◽  
S Gene ◽  
Furin Cleavage Site ◽  
Human Airway Epithelia

AbstractThe novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused the devastating ongoing coronavirus disease-2019 (COVID-19) pandemic which poses a great threat to global public health. The spike (S) polypeptide of SARS-CoV-2 consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary. The inclusion of the 4 amino acids (PRRA) at the S1/S2 boundary forms a furin cleavage site (FCS), 682RRAR↓S686, distinguishing SARS-CoV-2 from its closest relative, the SARS-CoV. Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, the virus acquired various deletions surrounding the FCS. In the present study, we studied the viral transcriptome in SARS-CoV-2 infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability at the S1/S2 boundary using RNA-seq. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the S1/S2 boundary of the S gene: one is a deletion of 12 amino acids, 678TNSPRRAR↓SVAS689, which contains the FCS, another is a deletion of 5 amino acids, 675QTQTN679, which is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions revealed that the selective pressure for the FCS maintains the S gene stability in HAE-ALI but with exceptions, in which the FCS deletions are remained at a high rate. Thus, our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is donor-dependent.

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

2020 ◽  
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.

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

Toxins ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 593 ◽  
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.

scholarly journals In Vitro Modeling of Human Bocavirus 1 Infection of Polarized Primary Human Airway Epithelia

2013 ◽  
Vol 87 (7) ◽  
pp. 4097-4102 ◽  
Author(s):  
X. Deng ◽  
Z. Yan ◽  
Y. Luo ◽  
J. Xu ◽  
F. Cheng ◽  
...  
Keyword(s):  
Human Bocavirus ◽  
Airway Epithelia ◽  
In Vitro Modeling ◽  
Human Airway ◽  
Human Airway Epithelia

scholarly journals Growth and characterization of different human rhinovirus C types in three-dimensional human airway epithelia reconstituted in vitro

Virology ◽  
2013 ◽  
Vol 446 (1-2) ◽  
pp. 1-8 ◽  
Author(s):  
Caroline Tapparel ◽  
Komla Sobo ◽  
Samuel Constant ◽  
Song Huang ◽  
Sandra Van Belle ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Human Rhinovirus ◽  
Airway Epithelia ◽  
Human Airway ◽  
Rhinovirus C ◽  
Human Airway Epithelia

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

npj Vaccines ◽  
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.

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 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. 

An In Vitro Model of Differentiated Human Airway Epithelia: Methods for Establishing Primary Cultures

2003 ◽  
pp. 115-137 ◽  
Author(s):  
Philip H. Karp ◽  
Thomas O. Moninger ◽  
S. Pary Weber ◽  
Tamara S. Nesselhauf ◽  
Janice L. Launspach ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Primary Cultures ◽  
Airway Epithelia ◽  
Human Airway ◽  
Vitro Model ◽  
Human Airway Epithelia

scholarly journals The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia

2011 ◽  
Vol 300 (1) ◽  
pp. L25-L31 ◽  
Author(s):  
Alejandro A. Pezzulo ◽  
Timothy D. Starner ◽  
Todd E. Scheetz ◽  
Geri L. Traver ◽  
Ann E. Tilley ◽  
...  
Keyword(s):  
Expression Profile ◽  
Primary Cultures ◽  
Liquid Interface ◽  
Transcriptional Profile ◽  
Surface Epithelium ◽  
Airway Epithelia ◽  
Human Airway ◽  
Air Liquid Interface ◽  
Human Airway Epithelia

Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.

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