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 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 A 20 Residues Motif Delineates the Furin Cleavage Site and its Physical Properties May Influence Viral Fusion

2009 ◽  
Vol 2 ◽  
pp. BCI.S2049 ◽  
Author(s):  
Sun Tian
Keyword(s):  
Amino Acids ◽  
Physical Properties ◽  
Cleavage Site ◽  
Binding Pocket ◽  
Core Region ◽  
Polar Regions ◽  
Viral Fusion ◽  
Furin Cleavage ◽  
The Core ◽  
Furin Cleavage Site

Furin is a proprotein convertase that proteolytically cleaves protein precursors to yield functional proteins. Efficient cleavage depends on the presence of a specific sequence motif on the substrate. Currently, the cleavage site motif is described as a four amino acid pattern: R-X-[K/R]-R⇓. However, not all furin cleavage recognition sites can be described by this pattern and not all R-X-[K/R]-R⇓ sites are cleaved by furin. Since many furin substrates are involved in the pathogenesis of viral infection and human diseases, it is important to accurately characterize the furin cleavage site motif. In this study, the furin cleavage site motif was characterized using statistical analysis. The data were interpreted within the 3D crystal structure of the furin catalytic domain. The results indicate that the furin cleavage site motif is comprised of about 20 residues, P14-P6′. Specific physical properties such as volume, charge, and hydrophilicity are required at specific positions. The furin cleavage site motif is divided into two parts: 1) one core region (8 amino acids, positions P6-P2′) packed inside the furin binding pocket; 2) two polar regions (8 amino acids, positions P7–P14; and 4 amino acids, positions P3′-P6′) located outside the furin binding pocket. The physical properties of the core region contribute to the binding strength of the furin substrate, while the polar regions provide a solvent accessible environment and facilitate the accessibility of the core region to the furin binding pocket. This furin cleavage site motif also revealed a dynamic relationship linking the evolution of physical properties in region P1′-P6′ of viral fusion peptides, furin cleavage efficacy, and viral infectivity.

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 SARS-CoV-2 bearing a mutation at the S1/S2 cleavage site exhibits attenuated virulence and confers protective immunity

2021 ◽  
Author(s):  
Michihito Sasaki ◽  
Shinsuke Toba ◽  
Yukari Itakura ◽  
Herman M. Chambaro ◽  
Mai Kishimoto ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Neutralizing Antibodies ◽  
Body Weight Loss ◽  
Airway Epithelial Cells ◽  
Spike Protein ◽  
Furin Cleavage ◽  
Growth Property ◽  
S Gene ◽  
Furin Cleavage Site ◽  
The Impact

AbstractSevere Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by host furin protease. Proteolytic cleavage activates the spike protein and influences both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogensis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited a low growth property. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, indicating the attenuated variant nature of S gene mutants. We reproduced the attenuated growth of S gene mutants in primary differenciated human airway epithelial cells. This transient infection was enough to induce protective neutralizing antibodies crossreacting with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of SARS-CoV-2 and highlights the potential benefits of S gene mutants as potential immunogens.

scholarly journals Characterization and Treatment of SARS-CoV-2 in Nasal and Bronchial Human Airway Epithelia

2020 ◽  
Vol 1 (4) ◽  
pp. 100059 ◽  
Author(s):  
Andrés Pizzorno ◽  
Blandine Padey ◽  
Thomas Julien ◽  
Sophie Trouillet-Assant ◽  
Aurélien Traversier ◽  
...  
Keyword(s):  
Airway Epithelia ◽  
Human Airway ◽  
Human Airway Epithelia

Distinct Patterns of Emergence of SARS-CoV-2 Spike Variants including N501Y in Clinical Samples in Columbus Ohio

2021 ◽  
Author(s):  
Huolin Tu ◽  
Matthew R Avenarius ◽  
Laura Kubatko ◽  
Matthew Hunt ◽  
Xiaokang Pan ◽  
...  
Keyword(s):  
Clinical Impact ◽  
Clinical Samples ◽  
Membrane Glycoprotein ◽  
Receptor Binding Domain ◽  
Furin Cleavage ◽  
S Gene ◽  
The United Kingdom ◽  
Furin Cleavage Site ◽  
Two Samples ◽  
The Uk

AbstractFollowing the worldwide emergence of the p.Asp614Gly shift in the Spike (S) gene of SARS-CoV-2, there have been few recurring pathogenic shifts occurring during 2020, as assessed by genomic sequencing. This situation has evolved in the last several months with the emergence of several distinct variants (first identified in the United Kingdom and South Africa) that manifest multiple changes in the S gene, particularly p.Asn501Tyr (N501Y), that likely have clinical impact. We report here the emergence in Columbus, Ohio in December 2020 of two novel SARS-CoV-2 clade 20G variants. One variant, that has become the predominant virus found in nasopharyngeal swabs in the December 2020-January 2021 period, harbors S p.Gln677His (Q677H), affecting a consensus QTQTN domain near the S1/S2 furin cleavage site, nucleocapsid (N) p.Asp377Tyr (D377Y) and membrane glycoprotein (M) p.Ala85Ser (A85S) mutations, with additional S mutations in subsets. The other variant present in two samples, contains S N501Y, which is a marker of the UK-B.1.1.7 (clade 20I/501Y.V1) strain, but lacks all other mutations from that virus. The Ohio variant is from a different clade and shares multiple mutations with the clade 20G viruses circulating in the area prior to December 2020. These two SARS-CoV-2 viruses, which we show are also present and evolving currently in several other parts of North America, add to the diversity of S gene shifts occurring worldwide. These and other shifts in this period of the pandemic support multiple independent acquisition of functionally significant and potentially complementing mutations affecting the S QTQTN site (Q675H or Q677H) and certain receptor binding domain mutations (e.g., E484K and N501Y).

scholarly journals Chronic β2AR stimulation limits CFTR activation in human airway epithelia

JCI Insight ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
John J. Brewington ◽  
Jessica Backstrom ◽  
Amanda Feldman ◽  
Elizabeth L. Kramer ◽  
Jessica D. Moncivaiz ◽  
...  
Keyword(s):  
Airway Epithelia ◽  
Human Airway ◽  
Human Airway Epithelia

scholarly journals Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

2021 ◽  
Vol 118 (47) ◽  
pp. e2109905118
Author(s):  
Liping Zhang ◽  
Matthew Mann ◽  
Zulfeqhar A. Syed ◽  
Hayley M. Reynolds ◽  
E. Tian ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Protein S ◽  
Viral Infectivity ◽  
The Novel ◽  
Furin Cleavage ◽  
Global Pandemic ◽  
Furin Cleavage Site ◽  
Respiratory Cells ◽  
Syncytia Formation

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a novel furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation in cells. Here, we show that O-glycosylation near the furin cleavage site is mediated by members of the GALNT enzyme family, resulting in decreased furin cleavage and decreased syncytia formation. Moreover, we show that O-glycosylation is dependent on the novel proline at position 681 (P681). Mutations of P681 seen in the highly transmissible alpha and delta variants abrogate O-glycosylation, increase furin cleavage, and increase syncytia formation. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that host O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the role of the P681 mutations found in the highly transmissible alpha and delta variants.

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