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

Wang, Pui; Lau, Siu-Ying; Deng, Shumin; Chen, Pin; Mok, Bobo Wing-Yee; Zhang, Anna Jinxia; Lee, Andrew Chak-Yiu; Chan, Kwok‐Hung; Song, Wenjun; To, Kelvin Kai‐Wang; Chan, Jasper Fuk‐Woo; Yuen, Kwok‐Yung; Chen, Honglin

doi:10.1101/2020.08.24.264192

Cited by 4 publications

(3 citation statements)

References 35 publications

(52 reference statements)

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“…Furthermore, the original 2003 SARS-CoV lacked a furin CS and was clearly transmitted between people, although this usually occurred during a symptomatic phase rather than asymptomatically or pre-symptomatically, thus allowing the virus outbreak to be controlled by public health measures (Liu, Gayle, Wilder-Smith, & Rocklov, 2020). Several recent studies have investigated the pathogenicity of naturally occurring or engineered deletion mutants in the hamster model, and shown that viruses lacking the furin CS are attenuated for replication and pathogenicity (Johnson et al, 2020; Lau et al, 2020; Wang et al, 2020; Wong et al, 2020). We, like others, did not find that SARS-CoV-2 induced significant clinical signs in ferrets, so were unable to assess the effect of the CS mutation on viral pathogenesis here.…”

Section: Discussionmentioning

confidence: 99%

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells

Peacock

Goldhill

Zhou

et al. 2020

Preprint

115

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SARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2' cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.

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Section: Discussionmentioning

confidence: 99%

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells

Peacock

Goldhill

Zhou

et al. 2020

Preprint

115

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“…Importantly, the deletion of QTQTN (mut-del2) diminished SARS-CoV-2 entry and infection in Vero-E6 cells (40). Furthermore, three FCS-related deletion mutants, ΔPRRA↓, ΔRAR↓SVAS, and ΔNSPRRAR↓SVA, have been shown to have reduced replication in vitro and lung disease in animal models (44,49,50), strongly supporting that the FCS is a virulencerelated motif. Since the ΔQTQTN also abolished the furin cleavage (40), we speculate mut-del2 mutant should have reduced lung disease in animals as well.…”

Section: Discussionmentioning

confidence: 88%

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

Zou

Xiong

Hao

et al. 2021

Preprint

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The 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.

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“…Importantly, the deletion of QTQTN (mut-del2) diminished SARS-CoV-2 entry and infection in Vero-E6 cells (41). Furthermore, three FCS-related deletion mutants, DPRRA;, DRAR;SVAS, and DNSPRRAR;SVA, have been shown to have reduced replication in vitro and lung disease in animal models (44,52,53), strongly supporting that the FCS is a virulence-related motif. Since the DQTQTN also abolished furin cleavage (41), we speculate that the mut-del2 mutant should have reduced lung disease in animals as well.…”

Section: Discussionmentioning

confidence: 89%

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

Zou

Xiong

Hao

et al. 2021

mBio

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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.

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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

Cited by 4 publications

References 35 publications

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells

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

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

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