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

Wei Zou,Ziying Yan,Jianming Qiu,Nathalie Baumlin,Elizabeth Yan Zhang,Siyuan Hao,Matthias Salathe,Michael D Kim,Min Xiong,Xiang-Jin Meng

doi:10.1128/mbio.01006-21

Wei Zou, Ziying Yan + Show 8 more

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https://doi.org/10.1128/mbio.01006-21

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Abstract

ABSTRACTThe 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.

Highlights

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
We identified two furin cleavage site (FCS) region deletions that are strikingly amplified in two HAE-ALI cultures after 2 to 3 weeks of infection, whereas these deletions were suppressed in nine other HAEALI cultures
That we have tested seven HAE-ALI cultures from seven healthy donors, we looked into the FCS deletions in four infected HAE-ALI cultures derived from bronchiolar epithelial cells isolated from four patients who had chronic obstructive pulmonary disease (COPD)

Summary

Introduction

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. Upon cell entry of the virus, the incoming positive-sense genomic RNA (1gRNA) subjects it to immediate translation of two large ORFs, ORF1a and ORF1b, for viral nonstructural proteins, which form the viral replication and transcription complex (RTC) [21]. One significant difference among S proteins of SARS-CoV-2, SARS-CoV, and other bat SARS-like coronaviruses, such as bat coronavirus BtCoV-RaTG13, is the addition of 4 amino acids, PRRA, at the S1/S2 boundary [25] This insertion forms a polybasic residue motif, assembling a furin cleavage site (FCS), RRAR;S, which is highly related to the furin cleavage consensus sequence RX[K/R]R (X, any amino acid) [28]. The absence of the FCS in the other betacoronaviruses suggests the insertion of PRRA is a key factor in the virulence of SARSCoV-2, which has been shown to broaden cell tropism, transmissibility, and pathogenicity of the virus [29,30,31]

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