Zou Wei, Xiong Min, Hao Siyuan, Zhang Elizabeth Yan, Baumlin Nathalie, Kim Michael D, Salathe Matthias, Yan Ziying, Qiu Jianming
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA.
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.
mBio. 2021 May 11;12(3):e01006-21. doi: 10.1128/mBio.01006-21.
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), RRAR↓S 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 (TNSPVAS) that contains the underlined FCS and a 5 amino acid deletion (QTQTN) 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. Polarized human airway epithelia at an air-liquid interface (HAE-ALI) are an 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 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 (ΔTNSPRRAR↓SVAS and ΔQTQTN) 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.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的刺突（S）多肽由S1和S2亚基组成，并在含有弗林蛋白酶切割位点（FCS）RRAR↓S的S1/S2边界处被细胞蛋白酶加工。在患者中已鉴定出FCS周围的各种缺失。当SARS-CoV-2在Vero细胞中繁殖时，它在FCS周围获得了缺失。我们研究了在气液界面（ALI）培养的Vero细胞衍生的SARS-CoV-2感染的原代人气道上皮细胞（HAE）中的病毒转录组，重点是FCS的病毒基因组稳定性。虽然我们总体上发现病毒转录组与感染Vero细胞产生的转录组相似，但我们在HAE-ALI中鉴定出高比例的突变病毒基因组和转录本。在FCS区域发现了两个高频缺失：一个包含下划线FCS的12个氨基酸缺失（TNSPVAS）和一个在FCS上游两个氨基酸处的5个氨基酸缺失（QTQTN）。对来自健康和肺病供体的11种感染的HAE-ALI培养物顶端释放的病毒粒子中FCS缺失动态的进一步研究表明，FCS的选择压力在9种HAE-ALI培养物中稳定地维持FCS，但有2个例外，其中在感染≥13天后，FCS缺失以>40%的高比例保留。我们的研究提供了证据，证明人气道上皮细胞的独特特性在人类气道感染期间FCS区域动态中的作用，这可能取决于供体。气液界面（HAE-ALI）的极化人气道上皮细胞是支持SARS-CoV-2有效感染的模型。SARS-CoV-2的刺突（S）蛋白在S1和S2结构域的边界处含有弗林蛋白酶切割位点（FCS），这使其与SARS-CoV不同。然而，在患者和细胞培养物中已鉴定出FCS缺失突变体，气道上皮细胞如何维持独特的FCS仍然未知。我们发现HAE-ALI培养物能够抑制在Vero细胞繁殖过程中选择的两个普遍存在的FCS缺失突变体（ΔTNSPRRAR↓SVAS和ΔQTQTN）。虽然在来自独立供体的11种测试的HAE-ALI培养物中的9种中观察到了这种抑制，但也发现了2个例外，它们保留了高比例的FCS缺失。我们的结果提供了证据，证明人气道上皮细胞在感染期间FCS进化中的供体依赖性特性。
