The polybasic cleavage site in the SARS-CoV-2 spike modulates viral sensitivity to Type I IFN and IFITM2
The polybasic cleavage site in the SARS-CoV-2 spike modulates viral sensitivity to Type I IFN and IFITM2
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DOI：
10.1101/2020.12.19.423592
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发表时间：
2020-12
期刊：
影响因子：
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通讯作者：
H. Winstone;M. Lista;A. Reid;S. Pickering;K. Doores;Chad M. Swanson;S. Neil
中科院分区：
文献类型：
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作者：
H. Winstone;M. Lista;A. Reid;S. Pickering;K. Doores;Chad M. Swanson;S. Neil
严重急性呼吸综合征相关冠状病毒1型和2型（SARS-CoV-1和-2）进入细胞需要病毒刺突糖蛋白（S）的连续蛋白酶加工。SARS-CoV-2 S在S1/S2边界处存在多碱基切割位点，这被认为是SARS-CoV-2与SARS-CoV-1相比传播性增加的一个因素，通过生产细胞中的弗林蛋白酶样蛋白酶而不是靶细胞中的内体组织蛋白酶促进S前体的成熟。我们研究了SARS-CoV-2进入途径中的多碱基切割位点的相关性，以及这对干扰素敏感性的后果，更具体地说，干扰素诱导的跨膜（IFITM）蛋白家族抑制不同包膜病毒的进入。我们发现SARS-CoV-2主要受IFITM 2的限制，这种限制的程度取决于病毒进入的途径。去除刺突蛋白中的切割位点使得SARS-CoV-2进入晚期内体中高度依赖于pH和组织蛋白酶，在晚期内体中，像SARS-CoV-1 S一样，它对IFITM 2限制更敏感。此外，我们发现，有效抑制SARS-CoV-2复制的I型，而不是II型干扰素减轻IFITM 2表达的靶向耗竭。我们建议，多碱裂解位点允许SARS-CoV-2介导的病毒进入一个pH值不依赖的方式，部分减轻对IFITM介导的限制，促进复制和传播。这表明靶向弗林蛋白酶介导的SARS-CoV-2 S裂解的治疗策略可能通过I型IFN的活性减少病毒复制。重要性S蛋白中的弗林蛋白酶切割位点是SARS-CoV-2的一个显著特征，并且已被认为是与SARS-CoV-1相比个体之间更高传播性的决定因素。对此的一种解释是，它允许在细胞表面处或附近更有效地激活融合，而不需要在靶细胞的内体中进行加工。在这里，我们表明，SARS-CoV-2被抑制的抗病毒膜蛋白IFITM 2，并加剧了敏感性的删除弗林蛋白酶切割位点，限制病毒进入低pH的车厢。此外，我们发现IFITM 2是I型干扰素抗SARS-CoV-2复制的抗病毒活性的显著效应子。我们认为弗林蛋白酶切割位点的一个作用是降低SARS-CoV-2对先天免疫限制的敏感性，因此可能代表COVID-19治疗开发的潜在治疗靶点。
The cellular entry of severe acute respiratory syndrome-associated coronaviruses types 1 and 2 (SARS-CoV-1 and -2) requires sequential protease processing of the viral spike glycoprotein (S). The presence of a polybasic cleavage site in SARS-CoV-2 S at the S1/S2 boundary has been suggested to be a factor in the increased transmissibility of SARS-CoV-2 compared to SARS-CoV-1 by facilitating maturation of the S precursor by furin-like proteases in the producer cells rather than endosomal cathepsins in the target. We investigate the relevance of the polybasic cleavage site in the route of entry of SARS-CoV-2 and the consequences this has for sensitivity to interferons, and more specifically, the IFN-induced transmembrane (IFITM) protein family that inhibit entry of diverse enveloped viruses. We found that SARS-CoV-2 is restricted predominantly by IFITM2 and the degree of this restriction is governed by route of viral entry. Removal of the cleavage site in the spike protein renders SARS-CoV-2 entry highly pH- and cathepsin-dependent in late endosomes where, like SARS-CoV-1 S, it is more sensitive to IFITM2 restriction. Furthermore, we find that potent inhibition of SARS-CoV-2 replication by type I but not type II IFNs is alleviated by targeted depletion of IFITM2 expression. We propose that the polybasic cleavage site allows SARS-CoV-2 to mediate viral entry in a pH-independent manner, in part to mitigate against IFITM-mediated restriction and promote replication and transmission. This suggests therapeutic strategies that target furin-mediated cleavage of SARS-CoV-2 S may reduce viral replication through the activity of type I IFNs. IMPORTANCE The furin cleavage site in the S protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell. Here we show that SARS-CoV-2 is inhibited by antiviral membrane protein IFITM2, and that the sensitivity is exacerbated by deletion of the furin cleavage site which restricts viral entry to low pH compartments. Furthermore, we find that IFITM2 is a significant effector of the antiviral activity of type I interferons against SARS-CoV-2 replication. We suggest one role of the furin cleavage site is to reduce SARS-CoV-2 sensitivity to innate immune restriction, and thus may represent a potential therapeutic target for COVID-19 treatment development.