Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion.
Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion.
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SARS-COV-2峰值的氟蛋白切割会促进，但对于感染和细胞融合并不是必不可少的。
DOI：
10.1371/journal.ppat.1009246
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发表时间：
2021-01
期刊：
影响因子：
6.7
通讯作者：
James LC
中科院分区：
文献类型：
--
作者：
Papa G;Mallery DL;Albecka A;Welch LG;Cattin-Ortolá J;Luptak J;Paul D;McMahon HT;Goodfellow IG;Carter A;Munro S;James LC
严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）通过与宿主细胞受体ACE 2结合并经历病毒-宿主膜融合来感染细胞。融合由蛋白酶TMPRSS 2触发，其处理病毒刺突（S）蛋白以显示融合肽。SARS-CoV-2在S1-S2边界进化出一个多碱基位点，该位点被认为是被弗林蛋白酶切割的，以便为TMPRSS 2加工准备S蛋白。在这里，我们表明，CRISPR-Cas9敲除弗林蛋白酶减少，但不能阻止传染性SARS-CoV-2病毒的产生。比较弗林蛋白酶敲除细胞中的S处理多碱基位点突变体揭示，虽然弗林蛋白酶的损失大大减少了S1-S2裂解，但并不能阻止它。SARS-CoV-2 S蛋白还介导细胞-细胞融合，可能使病毒传播病毒粒子独立。我们发现，在供体或受体细胞中弗林蛋白酶的损失减少，但不阻止，TMPRSS 2依赖的细胞-细胞融合，不像突变的多碱基位点，完全防止合胞体的形成。我们的研究结果表明，虽然弗林蛋白酶促进SARS-CoV-2感染性和细胞间传播，但这并不是必需的，这表明弗林蛋白酶抑制剂可能会减少但不会消除病毒传播。COVID-19大流行研究的主要焦点是SARS-CoV-2刺突蛋白，这是一种负责结合宿主细胞上ACE 2受体的病毒蛋白。在附着之前和之后，S需要被细胞蛋白酶（例如弗林蛋白酶、TMPRSS 2）激活，从而触发病毒进入靶细胞。SARS-CoV-2 S的独特特征是存在多碱基位点，这是一种由弗林蛋白酶识别的氨基酸基序，其切割激活S蛋白。深入的抗病毒研究一直集中在开发针对SARS-CoV-2感染的弗林蛋白酶抑制剂。在这里，我们表明，弗林蛋白酶不是绝对需要的SARS-CoV-2病毒的生产，这表明目前的SARS-CoV-2抗病毒治疗的基础上弗林蛋白酶靶向药物可能无法完全防止病毒感染。除了病毒进入过程外，S蛋白在触发细胞与细胞融合方面发挥着关键作用，可能使病毒在邻近细胞之间快速传播。我们表明，弗林蛋白酶介导的预激活的S蛋白在病毒感染的细胞是不必要的触发细胞-细胞融合，而多碱基位点和伴随存在的TMPRSS 2蛋白酶的宿主细胞膜上起着重要的作用，在编排的多核细胞的形成。我们的研究结果还表明TMPRSS 2蛋白酶在促进S介导的细胞-细胞融合中的关键作用，为利用抑制剂的鸡尾酒有效治疗SARS-CoV-2感染铺平了道路。
Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread. The main focus of research into the COVID-19 pandemic is the SARS-CoV-2 Spike (S) protein, which is the viral protein responsible for binding the ACE2 receptor on the host cell. Prior to and after attachment, the S needs to be activated by cellular proteases (e.g. furin, TMPRSS2), triggering the virus entry into the target cell. The unique feature of SARS-CoV-2 S is the presence of a multibasic site, an amino acid motif recognised by furin protease, and whose cleavage activates the S protein. Intensive antiviral research has been focused on developing furin inhibitors against SARS-CoV-2 infection. Here we show that furin is not absolutely required for SARS-CoV-2 virus production, suggesting that current SARS-CoV-2 antiviral therapies based on furin-targeting drugs may not completely prevent viral infection. Apart from the virus entry process, the S protein plays a key role in triggering cell-cell fusion, potentially allowing the quick spread of the virus among neighbouring cells. We show that furin-mediated pre-activation of S protein in virus infected cells is not necessary for triggering cell-cell fusion, while the multibasic site and the concomitant presence of TMPRSS2 protease on host cell membrane plays an important role in orchestrating the formation of multinucleated cells. Our results also suggest a key role of TMPRSS2 protease in promoting S-mediated cell-cell fusion paving the way for utilising a cocktail of inhibitors to efficiently treat SARS-CoV-2 infections.
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