Acquisition of Furin Cleavage Site and Further SARS-CoV-2 Evolution Change the Mechanisms of Viral Entry, Infection Spread, and Cell Signaling.
Acquisition of Furin Cleavage Site and Further SARS-CoV-2 Evolution Change the Mechanisms of Viral Entry, Infection Spread, and Cell Signaling.
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DOI：
10.1128/jvi.00753-22
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发表时间：
2022-08-10
影响因子：
5.4
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中科院分区：
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严重急性呼吸道综合征冠状病毒2型（SARS-CoV-2）在人群中的传播导致病毒进一步进化。在这种进化过程中出现的新变种更具传染性。我们的数据表明，新的变体已经从利用组织蛋白酶/内体和TMPRSS 2介导的进入机制转变为依赖于TMPRSS 2依赖性进入途径。因此，只有SARS-CoV-2的早期谱系能够感染缺乏TMPRSS 2表达的Vero/ACE 2细胞并形成合胞体。在S蛋白中存在完整的多碱基弗林蛋白酶切割位点（FCS）是细胞与细胞融合的关键要求。FCS的缺失使SARS-CoV-2在体外更具感染性，但使其不能形成合胞体。细胞与细胞融合可能代表了病毒传播的另一种方式，并且对培养基中存在的高水平中和单克隆抗体（MAb）和免疫血清具有抵抗力。在这项研究中，我们还注意到，SARS-CoV-2感染的细胞与完整的FCS或甲病毒复制子表达S蛋白（VEREp/S）释放高水平的游离S1亚基。释放的S1能够激活TLR 4受体并诱导促炎反应。因此，TLR 4的S1激活可能是SARS-CoV-2诱导的COVID-19疾病的重要贡献者，需要在COVID mRNA疫苗的设计中加以考虑。最后，VEREp/S-复制子显示出产生大量感染性的、形成合胞体的假病毒，因此可以代表用于筛选病毒进入和合胞体形成的抑制剂的替代实验系统。重要性本研究的结果表明，SARS-CoV-2的晚期谱系进化为更有效地利用TMPRSS 2介导的进入途径，并逐渐丧失利用组织蛋白酶/内体介导的进入的能力。通过SARS-CoV-2特异性S蛋白获得弗林蛋白酶切割位点（FCS）使该病毒成为合胞体的有效生产者。它们的形成也由ACE 2和TMPRSS 2的表达决定，并且对中和人MAb和免疫血清具有抗性。合胞体形成似乎是适应性免疫反应发展后感染传播的另一种手段。具有完整FCS的SARS-CoV-2感染的细胞分泌高水平的S1亚基。释放的S1证明了激活TLR 4受体和诱导促炎细胞因子的能力，这代表了SARS-CoV-2发病机制的标志。编码SARS-CoV-2S蛋白的甲病毒复制子可引起传播性的合胞体形成感染，可作为研究合胞体形成机制的实验工具。
Circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the human population leads to further viral evolution. The new variants that arise during this evolution are more infectious. Our data suggest that newer variants have shifted from utilizing both cathepsin/endosome- and TMPRSS2-mediated entry mechanisms to rely on a TMPRSS2-dependent entry pathway. Accordingly, only the early lineages of SARS-CoV-2 are capable of infecting and forming syncytia in Vero/ACE2 cells which lack TMPRSS2 expression. The presence of an intact multibasic furin cleavage site (FCS) in the S protein was a key requirement for cell-to-cell fusion. Deletion of FCS makes SARS-CoV-2 more infectious in vitro but renders it incapable of syncytium formation. Cell-to-cell fusion likely represents an alternative means of virus spread and is resistant to the presence of high levels of neutralizing monoclonal antibodies (MAbs) and immune sera in the media. In this study, we also noted that cells infected with SARS-CoV-2 with an intact FCS or alphavirus replicon expressing S protein (VEErep/S) released high levels of free S1 subunit. The released S1 is capable of activating the TLR4 receptor and inducing a pro-inflammatory response. Thus, S1 activation of TLR4 may be an important contributor to SARS-CoV-2-induced COVID-19 disease and needs to be considered in the design of COVID mRNA vaccines. Lastly, a VEErep/S-replicon was shown to produce large amounts of infectious, syncytium-forming pseudoviruses and thus could represent alternative experimental system for screening inhibitors of virus entry and syncytium formation. IMPORTANCE The results of this study demonstrate that the late lineages of SARS-CoV-2 evolved to more efficient use of the TMPRSS2-mediated entry pathway and gradually lost an ability to employ the cathepsins/endosome-mediated entry. The acquisition of a furin cleavage site (FCS) by SARS-CoV-2-specific S protein made the virus a potent producer of syncytia. Their formation is also determined by expression of ACE2 and TMPRSS2 and is resistant to neutralizing human MAbs and immune sera. Syncytium formation appears to be an alternative means of infection spread following the development of an adaptive immune response. Cells infected with SARS-CoV-2 with an intact FCS secrete high levels of the S1 subunit. The released S1 demonstrates an ability to activate the TLR4 receptor and induce pro-inflammatory cytokines, which represent a hallmark of SARS-CoV-2 pathogenesis. Alphavirus replicons encoding SARS-CoV-2 S protein cause spreading, syncytium-forming infection, and they can be applied as an experimental tool for studying the mechanism of syncytium formation.
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