Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells
Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells
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DOI：
10.1128/jvi.00683-18
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发表时间：
2018-10-01
影响因子：
5.4
通讯作者：
Kamitani, Wataru
中科院分区：
文献类型：
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作者：
Matsuyama, Shutoku;Shirato, Kazuya;Kamitani, Wataru
中东呼吸综合征冠状病毒（MERS-CoV）利用宿主细胞蛋白酶进入细胞。先前的报道表明，furin主要分布在高尔基体中并循环到细胞表面和内体，在受体结合后以蛋白水解的方式激活MERS-CoV刺突(S)蛋白，介导病毒与细胞膜的融合。在这项研究中，我们在抑制细胞蛋白酶后，使用基于实时pcr的病毒进入细胞试验，重新检测了MERS-CoV对呋喃的使用。我们发现furin抑制剂dec-RVKR-CMK可以阻断含有缺乏furin切割位点的S蛋白的MERS-CoV的进入；它甚至可以阻断进入富蛋白缺失的LoVo细胞。此外，dec-RVKR-CMK不仅能抑制furin的酶活性，还能抑制组织蛋白酶L、组织蛋白酶B、胰蛋白酶、木瓜蛋白酶和TMPRSS2的酶活性。此外，病毒进入细胞试验和细胞-细胞融合试验没有证据表明S蛋白被外源性呋喃激活。因此，我们认为furin在MERS-CoV进入细胞中不起作用，deco - rvkr - cmk对TMPRSS2和组织蛋白酶L的抑制作用是特异性的，而不是furin。先前使用furin抑制剂dec-RVKR-CMK的研究表明，MERS-CoV利用细胞蛋白酶furin在细胞进入过程中激活病毒糖蛋白。然而，我们发现dec-RVKR-CMK不仅抑制furin，还抑制其他蛋白酶。此外，我们没有发现MERS-CoV使用呋喃的证据。这些发现表明，以前基于dec-RVKR-CMK的病毒学领域的研究应该重新仔细审查。在这里，我们描述了适当的实验，可用于评估蛋白酶抑制剂对病毒进入细胞的影响。
Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin.IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.