Entry from the Cell Surface of Severe Acute Respiratory Syndrome Coronavirus with Cleaved S Protein as Revealed by Pseudotype Virus Bearing Cleaved S Protein
Entry from the Cell Surface of Severe Acute Respiratory Syndrome Coronavirus with Cleaved S Protein as Revealed by Pseudotype Virus Bearing Cleaved S Protein
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DOI：
10.1128/jvi.01412-08
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发表时间：
2008-12-01
影响因子：
5.4
通讯作者：
Taguchi, Fumihiro
中科院分区：
文献类型：
--
作者：
Watanabe, Rie;Matsuyama, Shutoku;Taguchi, Fumihiro
严重急性呼吸综合征冠状病毒(SARS-CoV)通过内体途径进入细胞；然而，当受体结合的病毒粒子尖峰(S)蛋白被胰酶作用时，它被认为直接从细胞表面进入，从而诱导S蛋白的切割并激活其融合潜能。这表明携带S蛋白裂解形式的SARS-CoV可以直接从细胞表面进入细胞，而不需要胰酶处理。为了探索这种可能性，我们在S蛋白的第798到801位氨基酸上引入了一个类似呋喃的切割序列，发现突变的S蛋白在细胞表面表达时被切割并诱导细胞融合，而不需要胰酶处理。此外，发现一种含有裂解的S蛋白的伪型病毒在溶酶体致病剂和蛋白酶抑制剂的存在下感染细胞，这两种药物都能通过内吞体阻断SARS-CoV的感染，而含有未裂解的野生型S蛋白的伪型病毒的感染则被这些药物阻断。从SARS-CoV S蛋白中提取的七肽重复序列可以有效地阻止细胞表面的感染，它可以通过切割的S蛋白阻止假型感染，但不能通过未切割的S蛋白阻止假型感染。这些结果表明，带有S裂解蛋白的SARS冠状病毒能够直接从细胞表面进入细胞，这与先前观察到的蛋白酶介导的SARS冠状病毒细胞表面进入一致。
Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is known to take an endosomal pathway for cell entry; however, it is thought to enter directly from the cell surface when a receptor-bound virion spike (S) protein is affected by trypsin, which induces cleavage of the S protein and activates its fusion potential. This suggests that SARS-CoV bearing a cleaved form of the S protein can enter cells directly from the cell surface without trypsin treatment. To explore this possibility, we introduced a furin-like cleavage sequence in the S protein at amino acids 798 to 801 and found that the mutated S protein was cleaved and induced cell fusion without trypsin treatment when expressed on the cell surface. Furthermore, a pseudotype virus bearing a cleaved S protein was revealed to infect cells in the presence of a lysosomotropic agent as well as a protease inhibitor, both of which are known to block SARS-CoV infection via an endosome, whereas the infection of pseudotypes with an uncleaved, wild-type S protein was blocked by these agents. A heptad repeat peptide, derived from a SARS-CoV S protein that is known to efficiently block infections from the cell surface, blocked the infection by a pseudotype with a cleaved S protein but not that with an uncleaved S protein. Those results indicate that SARS-CoV with a cleaved S protein is able to enter cells directly from the cell surface and agree with the previous observation of the protease-mediated cell surface entry of SARS-CoV.