Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry
Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry
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DOI：
10.1128/jvi.01387-16
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发表时间：
2017-01-01
影响因子：
5.4
通讯作者：
Matsuyama, Shutoku
中科院分区：
文献类型：
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作者：
Shirato, Kazuya;Kanou, Kazuhiko;Matsuyama, Shutoku
人类冠状病毒229E(Human CoronaVirus 229E，HCoV-229E)是普通感冒的病原体，它通过两条不同的途径进入宿主细胞：一条是由细胞表面的蛋白水解酶，特别是跨膜蛋白丝氨酸2(TMPRSS2)介导的，另一条是通过内体组织蛋白L(Enosomomal Cathepsin L)介导的。然而，目前还不清楚这些途径中的哪些实际上是HCoV-229E在人类呼吸道中利用的。在这里，我们研究了一种带有人类冠状病毒-229E刺突蛋白的假型病毒(S)在存在或不存在蛋白酶抑制剂的情况下进入细胞的机制。我们发现，与1966年分离并传代半个世纪的实验室毒株相比，临床分离的人类冠状病毒229E株不太可能利用组织蛋白酶L；相反，他们表现出对TMPRSS2的偏好。HCoV229E临床分离株S蛋白的两个氨基酸(R642M和N714K)突变改变了它们对组织蛋白酶L抑制剂的敏感性，提示这两个氨基酸与组织蛋白酶L的使用有关。在HeLa细胞中传代20代后，分离株利用组织蛋白酶的能力增强，与实验室株相当，这是由于S蛋白中的一个氨基酸替换(I577S)引起的。传代病毒在气液界面培养的分化的呼吸道上皮细胞中的复制能力降低。提示HCoV-229E通过内体途径感染人呼吸道上皮细胞，不利于HCoV-229E感染人呼吸道上皮细胞，因此临床分离株使用组织蛋白酶的能力较低。病毒刺突蛋白驱动病毒和内体膜的融合，以促进病毒基因组插入细胞质。人冠状病毒229E(HCoV-229E)利用内体组织蛋白L与受体结合后激活刺突蛋白。在这里，我们发现HCoV-229E临床分离株优先利用细胞表面蛋白TMPRSS2而不是内体组织蛋白L。内体是Toll样受体识别的主要部位，然后触发先天免疫反应；因此，HCoV-229E可能进化为通过TMPRSS2进入细胞而绕过内体。因此，该病毒使用一种简单的机制来逃避宿主的先天免疫系统。因此，针对冠状病毒介导的疾病，如严重急性呼吸综合征(SARS)和中东呼吸综合征(MERS)，治疗药物应该针对细胞表面TMPRSS2，而不是内体组织蛋白酶。
Human coronavirus 229E (HCoV-229E), a causative agent of the common cold, enters host cells via two distinct pathways: one is mediated by cell surface proteases, particularly transmembrane protease serine 2 (TMPRSS2), and the other by endosomal cathepsin L. Thus, specific inhibitors of these proteases block virus infection. However, it is unclear which of these pathways is actually utilized by HCoV-229E in the human respiratory tract. Here, we examined the mechanism of cell entry used by a pseudotyped virus bearing the HCoV-229E spike (S) protein in the presence or absence of protease inhibitors. We found that, compared with a laboratory strain isolated in 1966 and passaged for a half century, clinical isolates of HCoV-229E were less likely to utilize cathepsin L; rather, they showed a preference for TMPRSS2. Two amino acid substitutions (R642M and N714K) in the S protein of HCoV-229E clinical isolates altered their sensitivity to a cathepsin L inhibitor, suggesting that these amino acids were responsible for cathepsin L use. After 20 passages in HeLa cells, the ability of the isolate to use cathepsin increased so that it was equal to that of the laboratory strain; this increase was caused by an amino acid substitution (I577S) in the S protein. The passaged virus showed a reduced ability to replicate in differentiated airway epithelial cells cultured at an air-liquid interface. These results suggest that the endosomal pathway is disadvantageous for HCoV-229E infection of human airway epithelial cells; therefore, clinical isolates are less able to use cathepsin.IMPORTANCE Many enveloped viruses enter cells through endocytosis. Viral spike proteins drive the fusion of viral and endosomal membranes to facilitate insertion of the viral genome into the cytoplasm. Human coronavirus 229E (HCoV-229E) utilizes endosomal cathepsin L to activate the spike protein after receptor binding. Here, we found that clinical isolates of HCoV-229E preferentially utilize the cell surface protease TMPRSS2 rather than endosomal cathepsin L. The endosome is a main site of Toll-like receptor recognition, which then triggers an innate immune response; therefore, HCoV-229E presumably evolved to bypass the endosome by entering the cell via TMPRSS2. Thus, the virus uses a simple mechanism to evade the host innate immune system. Therefore, therapeutic agents for coronavirus-mediated diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), should target cell surface TMPRSS2 rather than endosomal cathepsin.