Shirato Kazuya, Kanou Kazuhiko, Kawase Miyuki, Matsuyama Shutoku
Laboratory of Acute Respiratory Viral Diseases and Cytokines, Department of Virology III, Murayama Branch, National Institute of Infectious Diseases, Tokyo, Japan.
Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo, Japan.
J Virol. 2016 Dec 16;91(1). doi: 10.1128/JVI.01387-16. Print 2017 Jan 1.
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.
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.
人冠状病毒229E（HCoV - 229E）是普通感冒的病原体，通过两种不同途径进入宿主细胞：一种由细胞表面蛋白酶介导，特别是跨膜蛋白酶丝氨酸2（TMPRSS2），另一种由内体组织蛋白酶L介导。因此，这些蛋白酶的特异性抑制剂可阻断病毒感染。然而，尚不清楚HCoV - 229E在人类呼吸道中实际利用的是这些途径中的哪一种。在此，我们研究了携带HCoV - 229E刺突（S）蛋白的假型病毒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，HCoV - 229E的临床分离株利用组织蛋白酶L的可能性较小；相反，它们表现出对TMPRSS2的偏好。HCoV - 229E临床分离株S蛋白中的两个氨基酸取代（R642M和N714K）改变了它们对组织蛋白酶L抑制剂的敏感性，表明这些氨基酸决定了对组织蛋白酶L的利用。在HeLa细胞中传代20次后，该分离株利用组织蛋白酶的能力增加，使其与实验室毒株相当；这种增加是由S蛋白中的一个氨基酸取代（I577S）引起的。传代后的病毒在气液界面培养的分化气道上皮细胞中的复制能力降低。这些结果表明，内体途径对HCoV - 229E感染人气道上皮细胞不利；因此，临床分离株利用组织蛋白酶的能力较弱。
许多包膜病毒通过内吞作用进入细胞。病毒刺突蛋白驱动病毒膜与内体膜融合，以促进病毒基因组插入细胞质。人冠状病毒229E（HCoV - 229E）在受体结合后利用内体组织蛋白酶L激活刺突蛋白。在此，我们发现HCoV - 229E的临床分离株优先利用细胞表面蛋白酶TMPRSS2而非内体组织蛋白酶L。内体是Toll样受体识别的主要部位，进而触发先天免疫反应；因此，HCoV - 229E可能通过TMPRSS2进入细胞从而绕过内体进行了进化。因此，该病毒利用一种简单机制逃避宿主先天免疫系统。因此，针对冠状病毒介导疾病（如严重急性呼吸综合征（SARS）和中东呼吸综合征（MERS））的治疗药物应靶向细胞表面的TMPRSS2而非内体组织蛋白酶。
