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Studies on the leukocyte-entry mechanism of the hand-foot-and-mouth disease virus

手足口病病毒进入白细胞机制的研究

基本信息

批准号：
21790452
负责人：
NISHIMURA Yorihiro
金额：
$ 2.75万
依托单位：
National Institute of Infectious Diseases
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2010
项目状态：
已结题

项目摘要

Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), a common febrile disease occurring mainly in young children. In this study, I identified human P-selectin glycoprotein ligand-1 (PSGL-1), a sialomucin membrane protein expressed on leukocytes that plays a critical role in early stages of inflammation, as a functional receptor for EV71. The N terminal region of PSGL-1 bound specifically to EV71. The stable PSGL-1 expression conferred EV71 entry, replication, and development of cytopathic effect to nonsusceptible mouse L929 cells. Five out of eight EV71 strains bound to soluble PSGL-1 and utilized intact PSGL-1 as the primary receptor for infection of Jurkat T cells. Three other EV71 strains did not utilize PSGL-1, suggesting the presence of strain-specific replication of EV71 in leukocytes. EV71 replicated in nonleukocyte cell lines in a PSGL-1-independent manner, indicating the presence of alternative receptor (s) for EV71.To elucidate the molecular basis of the PSGL-1-EV71 interaction, we generated a series of PSGL-1 mutants and identified the post-translational modifications that are critical for binding of PSGL-1 to EV71. Site-directed mutagenesis at one or more potential tyrosine sulfation sites in the N-terminal region of PSGL-1 significantly impaired PSGL-1 binding to EV71. Furthermore, an inhibitor of sulfation, sodium chlorate, blocked the PSGL-1-EV71 interaction and inhibited PSGL-1-mediated viral replication of EV71 in Jurkat T cells in a dose-dependent manner. Thus, the results presented in this study reveal that tyrosine sulfation, but not O-glycosylation, in the N-terminal region of PSGL-1 may facilitate virus entry and replication of EV71 in leukocytes.Thus, the identification of PSGL-1 as a receptor for EV71 sheds new light on a role for PSGL-1-positive leukocytes in cell tropism and pathogenesis during the course of HFMD and a variety of other EV71 -mediated diseases.

肠道病毒71型(EV71)是手足口病(HFMD)的主要病原体，手足口病是一种常见的发热性疾病，主要发生在幼儿中。在这项研究中，我确定人P-选择素糖蛋白配体-1(PSGL-1)是EV71的功能性受体，它是一种表达在白细胞上的唾液粘蛋白膜蛋白，在炎症的早期阶段发挥关键作用。PSGL-1的N末端区域与EV71特异结合。稳定的PSGL-1表达使EV71进入、复制和对不敏感的小鼠L929细胞产生细胞病变效应。在8株EV71菌株中，有5株与可溶性PSGL-1结合，并利用完整的PSGL-1作为感染Jurkat T细胞的主要受体。另外三个EV71菌株不利用PSGL-1，这表明EV71在白细胞中存在菌株特异性复制。EV71以不依赖于PSGL-1的方式在非白细胞细胞系中复制，表明存在EV71的替代受体(S)。为了阐明PSGL-1与EV71相互作用的分子基础，我们产生了一系列PSGL-1突变体，并鉴定了对PSGL-1与EV71结合至关重要的翻译后修饰。PSGL-1 N-末端的一个或多个潜在酪氨酸硫化位点的定点突变显著削弱了PSGL-1与EV71的结合。此外，硫酸盐抑制剂氯酸钠以剂量依赖的方式阻断PSGL-1与EV71的相互作用，并抑制PSGL-1介导的EV71在Jurkat T细胞中的复制。因此，本研究的结果表明，PSGL-1 N-末端的酪氨酸硫酸基化而不是O-糖基化可能促进EV71在白细胞中的病毒进入和复制。因此，PSGL-1作为EV71的受体的鉴定为揭示PSGL-1阳性的白细胞在手足口病和其他EV71介导的疾病过程中的细胞趋向性和发病机制中的作用提供了新的线索。