Department of Virology II, National Institute of Infectious Diseases, Musashimurayama-shi, Tokyo, Japan.
PLoS Pathog. 2010 Nov 4;6(11):e1001174. doi: 10.1371/journal.ppat.1001174.
Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease, a common febrile disease in children; however, EV71 has been also associated with various neurological diseases including fatal cases in large EV71 outbreaks particularly in the Asia Pacific region. Recently we identified human P-selectin glycoprotein ligand-1 (PSGL-1) as a cellular receptor for entry and replication of EV71 in leukocytes. PSGL-1 is a sialomucin expressed on the surface of leukocytes, serves as a high affinity counterreceptor for selectins, and mediates leukocyte rolling on the endothelium. The PSGL-1-P-selectin interaction requires sulfation of at least one of three clustered tyrosines and an adjacent O-glycan expressing sialyl Lewis x in an N-terminal region of PSGL-1. 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. We expressed the PSGL-1 mutants in 293T cells and the transfected cells were assayed for their abilities to bind to EV71 by flow cytometry. We found that O-glycosylation on T57, which is critical for PSGL-1-selectin interaction, is not necessary for PSGL-1 binding to EV71. On the other hand, 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.
肠道病毒 71 型（EV71）是引起手足口病的主要病原体之一，手足口病是儿童常见的发热性疾病；然而，EV71 也与各种神经系统疾病有关，包括在亚太地区大规模 EV71 爆发时的致命病例。最近，我们确定了人类 P 选择素糖蛋白配体-1（PSGL-1）作为 EV71 进入和复制白细胞的细胞受体。PSGL-1 是一种在白细胞表面表达的唾液酸糖蛋白，作为选择素的高亲和力互补受体，并介导白细胞在内皮细胞上的滚动。PSGL-1-P 选择素相互作用需要 PSGL-1 N 端区域中至少一个三个簇酪氨酸和相邻 O-聚糖的硫酸化，以表达唾液酸化 Lewis x。为了阐明 PSGL-1-EV71 相互作用的分子基础，我们生成了一系列 PSGL-1 突变体，并确定了与 PSGL-1 结合 EV71 相关的翻译后修饰。我们在 293T 细胞中表达 PSGL-1 突变体，并通过流式细胞术检测转染细胞与 EV71 结合的能力。我们发现，对于 PSGL-1-选择素相互作用至关重要的 T57 O-糖基化对于 PSGL-1 结合 EV71 并非必需。另一方面，在 PSGL-1 N 端区域的一个或多个潜在酪氨酸硫酸化位点进行定点突变会显著降低 PSGL-1 与 EV71 的结合。此外，硫酸化抑制剂，氯酸钠，以剂量依赖的方式阻断 PSGL-1-EV71 相互作用并抑制 Jurkat T 细胞中 PSGL-1 介导的 EV71 病毒复制。因此，本研究结果表明，PSGL-1 N 端区域的酪氨酸硫酸化，而不是 O-糖基化，可能有助于病毒进入和白细胞中 EV71 的复制。
