Icahn School of Medicine at Mount Sinai, New York, NY, USA.
J Virol. 2013 Mar;87(6):3324-34. doi: 10.1128/JVI.01598-12. Epub 2013 Jan 9.
Zaire Ebola virus (EBOV) is a zoonotic pathogen that causes severe hemorrhagic fever in humans. A single viral glycoprotein (GP) mediates viral attachment and entry. Here, virus-like particle (VLP)-based entry assays demonstrate that a GP mutant, GP-F88A, which is defective for entry into a variety of human cell types, including antigen-presenting cells (APCs), such as macrophages and dendritic cells, can mediate viral entry into mouse CD11b(+) APCs. Like that of wild-type GP (GP-wt), GP-F88A-mediated entry occurs via a macropinocytosis-related pathway and requires endosomal cysteine proteases and an intact fusion peptide. Several additional hydrophobic residues lie in close proximity to GP-F88, including L111, I113, L122, and F225. GP mutants in which these residues are mutated to alanine displayed preferential and often impaired entry into several cell types, although not in a species-specific manner. Niemann-Pick C1 (NPC1) protein is an essential filovirus receptor that binds directly to GP. Overexpression of NPC1 was recently demonstrated to rescue GP-F88A-mediated entry. A quantitative enzyme-linked immunosorbent assay (ELISA) demonstrated that while the F88A mutation impairs GP binding to human NPC1 by 10-fold, it has little impact on GP binding to mouse NPC1. Interestingly, not all mouse macrophage cell lines permit GP-F88A entry. The IC-21 cell line was permissive, whereas RAW 264.7 cells were not. Quantitative reverse transcription (RT)-PCR assays demonstrate higher NPC1 levels in GP-F88A permissive IC-21 cells and mouse peritoneal macrophages than in RAW 264.7 cells. Cumulatively, these studies suggest an important role for NPC1 in the differential entry of GP-F88A into mouse versus human APCs.
扎伊尔埃博拉病毒（EBOV）是一种人畜共患病原体，可导致人类严重出血热。单一病毒糖蛋白（GP）介导病毒附着和进入。在这里，基于病毒样颗粒（VLP）的进入测定表明，一种 GP 突变体，GP-F88A，其进入各种人类细胞类型（包括抗原呈递细胞（APC），如巨噬细胞和树突状细胞）的能力是有缺陷的，但它可以介导病毒进入小鼠 CD11b（+）APC。与野生型 GP（GP-wt）一样，GP-F88A 介导的进入是通过吞噬作用相关途径发生的，需要内体半胱氨酸蛋白酶和完整的融合肽。几个额外的疏水性残基靠近 GP-F88，包括 L111、I113、L122 和 F225。这些残基突变为丙氨酸的 GP 突变体显示出对几种细胞类型的优先和经常受损的进入，尽管不是以种属特异性的方式。尼曼-匹克 C1（NPC1）蛋白是一种必需的丝状病毒受体，直接与 GP 结合。最近证明 NPC1 的过表达可以挽救 GP-F88A 介导的进入。定量酶联免疫吸附测定（ELISA）表明，虽然 F88A 突变使 GP 与人类 NPC1 的结合降低了 10 倍，但对 GP 与小鼠 NPC1 的结合影响很小。有趣的是，并非所有小鼠巨噬细胞系都允许 GP-F88A 进入。IC-21 细胞系是允许的，而 RAW 264.7 细胞则不允许。定量逆转录（RT）-PCR 测定表明，在允许 GP-F88A 进入的 IC-21 细胞和小鼠腹腔巨噬细胞中 NPC1 的水平高于 RAW 264.7 细胞。总而言之，这些研究表明 NPC1 在 GP-F88A 进入小鼠与人类 APC 中的差异中起重要作用。
