Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry
Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry
复制标题
DOI：
10.1073/pnas.0306446101
复制
发表时间：
2004-03-23
影响因子：
11.1
通讯作者：
Bates, P
中科院分区：
文献类型：
--
作者：
Simmons, G;Reeves, JD;Bates, P
严重急性呼吸综合征相关冠状病毒(SARS-CoV)是一种迅速出现的病原体，对公众健康具有潜在的严重后果。在这里，我们描述了不仅导致SARS-CoV Spike(S)蛋白在细胞表面高效表达的条件，而且导致其整合到慢病毒颗粒中，慢病毒颗粒可以用于以S糖蛋白依赖的方式转导细胞。我们发现，虽然一些灵长类细胞系，包括Vero E6,293T和Huh-7细胞，可以被SARS冠状病毒S糖蛋白伪病毒有效地转导，但其他细胞系对病毒的进入要么是抵抗的，要么是非常不允许的。假病毒粒子的感染可以被几种溶酶体亲和剂抑制，这表明需要内体的酸化才能有效地进入S介导的病毒。此外，我们还建立了一种细胞-细胞融合实验，可以用来监测S糖蛋白依赖的膜融合。虽然蛋白分解并不能增强无细胞假病毒粒子的感染性，但细胞-细胞融合需要胰酶的激活。此外，S糖蛋白介导的细胞-细胞融合不需要明显的pH。总之，这些研究描述了可用于研究SARS-CoV S糖蛋白结构和功能的重要工具，包括可用于识别SARS-CoV进入靶细胞的抑制物的方法。
Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoprotein-dependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addition, we were able to develop a cell-cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell-cell fusion. Additionally, there was no apparent pH requirement for S glycoprotein-mediated cell-cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.