Diaz-Griffero Felipe, Jackson Antony P, Brojatsch Jürgen
Skirball Institute of Biomolecular Medicine, New York University School of Medicine, NY 10003, USA.
Virology. 2005 Jun 20;337(1):45-54. doi: 10.1016/j.virol.2005.02.027.
Avian leukosis virus (ALV) requires endocytosis and a low pH step for successful viral entry. Here we report that transient treatment with lysosomotropic agents was not sufficient to block ALV subgroup B (ALV-B) entry, while it completely inhibited uptake of the pH-dependent Semliki Forest virus. Extended incubations with lysosomotropic agents were required to block ALV-B entry, suggesting that ALV particles are stable in endosomal compartments. We analyzed endocytic pathways involved in the uptake of ALV-B into target cells. The ALV-B receptor TVB(S3) was not associated with detergent-resistant membranes (DRMs) in the presence or absence of ALV-B particles. This result suggested that DRM-associated endocytic pathways were not required for ALV-B entry. Using several approaches, we found that clathrin mediates endocytosis of ALV-B particles into target cells. By means of confocal microscopy, we established that the ALV-B receptor TVB(S3) colocalized with clathrin in TVB(S3)-expressing quail QT-6 cells. In addition, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, blocked uptake of soluble ALV-B Env into chicken embryo fibroblasts. To examine ALV-B uptake into clathrin-negative cells, we used a chicken DT40 B cell line containing a tetracycline-regulatable clathrin gene. Clathrin depletion significantly reduced ALV-B entry into the chicken DT40 cell line. Taken together, our results suggest that clathrin is involved in uptake of ALV-B particles into target cells.
禽白血病病毒（ALV）成功进入细胞需要内吞作用和低pH步骤。在此我们报告，用溶酶体促渗剂进行短暂处理不足以阻断B亚群禽白血病病毒（ALV-B）的进入，而它能完全抑制pH依赖性的Semliki森林病毒的摄取。需要用溶酶体促渗剂进行长时间孵育才能阻断ALV-B的进入，这表明ALV颗粒在内体区室中是稳定的。我们分析了参与ALV-B进入靶细胞的内吞途径。无论有无ALV-B颗粒，ALV-B受体TVB(S3)都不与抗去污剂膜（DRM）相关联。这一结果表明，ALV-B进入不需要与DRM相关的内吞途径。通过几种方法，我们发现网格蛋白介导ALV-B颗粒进入靶细胞内。借助共聚焦显微镜，我们确定在表达TVB(S3)的鹌鹑QT-6细胞中，ALV-B受体TVB(S3)与网格蛋白共定位。此外，网格蛋白介导的内吞作用抑制剂氯丙嗪可阻断可溶性ALV-B Env进入鸡胚成纤维细胞。为了检测ALV-B进入网格蛋白阴性细胞的情况，我们使用了一个含有四环素可调控网格蛋白基因的鸡DT40 B细胞系。网格蛋白缺失显著降低了ALV-B进入鸡DT40细胞系的效率。综上所述，我们的结果表明网格蛋白参与了ALV-B颗粒进入靶细胞的过程。
