Chen Chen, Zhuang Xiaowei
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11790-5. doi: 10.1073/pnas.0803711105. Epub 2008 Aug 8.
During clathrin-mediated endocytosis, adaptor proteins recognize specific internalization signals on cargo receptors, either recruiting cargos into clathrin-coated pits (CCPs) or initiating clathrin-coat assembly around the cargo molecules. Here, we identify epsin 1, a clathrin-, ubiquitin-, and phospholipid-interacting protein, as a cargo-specific adaptor for influenza virus entry through the clathrin-mediated pathway. Using live-cell imaging to monitor the entry of individual virus particles, we observed recruitment of epsin 1 to the binding sites of influenza viruses in synchrony with the assembly of CCPs. Epsin 1 knockdown by siRNA significantly inhibited the clathrin-mediated endocytosis of the influenza virus and caused the majority of the virus particles to enter through a clathrin-independent pathway. The same treatment did not affect the entry of several classical ligands for clathrin-mediated endocytosis, including transferrin, LDL, and EGF. Overexpression of the dominant-negative epsin 1 mutant lacking the ubiquitin-interaction motifs nearly completely blocked the clathrin-mediated entry of the influenza virus without affecting transferrin uptake. These results suggest that epsin 1 functions as a cargo-specific adaptor for the clathrin-mediated entry of the influenza virus.
在网格蛋白介导的内吞作用过程中，衔接蛋白识别货物受体上的特定内化信号，要么将货物招募到网格蛋白包被小窝（CCP）中，要么在货物分子周围启动网格蛋白包被的组装。在此，我们鉴定出 epsin 1，一种与网格蛋白、泛素和磷脂相互作用的蛋白，作为流感病毒通过网格蛋白介导途径进入细胞的货物特异性衔接蛋白。利用活细胞成像监测单个病毒颗粒的进入过程，我们观察到 epsin 1 与 CCP 的组装同步被招募到流感病毒的结合位点。通过 siRNA 敲低 epsin 1 显著抑制了流感病毒的网格蛋白介导的内吞作用，并导致大多数病毒颗粒通过非网格蛋白依赖途径进入细胞。相同处理对几种网格蛋白介导内吞作用的经典配体（包括转铁蛋白、低密度脂蛋白和表皮生长因子）的进入没有影响。缺乏泛素相互作用基序的显性负性 epsin 1 突变体的过表达几乎完全阻断了流感病毒的网格蛋白介导的进入，而不影响转铁蛋白的摄取。这些结果表明 epsin 1 作为流感病毒网格蛋白介导进入细胞的货物特异性衔接蛋白发挥作用。
