Rab7 Associates with Early Endosomes to Mediate Sorting and Transport of Semliki Forest Virus to Late Endosomes

Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.

塞姆利基森林病毒（Semliki Forest Virus, SFV）通过网格蛋白介导的内吞作用（clathrin-mediated endocytosis）被细胞摄取，并经早期内体（early endosomes）转运至晚期内体（late endosomes）与溶酶体（lysosomes）。研究人员分别通过未转染细胞中的免疫荧光技术（immunofluorescence），以及转染了绿色荧光蛋白（Green Fluorescent Protein, GFP）与红色荧光蛋白（Red Fluorescent Protein, RFP）标记的Rab5、Rab7、Rab4及Arf1的活细胞，采用视频增强型三色荧光显微镜（video-enhanced triple-color fluorescence microscopy）追踪单个荧光标记SFV颗粒所经历的胞内通路。病毒从Rab5阳性的早期内体，迁移至同时含有Rab5与Rab7的早期内体群体（约占总早期内体的10%）。SFV被隔离至Rab7结构域中，当这些结构域脱离形成不含Rab5、Rab4、早期内体抗原1（Early Endosome Antigen 1, EEA1）、Arf1及转铁蛋白的独立转运载体时，病毒便从早期内体中被分选出来。该过程不依赖于Arf1与早期内体的酸性pH环境。诺考达唑（Nocodazole）处理实验表明，转运载体的释放需微管提供辅助。组成型失活的Rab7T22N的表达会导致SFV在早期内体中积累。本研究最终得出结论：Rab7被招募至早期内体，在此形成独特的结构域，介导货物分选以及靶向晚期内体的转运囊泡的形成。