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

TLDR

Semliki Forest virus enters cells via clathrin‑mediated endocytosis and traffics from early endosomes to late endosomes and lysosomes. The study tracked individual fluorescent SFV particles using immunofluorescence and live‑cell triple‑color fluorescence microscopy with GFP‑ and RFP‑tagged Rab5, Rab7, Rab4, and Arf1. SFV moves from Rab5‑positive early endosomes to a small Rab5/Rab7‑positive subpopulation, where Rab7 domains sequester the virus and, upon detachment as Rab5‑free carriers, transport it to late endosomes, a sorting process that is Arf1‑ and pH‑independent, microtubule‑assisted, and disrupted by inactive Rab7, demonstrating that Rab7 recruitment to early endosomes creates domains that mediate cargo sorting and late‑endosome vesicle formation.

Abstract

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.