Time-Resolved Imaging of Endosome Acidification and Single Retrovirus Fusion with Endosomes

Avian Sarcoma and Leukosis Virus (ASLV) enters cells by receptor-mediated endocytosis followed by low pH-dependent fusion with endosomes. We examined the pH dynamics in virus-carrying endosomes and the relationship between endosome acidification and ASLV fusion in cells expressing alternative receptor isoforms. The pH drop in endosomal lumen was measured by incorporating a genetically encoded pH sensor into the viral membrane. The subsequent single virus fusion was visualized as the release of a fluorescent viral content marker into the cytosol. To our knowledge, this is the first implementation of real-time measurements of endosomal pH dynamics and single virus fusion in the same experiment. We found that the pH in early acidic compartments harboring the virus was broadly distributed, ranging from 5.6 to 6.5. Surprisingly, after initial acidification, the endosomal pH did not decrease further for at as long as we could track the viral particles (up to 20 min). Analysis of the relationship between the endosome motility and lumenal pH showed that cells expressing the transmembrane isoform of the receptor (TVA950) preferentially sorted ASLV to slowly moving, less acidic endosomes. In contrast, ASLV showed no preference for fast vs. slow vesicles in cells expressing the GPI-anchored isoform (TVA800). The rate of fusion (distribution of time intervals between acidification and fusion) was significantly shorter and fusion pores were larger in mobile endosomes compared to more stationary compartments. In addition, TVA950 supported faster fusion than TVA800 in spite of the same average pH values in mobile compartments of these cells. Taken together, our results revealed that the ASLV can be selectively sorted to distinct endosomal pools and that the virus fuses with different kinetics and efficiency with these intracellular compartments. This work was partially supported by NIH AI053668 grant to G.B.M.