Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes

Abstract

Diverse enveloped viruses enter host cells through endocytosis and fuse with endosomal membranes upon encountering acidic pH. Currently, the pH dynamics in virus-carrying endosomes and the relationship between acidification and viral fusion are poorly characterized. Here, we examined the entry of avian retrovirus that requires two sequential stimuli-binding to a cognate receptor and low pH-to undergo fusion. A genetically encoded sensor incorporated into the viral membrane was used to measure the pH in viruscarrying endosomes. Acid-induced virus fusion was visualized as the release of a fluorescent viral content marker into the cytosol. The pH values in early acidic endosomes transporting the virus ranged from 5.6 to 6.5 but were relatively stable over time for a given vesicle. Analysis of viral motility and luminal pH showed that cells expressing the transmembrane isoform of the receptor (TVA950) preferentially sorted the virus into slowly trafficking, less acidic endosomes. In contrast, viruses internalized by cells expressing the GPI-anchored isoform (TVA800) were uniformly distributed between stationary and mobile compartments. We found that the lag times between acidification and fusion were significantly shorter and fusion pores were larger in dynamic endosomes than in more stationary compartments. Despite the same average pH within mobile compartments of cells expressing alternative receptor isoforms, TVA950 supported faster fusion than TVA800 receptor. Collectively, our results suggest that fusion steps downstream of the low-pH trigger are modulated by properties of intracellular compartments harboring the virus.confocal imaging | nano-pH-meter | single virus tracking | FRET M any enveloped viruses use endocytosis and vesicular trafficking to enter host cells, where acidification of an endosomal lumen serves as a trigger for viral fusion (1, 2). Viral fusion proteins are fine-tuned to respond to different pH values. Those that are activated at less acidic pH (∼6.0) are thought to mediate fusion with early endosomes, whereas those with a lower pH threshold (∼5.0) appear to direct the virus entry from late endosomes (1, 3). However, recent evidence implies that factors other than low pH, such as specific endosome-resident lipids, can determine the intracellular compartments from which the viral capsid is released into the cytosol (2, 4, 5). Progress in understanding the complex regulation of virus-endosome fusion has been hindered by poor accessibility of intracellular compartments and lack of direct techniques for monitoring this process in situ.Single-virus imaging is a powerful tool for gaining critical insights into virus entry (reviewed in ref. 6), but detection of virus-endosome fusion (here defined as the release of viral content into the cytoplasm) is technically challenging (7,8). To understand the relationship between the pH trigger and virus-endosome fusion, it is essential to visualize these events in real time. A ratiometric method developed by the Zhuang group (9, 10) permits monitoring endos...

pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion

“…2B). This striking phenotype implies that internalized ASLV quickly enters into mildly acidic compartments, but completes acid-dependent steps of fusion after 10 -20 min, which is at least an order of magnitude slower than the low pH-triggered ASLV fusion with CV-1-derived cells (14).…”

“…Given the relatively high pH threshold for ASLV fusion (14,46,57), we have originally assumed that quenching the EcpH probe reports entry into early acidic compartments that support viral fusion (31). To test this notion, we estimated the pH that effectively quenches the EcpH signal of single virions.…”

“…These features make ASLV amenable to biophysical studies of virus entry at a single particle level. We have previously investigated the entry/fusion mechanism of ASLV by single virus imaging and delineated the relationship between endosomal pH and the probability and kinetics of ASLV fusion (13,14,32 …”

“…It is thus likely that cell type-dependent regulation of endosomal pH modulates the efficiency and kinetics of virus fusion. To date, however, only a few studies have directly examined the link between the pH in virus-carrying endosomes and the efficiency/kinetics of subsequent viral fusion (12)(13)(14)(15).…”

Nanoscale virus biosensors: state of the art

“…182 Another option for virus identification by optical fluorescence detection is the application of the retroviruses tagged with a genetically encoded pH-sensor and a fluorescent content marker enabled simultaneous measurements of the pH drop within virus-carrying vesicles and the resulting virus-endosome fusion. 183 Label-free chemiresistive sensors based on a polypyrrole nanoribbon were batch-fabricated by a lithographically patterned nanowire electrodeposition technique. A plant-pathogen-specific antibody was covalently conjugated on the surface of the structure via N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide/Nhydrosuccinimide cross-linking.…”