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Abstract
Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH4Cl. Removal of NH4Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH4Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude that the NH4Cl block traps the virus within intralumenal vesicles of late endosomes in cells expressing the GPI-anchored receptor. Viruses surrounded by more than one endosomal membrane release their core into the cytoplasm in two steps – fusion with an intralumenal vesicle followed by a yet unknown temperature-dependent step that liberates the core from late endosomes.
Highlights
A large number of enveloped and non-enveloped viruses enter cells through endocytosis [1]
Through tracking individual sub-viral particles released from endosomes, we found that the full length receptor mediated core delivery into the cytoplasm
A quick drop of endosomal pH caused by the NH4Cl removal serves as a standardized trigger for ASLV-endosome fusion, irrespective of the distinct trafficking itineraries for this virus through TVA800 and TVA950 isoforms [16,17]
Summary
A large number of enveloped and non-enveloped viruses enter cells through endocytosis [1]. Preferential entry from early or late endosomes is achieved through adjusting the pH threshold for triggering fusion [2,3,4] or by relying on endosome-specific factors, such as lipids [5,6,7] or lysosomal enzymes [8,9]. Viruses, which are activated by mildly acidic pH in early endosomes, may require late endosome-resident factors to complete their fusion process and release the nucleocapsid [5,7]. The asynchronous and often rate-limiting steps of virus internalization and trafficking hamper the studies of endosomal fusion and its regulation. In order to gain mechanistic insights into the virusendosome fusion, it is essential to isolate the virus fusion step from the upstream asynchronous processes and to control the timing of low pH exposure and acidity of endosomal compartments
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