Precise triggering and chemical control of single-virus fusion within endosomes.

Haldar, Sourav; Okamoto, Kenta; Dunning, Rebecca A; Kasson, Peter M

Many enveloped viruses infect cells within endocytic compartments. The pH drop that accompanies endosomal maturation, often in conjunction with proteolysis, triggers viral proteins to insert into the endosomal membrane and drive fusion. Fusion dynamics have been studied by tracking viruses within living cells, which limits the precision with which fusion can be synchronized and controlled, and reconstituting viral fusion to synthetic membranes, which introduces non-physiological membrane curvature and composition. To overcome these limitations, we report chemically controllable triggering of single-virus fusion within endosomes. We isolate influenza (A/Aichi/68; H3N2) virus:endosome conjugates from cells, immobilize them in a microfluidic flow cell, and rapidly and controllably trigger fusion. Observed lipid-mixing kinetics are surprisingly similar to influenza fusion with model membranes of opposite curvature: 80% of single-virus events have indistinguishable kinetics. This result suggests that endosomal membrane curvature is not a key permissive feature for viral entry, at least lipid mixing. The assay preserves endosomal membrane asymmetry and protein composition, providing a platform to test how cellular restriction factors and altered endosomal trafficking affect viral membrane fusion.IMPORTANCE Many enveloped viruses infect cells via fusion to endosomes, but controlling this process within living cells has been challenging. Here, we study the fusion of influenza virions to endosomes in a chemically controllable manner. Extracting virus-endosome conjugates from cells and exogenously triggering fusion permits precise study of virus-endosome fusion kinetics. Surprisingly, endosomal curvature does not grossly alter fusion kinetics, although membrane deformability does. This supports a model for influenza virus entry where cells restrict or permit membrane fusion by changing deformability, for instance using interferon-induced proteins.