Enveloped viruses release their genome into the cytosol upon membrane fusion of the virus most commonly to the limiting membrane of the endosomal compartment. This process tends to be asynchronous, happening by chance and at different times and locations within the endosomal population. It would be ideal if one could circumvent this inherent stochasticity and instead drive the fusion reaction ‘on command’, as this trick would then allow direct visualization of single fusion and escape event, hence providing a tool to explore mechanistic questions associated with virus penetration. We have therefore used opto-genetics to create a light-activated Bafilomycin-insensitive proton pump specifically targeted to the limiting membrane of endosomes. Its photoactivation allows the synchronous pH drop in endosomes in cells incubated with Bafilomycin, an inhibitor specific for the endosomal proton pump. Using a lattice light sheet microscope to visualize in real time the complete cellular volume, we are now analyzing the dynamics of viral fusion of single particles to endosomal membranes and the kinetics of capsid release into the cytosol, key steps for successful infection.
Genscript (Nanjing) LTD.