Microscopic Observation of Membrane Fusion between Giant Liposomes and Baculovirus Budded Viruses Activated by the Release of a Caged Proton.

Baculovirus (Autographa californica multiple nucleopolyhedrovirus, AcMNPV) is an envelope virus possessing a fusogenic protein, GP64, which can be activated under weak acidic conditions close to those in endosomes. When the budded viruses (BVs) are bathed at pH 4.0 to 5.5, they can bind to liposome membranes with acidic phospholipids, and this results in membrane fusion. In the present study, using the caged-proton reagent 1-(2-nitrophenyl)ethyl sulfate, sodium salt (NPE-caged-proton), which can be uncaged by irradiation with ultraviolet light, we triggered the activation of GP64 by lowering the pH and observed membrane fusion on giant liposomes (giant unilamellar vesicles, GUVs) by visualizing the lateral diffusion of fluorescence emitted from a lipophilic fluorochrome (octadecyl rhodamine B chloride, R18) that stained viral envelopes of BVs. In this fusion, entrapped calcein did not leak from the target GUVs. The behavior of BVs prior to the triggering of membrane fusion by the uncaging reaction was closely monitored. BVs appeared to accumulate around a GUV with DOPS, implying that BVs preferred phosphatidylserine. The monitoring of viral fusion triggered by the uncaging reaction could be a valuable tool for revealing the delicate behavior of viruses affected by various chemical and biochemical environments.

Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases.