Abstract
To help understand the dynamic nature of membrane fusion induced by the human immunodeficiency virus-1 (HIV-1) envelope protein, we developed a new cell-based real-time assay system employing a pair of novel reporter proteins. The reporter proteins consist of a pair of split Renilla luciferase (spRL) fused to split green fluorescent protein (spGFP). The spGFP modules were chosen not only to compensate weak self-association of spRL but also to provide visual reporter signals during membrane fusion. Use of this reporter together with a membrane permeable substrate for Renilla luciferase achieved a simple real-time monitoring of membrane fusion using live cells. We analyzed the HIV-1 envelope mutants whose membrane-spanning domains were replaced with that of glycophorin A or vesicular stomatitis virus G-protein. These mutants showed a slower kinetics of membrane fusion. The analysis of membrane fusion in the presence of fusion inhibitors, soluble CD4 and C34, revealed that these replacements prolonged the period during which the mutants were sensitive to the inhibitors, as compared with the wild type. These results suggest that the mutations within the membrane-spanning domains exerted an allosteric effect on the HIV-1 envelope protein, probably affecting the receptor-induced conformational changes of the ectodomain of the protein.
Highlights
To achieve a real-time analysis of membrane fusion, an appropriate assay system that is quick enough to generate a reporter signal is needed
The added split GFP (spGFP) portion [14] serves as a strong association module and provides a visible green signal upon membrane fusion. We named this new reporter protein dual split protein (DSP) because it recovers dual functions of RL and GFP upon reassociation. This dual functional reporter system was applied to analyzing mutant human immunodeficiency virus-1 (HIV-1) envelope protein (Env) whose membranespanning domain (MSD) was replaced with heterologous MSD derived from other membrane proteins such as glycophorin A (GpA) or vesicular stomatitis virus G protein (VSV-G) [15]
Design and Complementation Analyses of Fusion Proteins— Our assay system of membrane fusion relies on the recovery of the activity of the split enzyme, split Renilla luciferase (spRL), via reassociation induced by membrane fusion
Summary
Construction of the Plasmids for Split and Dual Split Protein Expression—The expression vectors were derivatives of phRLCMV (Promega). Live-cell Monitoring of Membrane Fusion Using the Dual Split Proteins and a Membrane-permeable Substrate—The 293FT cells with more than 50% confluency in the 96-well plate were transfected with both the DSP1–7 and the HIV-1 Env expression vectors. The latter vector, pNHcRedEluc [10], contains the wild type or mutant envelope gene derived from the HXB2 strain. The fusion assay by the co-culturing was performed as described above, except that the culture was first incubated for 2 h at 25 °C with 5% CO2 to induce TAS After this low temperature treatment, the temperature was shifted to 37 °C, and the RL activity was measured in a time-dependent manner
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