Measurement of membrane fusion activity from viral membrane fusion proteins based on a fusion-dependent promoter induction system in insect cells

Abstract

A number of viral membrane fusion proteins can be expressed alone on the surface of host cells, and then triggered to induce cell-to-cell fusion or syncytium formation. Although rapid and easily observed, syncytium formation is not easily quantified and differences in fusion activity are not easily distinguished or measured. To address this problem, we developed a rapid and quantitative cell-to-cell fusion system that is useful for comparative analysis and may be suitable for high throughput screening. In this system, expression of a reporter protein, enhanced green fluorescent protein (EGFP), is dependent on cell-to-cell fusion. Spodoptera frugiperda (Sf9) insect cells expressing a chimeric Lac repressor-IE1 protein were fused to Sf9 cells containing an EGFP reporter construct under the control of a responsive lac operator-containing promoter. Membrane fusion efficiency was measured from the resulting EGFP fluorescence activity. Sf9 cells expressing the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) GP64 envelope fusion protein were used as a model to test this fusion assay. Subtle changes in fusion activities of GP64 proteins containing single amino acid substitutions in a putative membrane fusion domain were distinguished, and decreases in EGFP fluorescence corresponded to decreases in the hydrophobicity in the small putative membrane fusion domain.