Reconstitution of membranes with individual paramyxovirus glycoproteins and phospholipid in cholate solution

Abstract

A method has been developed for reconstitution of biologically active membranes with individual Sendai virus glycoproteins (HN and F) and phosphatidylcholine. The glycoproteins were isolated in the presence of Triton X-100 and transferred into cholate solution by sedimentation into a sucrose gradient containing 2% cholate. Membranes were then reconstituted with HN or F and phosphatidylcholine by removal of cholate by dialysis. Experiments with radioactively labeled detergents showed that the removal of Triton X-100 by sedimentation into cholate and the removal of cholate by dialysis were essentially complete. With the F protein, vesicles 400–800 Å in diameter and filaments 600–800 Å in length were formed, depending on the proportions of lipid and protein in the initial mixture. Both structures were covered with glycoprotein spikes. With the HN protein, only vesicles were formed, and the densities of the spikes on the surface was dependent on the initial lipid to protein ratio. Membranes reconstituted from HN protein exhibited hemagglutinating and neuraminidase activities. Reconstituted particles containing the F protein exhibited hemolytic activity when a mechanism was provided to attach the F protein-lipid complex to the cell, i.e., by the addition of wheat germ agglutinin. These results have confirmed the role of the F protein in membrane fusion and have shown that the requirements for F protein activity are the previously demonstrated proteolytic processing of F, the insertion of the F protein into lipid, and the presence of an attachment mechanism.