Functional reconstitution of the integral membrane proteins of influenza virus into phospholipid liposomes.
The integral membrane proteins of influenza virus, a hemagglutinin and a neuraminidase, have been incorporated into liposomes composed of either phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylethanolamine (2:1 w/w) using detergent dialysis. The virus spike glycoproteins for reconstitution were selectively solubilized by using cetyltrimethylammonium bromide to leave a "core particle", which lacked a lipid bilayer but possessed quaternary structure as observed by electron microscopy. The viral spike proteins were combined with exogenous phospholipid in excess sodium cholate followed by exhaustive dialysis for 150 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that only the viral glycoproteins were associated with all the complexes formed. The level of sodium cholate remaining after dialysis was shown to be reduced to less than 1 molecule per 80 protein molecules. Viral proteins reconstituted into dimyristoylphosphatidylcholine liposomes were shown to have retained hemagglutination, low-pH-dependent hemolysis, and neuraminidase activities and were associated with a lipid bilayer in two types of complexes with average lipid to protein mole ratios after sucrose density gradient purification of either 590:1 or 970:1. The bilayer vesicles formed were of similar sizes and were shown by negative-stain electron microscopy to be 150-300 nm in diameter with well-defined spikes on their surface. Reconstituted liposomes of dimyristoylphosphatidylcholine were found to be unstable with respect to their trapped volume and therefore were unsuitable for fusion studies, unlike complexes formed with phosphatidylcholine or a mixture of phosphatidylcholine/phosphatidylethanolamine derived from hen eggs.(ABSTRACT TRUNCATED AT 250 WORDS)
Sizer
P J
PJ
Department of Biochemistry, University of Oxford, U.K.
Miller
A
A
Watts
A
A
D004134:Dimyristoylphosphatidylcholine; D006384:Hemagglutination; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D009980:Influenza A virus / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000378:metabolism*; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D014764:Viral Proteins / Q000378:metabolism*
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D008081:Liposomes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D014764:Viral Proteins; D004134:Dimyristoylphosphatidylcholine