Application of fluorescence photobleaching recovery to assess complex formation between the two envelope proteins of Sendai virus in membranes of fused human erythrocytes.
Fusion of human erythrocytes by Sendai virions is accompanied by lateral mobilization of the viral envelope proteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase protein) in the target cell membrane; the dynamic parameters characterizing the lateral diffusion of F and HN in the fused cell membrane are identical [Henis, Y. I., & Gutman, O. (1987) Biochemistry 26, 812-819; Aroeti, B., & Henis, Y. I. (1988) Biochemistry 27, 5654-5661]. This identity raised the possibility that F and HN diffuse together in the cell membrane in mutual heterocomplexes. In order to investigate the possible formation of F-HN complexes in the target cell membrane, which could be important for the fusion process mediated by the viral envelope proteins, we combined fluorescence photobleaching recovery (FPR) measurements of the lateral mobility of the viral glycoproteins with antibody-mediated cross-linking of F or HN. After fusion, one viral glycoprotein type was immobilized by cross-linking with highly specific bivalent polyclonal IgG. The other glycoprotein type was labeled with fluorescence monovalent Fab' fragments that do not induce cross-linking, and its mobility was measured by FPR. Neither the mobile fraction nor the lateral diffusion coefficient of the Fab'-labeled viral glycoproteins was affected by immobilization of the second viral envelope protein, demonstrating that F and HN diffuse independently in the target cell membrane and are not associated in mutual complexes.
Katzir
Z
Z
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel.
Gutman
O
O
Henis
Y I
YI
D000818:Animals; D000936:Antigen-Antibody Complex / Q000032:analysis; D002459:Cell Fusion; D002642:Chick Embryo; D004910:Erythrocyte Membrane / Q000502:physiology*; D015707:HN Protein / Q000378:metabolism; D006389:Hemagglutinins, Viral; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008970:Molecular Weight; D010222:Parainfluenza Virus 1, Human / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D014771:Virion / Q000378:metabolism*
D000936:Antigen-Antibody Complex; D015707:HN Protein; D006389:Hemagglutinins, Viral; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D014759:Viral Envelope Proteins