Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion

It has been proposed that membrane fusion events such as virus-cell fusion proceed through a hemifusion intermediate, a state where lipids but not contents of the fusing compartments mix. We engineered the influenza hemagglutinin (HA) such that it would be anchored in membranes via a glycosylphosphatidylinositol (GPI) tail. GPI-anchored HA forms a trimer that can bind red blood cells (RBCs) and change conformation under fusion-inducing conditions. Using RBCs labeled with fluorescent lipid or fluorescent soluble content probes, we found that GPI-anchored HA mediated lipid mixing with similar time course and efficiency as wt-HA, yet did not mediate transfer of soluble contents. Hence, GPI-anchored HA appears to initiate, but not complete, a fusion reaction. We interpret our results as evidence for uncoupling a physiological fusion reaction, for trapping a hemifusion intermediate, and for assigning a role to a transmembrane domain in a fusion event.

[1]  C. Di Simone,et al.  Membrane fusion of mumps virus with ghost erythrocytes and CV-1 cells. , 1992, Virology.

[2]  J. Heuser,et al.  Arrest of membrane fusion events in mast cells by quick-freezing , 1980, The Journal of cell biology.

[3]  R. Lamb,et al.  Alterations to influenza virus hemagglutinin cytoplasmic tail modulate virus infectivity , 1992, Journal of virology.

[4]  M. Gething,et al.  Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus , 1986, The Journal of cell biology.

[5]  R. Scheller,et al.  Synaptic vesicle membrane proteins interact to form a multimeric complex , 1992, The Journal of cell biology.

[6]  R. Doms,et al.  Protein-mediated membrane fusion. , 1989, Annual review of biophysics and biophysical chemistry.

[7]  Deborah A. Brown,et al.  Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface , 1992, Cell.

[8]  T. Reese,et al.  Beginning of exocytosis captured by rapid-freezing of Limulus amebocytes , 1981, The Journal of cell biology.

[9]  E. Chen,et al.  A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast , 1989, Nature.

[10]  F. Booy,et al.  Functional reconstitution of influenza virus envelopes. , 1987, The EMBO journal.

[11]  M. Edidin.  Patches, posts and fences: proteins and plasma membrane domains. , 1992, Trends in cell biology.

[12]  J. Skehel,et al.  Studies of influenza haemagglutinin-mediated membrane fusion. , 1986, Virology.

[13]  T. Wolfsberg,et al.  A potential fusion peptide and an integrin ligand domain in a protein active in sperm–egg fusion , 1992, Nature.

[14]  P. Choppin,et al.  Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. , 1975, Virology.

[15]  N. Gilula,et al.  Membrane events involved in myoblast fusion , 1979, The Journal of cell biology.

[16]  J. Rothman,et al.  Molecular dissection of the secretory pathway , 1992, Nature.

[17]  J. White,et al.  Fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density. , 1990, Biochemistry.

[18]  I. Wilson,et al.  Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin , 1992, Journal of virology.

[19]  Y. Henis,et al.  GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity , 1993, The Journal of cell biology.

[20]  Nichols Jw,et al.  Use of resonance energy transfer to study the kinetics of amphiphile transfer between vesicles. , 1982 .

[21]  G. Semenza,et al.  Hydrophobic binding of the ectodomain of influenza hemagglutinin to membranes occurs through the "fusion peptide". , 1989, The Journal of biological chemistry.

[22]  J. Skehel,et al.  The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. , 1987, Annual review of biochemistry.

[23]  J. Sambrook,et al.  Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway , 1986, The Journal of cell biology.

[24]  M. L. Nogueira,et al.  Membrane fusion during secretion. A hypothesis based on electron microscope observation of Phytophthora Palmivora zoospores during encystment , 1977, The Journal of cell biology.

[25]  D. Alford,et al.  An architecture for the fusion site of Influenza hemagglutinin , 1990, FEBS letters.

[26]  J. M. Fernández,et al.  Release of secretory products during transient vesicle fusion , 1993, Nature.

[27]  J. Skehel,et al.  Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity , 1992, Cell.

[28]  G. Palade,et al.  STRUCTURAL MODULATIONS OF PLASMALEMMAL VESICLES , 1968, The Journal of cell biology.

[29]  W. Almers,et al.  Patch clamp studies of single cell-fusion events mediated by a viral fusion protein , 1989, Nature.

[30]  W. Almers,et al.  Membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion , 1993, The Journal of cell biology.

[31]  D. Georgescauld,et al.  Membrane fusion without cytoplasmic fusion (hemi-fusion) in erythrocytes that are subjected to electrical breakdown. , 1991, Biochimica et biophysica acta.

[32]  F. Szoka,et al.  H+- and Ca2+-induced fusion and destabilization of liposomes. , 1985, Biochemistry.

[33]  F. Richards,et al.  The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion. , 1991, The Journal of biological chemistry.

[34]  M. Roth,et al.  A chimeric avian retrovirus containing the influenza virus hemagglutinin gene has an expanded host range , 1992, Journal of virology.

[35]  Paul Tempst,et al.  SNAP receptors implicated in vesicle targeting and fusion , 1993, Nature.

[36]  I. Wilson,et al.  Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin , 1987, The Journal of cell biology.

[37]  M. Gething,et al.  Haemagglutinin of influenza virus expressed from a cloned gene promotes membrane fusion , 1982, Nature.

[38]  R. Rand,et al.  Divalent cation induced fusion and lipid lateral segregation in phosphatidylcholine-phosphatidic acid vesicles. , 1986, Biochemistry.

[39]  H. Wunderli-Allenspach,et al.  Kinetics of fusion and lipid transfer between virus receptor containing liposomes and influenza viruses as measured with the octadecylrhodamine B chloride assay. , 1990, Biochemistry.

[40]  H. Guy,et al.  Analyzing the fusion process of influenza hemagglutinin by mutagenesis and molecular modeling. , 1992, Biophysical Journal.

[41]  W. Almers,et al.  The first milliseconds of the pore formed by a fusogenic viral envelope protein during membrane fusion. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[42]  R. Schekman.  Genetic and biochemical analysis of vesicular traffic in yeast , 1992, Current Biology.

[43]  A. Helenius,et al.  Intermediates in influenza induced membrane fusion. , 1990, The EMBO journal.

[44]  R. Doms,et al.  Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change. , 1985, The Journal of biological chemistry.

[45]  I D Kuntz,et al.  Inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinones. , 1993, Biochemistry.

[46]  P. S. Kim,et al.  A spring-loaded mechanism for the conformational change of influenza hemagglutinin , 1993, Cell.