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1.
J Membr Biol ; 199(1): 1-14, 2004 May 01.
Article in English | MEDLINE | ID: mdl-15366419

ABSTRACT

The main steps of viral membrane fusion are local membrane approach, hemifusion, pore formation, and pore enlargement. Experiments and theoretical analyses have helped determine the relative energies required for each step. Key protein structures and conformational changes of the fusion process have been identified. The physical deformations of monolayer bending and lipid tilt have been applied to the steps of membrane fusion. Experiment and theory converge to strongly indicate that, contrary to former conceptions, the fusion process is progressively more energetically difficult: hemifusion has a relatively low energy barrier, pore formation is more energy-consuming, and pore enlargement is the most difficult to achieve.


Subject(s)
Cell Membrane/metabolism , Lipid Bilayers/metabolism , Membrane Fusion/physiology , Membrane Lipids/metabolism , Viral Fusion Proteins/metabolism , Lipid Metabolism , Models, Theoretical
2.
Biophys J ; 87(5): 3291-8, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15339808

ABSTRACT

The fusion protein of avian sarcoma and leukosis virus is likely to fold into a six-helix bundle as part of its final configuration. A peptide, R99, inhibits fusion, probably by binding into the grooves of the triple-stranded coiled coil that becomes the central core of the six-helix bundle. The stages at which the envelope protein (Env) of avian sarcoma and leukosis virus subgroup A folds into a bundle during low pH-induced fusion were determined. Effector cells expressing Env were bound to target cells expressing the cognate receptor Tva, and intermediates of fusion were created. R99 was added and the extent of fusion inhibition was used to distinguish between a prebundle state with exposed grooves and a state in which the grooves were no longer exposed. The native conformation of Env was not sensitive to R99. But adding a soluble form of Tva to effector cells conferred sensitivity. Acidic pH applied at low temperature created an intermediate state of local hemifusion. Surprisingly, R99 caused these locally hemifused membranes to separate. This indicates that the grooves of Env were still exposed, that prebundle configurations of Env stabilized hemifused states, and that binding of R99 altered the conformation of Env. In the presence of an inhibitory lipid that blocks fusion before hemifusion, applying low pH at 37 degrees C created an intermediate in which R99 was without effect. This suggests that the six-helix bundle can form before hemifusion and that subsequent conformational changes, such as formation of the trimeric hairpin, are responsible for pore formation and/or growth.


Subject(s)
Avian Sarcoma Viruses/metabolism , Cell Membrane Permeability/physiology , Gene Products, env/metabolism , Membrane Fusion/drug effects , Membrane Fusion/physiology , Peptides/pharmacology , 3T3 Cells , Animals , Cell Membrane Permeability/drug effects , Dose-Response Relationship, Drug , Hydrogen-Ion Concentration , Mice , Models, Biological , Protein Conformation/drug effects , Protein Folding , Protein Structure, Secondary/drug effects
3.
J Virol ; 78(7): 3753-62, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15016895

ABSTRACT

Binding of avian sarcoma and leukosis virus (ASLV) to its cognate receptor on the cell surface causes conformational changes in its envelope protein (Env). It is currently debated whether low pH is required for ASLV infection. To elucidate the role of low pH, we studied the association between ASLV subgroup B (ASLV-B) and liposomes and fusion between effector cells expressing Env from ASLV-A and ASLV-B and target cells expressing cognate receptors. Neither EnvA nor EnvB promoted cell-cell fusion at neutral pH, but lowering the pH resulted in quick and extensive fusion. As expected for a low-pH-triggered reaction, fusion was a steep function of pH. Steps that required low pH were identified. Binding a soluble form of the receptor caused ASLV-B to hydrophobically associate with liposome membranes at neutral pH, indicating that low pH is not required for insertion of Env's fusion peptides into membranes. But both cell-cell hemifusion and fusion pore formation were pH dependent. It is proposed that fusion peptide insertion stabilizes the conformation of ASLV Env into a form that can be acted upon by low pH. At this point, but not before, low pH can induce fusion and is in fact required for fusion to occur. However, low pH is no longer necessary after formation of the initial fusion pore: pore enlargement does not require low pH.


Subject(s)
Avian Leukosis Virus/metabolism , Avian Sarcoma Viruses/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , Membrane Fusion , Animals , Avian Leukosis Virus/physiology , Avian Sarcoma Viruses/physiology , Cell Fusion , Cell Line , Chickens , Cold Temperature , Fibroblasts , Gene Products, env/metabolism , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Liposomes/metabolism , Models, Biological , Receptors, Virus/metabolism , Substrate Specificity
4.
Virology ; 316(2): 234-44, 2003 Nov 25.
Article in English | MEDLINE | ID: mdl-14644606

ABSTRACT

For influenza virus hemagglutinin, an N-cap structure, created at low pH, interacts with membrane-proximal residues (173-178), bringing fusion peptides and membrane-spanning domains close together. Mutational analysis was used to define the role of these interactions in membrane fusion. For all N-cap mutants, both lipid and aqueous dye spread was greatly reduced. Mutation at residues that interact with the N-cap did not reduce levels of fusion, except for substitutions made at residue I173. For N-cap and I173 mutants, the addition of chlorpromazine greatly promoted transfer of aqueous dye. Electrical capacitance measurements confirmed that fusion pores usually did not form for the I173 mutants. Thus, neither N-cap formation nor interactions with segment 173-178 are needed for hemifusion, but are required for reliable formation and enlargement of the fusion pore. It is proposed that binding of I173 into a deep hydrophobic cavity within the coiled-coil promotes the transition from hemifusion to fusion.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Membrane Fusion , Cell Line , Humans , Hydrogen-Ion Concentration , Microscopy, Fluorescence , Mutation , Protein Conformation , Viral Fusion Proteins/chemistry
5.
J Virol ; 77(10): 5829-36, 2003 May.
Article in English | MEDLINE | ID: mdl-12719576

ABSTRACT

A mutant human immunodeficiency virus (HIV) envelope protein (Env) with an engineered disulfide bond between the gp120 and gp41 subunits (SOS-Env) was expressed on cell surfaces. With the disulfide bond intact, these cells did not fuse to target cells expressing CD4 and CCR5, but the fusion process did advance to an intermediate state: cleaving the disulfide bond with a reducing agent after but not before binding to target cells allowed fusion to occur. Through the use of an antibody directed against CCR5, it was found that at the intermediate stage, SOS-Env had associated with coreceptors. Reducing the disulfide bond after this intermediate had been reached resulted in hemifusion at low temperature and fusion at physiological temperature. The addition of C34 or N36, peptides that prevent six-helix bundle formation, at the hemifused state blocked the fusion that would have resulted after raising the temperature. Thus, Env has not yet folded into six-helix bundles after hemifusion has been achieved. Because SOS-Env binds CCR5, it is suggested that the conformational changes in wild-type Env that result from this binding cause disengagement of gp120 from gp41 in the region of the engineered bond. It is proposed that this disengagement is the event that directly frees gp41 to undergo the conformational changes that lead to fusion. The intermediate state achieved prior to reduction of the disulfide bond was stable. The capture of this configuration of Env could yield a suitable antigen for vaccine development, and it may also be a target for pharmacological intervention against HIV-1 entry.


Subject(s)
Disulfides/metabolism , Gene Products, env/metabolism , HIV-1/pathogenicity , Membrane Fusion , Receptors, HIV/metabolism , CD4 Antigens/metabolism , Cell Line , Disulfides/chemistry , Disulfides/pharmacology , Dithiothreitol/pharmacology , Gene Products, env/genetics , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/metabolism , HIV-1/metabolism , Humans , Membrane Fusion/drug effects , Oxidation-Reduction , Protein Conformation , Receptors, CCR5/metabolism , Reducing Agents/pharmacology
7.
Biophys J ; 80(2): 812-21, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11159448

ABSTRACT

Cells expressing wild-type influenza virus hemagglutinin (HA) or HA with a point mutation within the transmembrane domain (G520L) were bound to red blood cells and exposed to low pH for short times at suboptimal temperatures followed by reneutralization. This produced intermediate states of fusion. The ability of intermediate states to proceed on to fusion when temperature was raised was compared kinetically. In general, for wild-type HA, fusion occurred more quickly by directly lowering pH at 37 degrees C in the bound state than by raising temperature at the intermediate stage. When pH was lowered for 1-2 min, kinetics of fusion upon raising temperature of an intermediate slowed the longer the intermediate was maintained at neutral pH. But for a more sustained (10 min) acidification, kinetics was independent of the time the intermediate was held at neutral pH before triggering fusion by raising temperature. In contrast, generating intermediates in the same way with G520L yielded kinetics of fusion that did not depend on the time intermediates were maintained after reneutralization. For both HA and G520L, the extents of fusion did not depend on the temperature at which pH was lowered, but fusion from the intermediate was extremely sensitive to the temperature to which the cells were raised. The measured kinetics and temperature dependencies suggest that the rate-limiting step of fusion occurs subsequent to formation of any of the intermediates; the conformational change of HA into its final configuration may be the rate-limiting step.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/physiology , Membrane Fusion/physiology , Animals , Biophysical Phenomena , Biophysics , Cell Line , Erythrocyte Membrane/chemistry , Erythrocyte Membrane/physiology , Hemagglutinin Glycoproteins, Influenza Virus/genetics , In Vitro Techniques , Kinetics , Point Mutation , Temperature
8.
Mol Biol Cell ; 11(11): 3765-75, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11071905

ABSTRACT

A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23 degrees C rather than 37 degrees C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37 degrees C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4 degrees C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Point Mutation , Animals , Cell Fusion , Cells, Cultured , Chlorpromazine/pharmacology , Erythrocytes/cytology , Erythrocytes/drug effects , Leucine , Osmotic Pressure , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Temperature , Time Factors
9.
J Cell Biol ; 151(2): 413-23, 2000 Oct 16.
Article in English | MEDLINE | ID: mdl-11038187

ABSTRACT

Many viral fusion proteins exhibit a six-helix bundle as a core structure. HIV Env-induced fusion was studied to resolve whether membrane merger was due to the transition into the bundle configuration or occurred after bundle formation. Suboptimal temperature was used to arrest fusion at an intermediate stage. When bundle formation was prevented by adding inhibitory peptides at this stage, membranes did not merge upon raising temperature. Inversely, when membrane merger was prevented by incorporating lysophosphatidylcholine (LPC) into cell membranes at the intermediate, the bundle did not form upon optimizing temperature. In the absence of LPC, the six-helix bundle did not form when the temperature of the intermediate was raised for times too short to promote fusion. Kinetic measures showed that after the temperature pulse, cells had not advanced further toward fusion. The latter results indicate that bundle formation is the rate-limiting step between the arrested intermediate and fusion. Electrical measures showed that the HIV Env-induced pore is initially large and grows rapidly. It is proposed that bundle formation and fusion are each contingent on the other and that movement of Env during its transition into the six-helix bundle directly induces the lipid rearrangements of membrane fusion. Because peptide inhibition showed that, at the intermediate stage, the heptad repeats of gp41 have become stably exposed, creation of the intermediate could be of importance in drug and/or vaccine development.


Subject(s)
HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/metabolism , HIV-1 , Membrane Fusion , CD4-Positive T-Lymphocytes/virology , Lysophosphatidylcholines/pharmacology , Membrane Fusion/drug effects , Models, Biological , Motion , Protein Conformation , Receptors, CXCR4 , Temperature , Thermodynamics
10.
Mol Biol Cell ; 11(4): 1143-52, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10749920

ABSTRACT

GPI-linked hemagglutinin (GPI-HA) of influenza virus was thought to induce hemifusion without pore formation. Cells expressing either HA or GPI-HA were bound to red blood cells, and their fusion was compared by patch-clamp capacitance measurements and fluorescence microscopy. It is now shown that under more optimal fusion conditions than have been used previously, GPI-HA is also able to induce fusion pore formation before lipid dye spread, although with fewer pores formed than those induced by HA. The GPI-HA pores did not enlarge substantially, as determined by the inability of a small aqueous dye to pass through them. The presence of 1,1'-dioctadecyl-3, 3,3',3'-tetramethylindocarbocyanine perchlorate or octadecylrhodamine B in red blood cells significantly increased the probability of pore formation by GPI-HA; the dyes affected pore formation to a much lesser degree for HA. This greater sensitivity of pore formation to lipid composition suggests that lipids are a more abundant component of a GPI-HA fusion pore than of an HA pore. The finding that GPI-HA can induce pores indicates that the ectodomain of HA is responsible for all steps up to the initial membrane merger and that the transmembrane domain, although not absolutely required, ensures reliable pore formation and is essential for pore growth. GPI-HA is the minimal unit identified to date that supports fusion to the point of pore formation.


Subject(s)
Glycosylphosphatidylinositols/physiology , Hemagglutinin Glycoproteins, Influenza Virus/physiology , Membrane Fusion/physiology , Animals , CHO Cells , Cell Membrane/physiology , Coloring Agents , Cricetinae , Erythrocytes/physiology , Glycosylphosphatidylinositols/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Humans , Hydrogen-Ion Concentration , In Vitro Techniques , Microscopy, Video , Patch-Clamp Techniques , Protein Structure, Tertiary , Temperature
11.
J Virol ; 74(1): 447-55, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10590134

ABSTRACT

Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*-a construct of Env with the last 16 amino acid residues (617 to 632; the R peptide) deleted from its C terminus to match the proteolytically cleaved Env produced during viral budding-resulted in high levels of fusion. Env 601*, lacking the entire cytoplasmic tail (CT) (identified by hydrophobicity), also led to fusion. Truncation of an additional six residues (Env 595*) abolished fusion. The kinetics of forming fusion pores did not depend on whether cells were first prebound at 4 degrees C and the time until fusion measured after the temperature was raised to 37 degrees C or whether cells were first brought into contact at 37 degrees C and the time until fusion immediately measured. This similarity in kinetics indicates that binding is accomplished quickly compared to subsequent steps in fusion. The fusion pores formed by Env 601* and Env 616* had the same initial size and enlarged in similar manners. Thus, once the R peptide is removed, the CT is not needed for fusion and does not affect formed pores. However, residues 595 to 601 are required for fusion. It is suggested here that the ectodomain and membrane-spanning domain of Env are directly responsible for fusion and that the R peptide affects their configurations at some point during the fusion process, thereby indirectly controlling fusion.


Subject(s)
Cell Fusion , Cytoplasm/metabolism , Gene Products, env/physiology , Moloney murine leukemia virus/metabolism , 3T3 Cells , Animals , Cell Line , Humans , Mice
12.
Biophys J ; 77(6): 3144-51, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10585935

ABSTRACT

The chronological relation between the establishment of lipid continuity and fusion pore formation has been investigated for fusion of cells expressing hemagglutinin (HA) of influenza virus to planar bilayer membranes. Self-quenching concentrations of lipid dye were placed in the planar membrane to monitor lipid mixing, and time-resolved admittance measurements were used to measure fusion pores. For rhodamine-PE, fusion pores always occurred before a detectable amount of dye moved into an HA-expressing cell. However, with DiI in the planar membrane, the relationship was reversed: the spread of dye preceded formation of small pores. In other words, by using DiI as probe, hemifusion was clearly observed to occur before pore formation. For hemifused cells, a small pore could form and subsequently fully enlarge. In contrast, for cells that express a glycosylphosphatidylinositol-anchored ectodomain of HA, hemifusion occurred, but no fully enlarged pores were observed. Therefore, the transmembrane domain of HA is required for the formation of fully enlarging pores. Thus, with the planar bilayer membranes as target, hemifusion can precede pore formation, and the occurrence of lipid dye spread does not preclude formation of pores that can enlarge fully.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/physiology , Membrane Fusion/physiology , 3T3 Cells , Animals , Biophysical Phenomena , Biophysics , Carbocyanines , Fluorescent Dyes , Lipid Bilayers , Mice , Microscopy, Fluorescence
13.
Mol Biol Cell ; 10(8): 2759-69, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10436026

ABSTRACT

We showed previously that substitution of the first residue of the influenza hemagglutinin (HA) fusion peptide Gly1 with Glu abolishes fusion activity. In the present study we asked whether this striking phenotype was due to the charge or side-chain volume of the substituted Glu. To do this we generated and characterized six mutants with substitutions at position 1: Gly1 to Ala, Ser, Val, Glu, Gln, or Lys. We found the following. All mutants were expressed at the cell surface, could be cleaved from the precursor (HA0) to the fusion permissive form (HA1-S-S-HA2), bound antibodies against the major antigenic site, bound red blood cells, and changed conformation at low pH. Only Gly, Ala, and Ser supported lipid mixing during fusion with red blood cells. Only Gly and Ala supported content mixing. Ser HA, therefore, displayed a hemifusion phenotype. The hemifusion phenotype of Ser HA was confirmed by electrophysiological studies. Our findings indicate that the first residue of the HA fusion peptide must be small (e.g., Gly, Ala, or Ser) to promote lipid mixing and must be small and apolar (e.g., Gly or Ala) to support both lipid and content mixing. The finding that Val HA displays no fusion activity underscores the idea that hydrophobicity is not the sole factor dictating fusion peptide function. The surprising finding that Ser HA displays hemifusion suggests that the HA ectodomain functions not only in the first stage of fusion, lipid mixing, but also, either directly or indirectly, in the second stage of fusion, content mixing.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/genetics , Membrane Fusion , Point Mutation , Viral Fusion Proteins/genetics , 3T3 Cells/metabolism , 3T3 Cells/virology , Amino Acid Substitution , Animals , COS Cells/metabolism , COS Cells/virology , Cell Membrane/virology , Chlorpromazine/pharmacology , Electrophysiology , Erythrocytes/drug effects , Erythrocytes/metabolism , Erythrocytes/virology , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Lipid Metabolism , Mice , Patch-Clamp Techniques , Phenotype , Protein Conformation , Viral Fusion Proteins/chemistry
14.
Biophys J ; 77(2): 943-52, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10423439

ABSTRACT

The effects of membrane tension on fusion between cells expressing the hemagglutinin (HA) of influenza virus and red blood cells were studied by capacitance measurements. Inflation of an HA-expressing cell was achieved by applying a positive hydrostatic pressure to its interior through a patch-clamp pipette in the whole-cell configuration. Inflating cells to the maximum extent possible without lysis created a membrane tension and completely inhibited low-pH-induced fusion at room temperature. Fully inflated cells that were subsequently deflated to normal size resumed the ability to fuse in response to low pH. At the higher temperature of 32 degrees C, fusion conditions were sufficiently optimal that full inflation did not hinder fusion, and once formed, pores enlarged more rapidly than those of never inflated cells. It is suggested that under fusogenic conditions HA causes the formation of a dimple within the membrane in which it resides, and that membrane tension hinders fusion by preventing the formation of dimples. Because dimpling bends the bilayer portion of bound membranes so that they come into intimate contact, the damping of dimpling would suppress this initial step in the fusion process.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/physiology , Membrane Fusion/physiology , 3T3 Cells , Animals , CHO Cells , Cell Size , Cricetinae , Erythrocytes/physiology , Gene Expression , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hydrogen-Ion Concentration , Hydrostatic Pressure , Kinetics , Mice , Models, Biological , Surface Tension
15.
Mol Biol Cell ; 10(6): 1821-36, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10359599

ABSTRACT

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Membrane Fusion/physiology , Amino Acid Sequence , Cell Membrane , Cytoplasm , Erythrocytes/metabolism , Erythrocytes/virology , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Kinetics , Molecular Sequence Data , Point Mutation , Protein Folding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
16.
Methods ; 16(2): 215-26, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9790869

ABSTRACT

The process of membrane fusion has been profitably studied by fusing cells that express fusion proteins on their surfaces to the membranes of target cells. Primary methods for monitoring the occurrence of fusion between cells are measurement of formation of heterokaryons, measurement of activation of reporter genes, measurement of transfer of lipidic and aqueous fluorescent dyes, and electrophysiological recording of fusion pores. Fluorescence and electrical methods have been well developed for fusion of a nucleated cell expressing viral fusion proteins to red blood cell targets. These techniques are now being extended to the study of fusion between two nucleated cells. Microscopic observation of spread of fluorescent dyes from one cell to another is a sensitive and convenient means of detecting fusion on the level of single events. In such studies, both the membrane and the aqueous continuities that occur as a result of fusion can be measured in the same experiment. By following spread of aqueous dyes of different sizes from one cell to another, the growth of a fusion pore can also be followed. By labeling cells with fluorescent probes, a state of hemifusion can be identified if probes in outer membrane leaflets transfer but probes in inner leaflets or aqueous spaces do not. Electrical measurements-both capacitance and double-whole-cell voltage-clamp techniques-are the most sensitive methods yet developed for detecting the formation of pores and for quantifying their growth. These powerful single-event methodologies should be directly applicable to further advances in expressing nonviral fusion proteins on cell surfaces.


Subject(s)
Cell Membrane/physiology , Cytological Techniques , Membrane Fusion , Cell Fusion/physiology , Erythrocytes/physiology , Gene Expression Regulation , Genes, Reporter , Hybrid Cells , Microscopy, Fluorescence , Molecular Probes , Patch-Clamp Techniques , Viral Fusion Proteins/metabolism
17.
J Gen Physiol ; 112(4): 409-22, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9758860

ABSTRACT

Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that these probes facilitate the formation of negative curvature structures. The probes are known to translocate from one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of the cis monolayer (facing the cells) or the trans monolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the open times of flickering fusion pores were shorter when probes were in trans monolayers and longer when in cis monolayers compared with times when probe was symmetrically distributed. Open times were the same for probe symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of the spontaneous curvature between the trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in trans leaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a "flat" structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/physiology , Lipid Bilayers/metabolism , Membrane Fusion/physiology , Orthomyxoviridae/physiology , Porins/physiology , 3T3 Cells/chemistry , 3T3 Cells/physiology , Animals , Carbocyanines/pharmacology , Fluorescent Dyes/pharmacology , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Kinetics , Membrane Fusion/drug effects , Membrane Proteins/physiology , Mice , Porins/chemistry , Porins/drug effects , Protein Conformation , Rhodamines/pharmacology
18.
Trends Microbiol ; 5(9): 349-55, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9294890

ABSTRACT

Diverse enveloped viruses enter host cells by fusing their envelopes with cell membranes. The mechanisms of merger of lipid bilayers of two membranes mediated by influenza hemagglutinin and other viral fusion proteins apparently involve local lipidic connections that evolve into a bilayer septum in which a pore forms and expands.


Subject(s)
Cell Membrane/metabolism , Cell Membrane/virology , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Cells, Cultured , Lipid Metabolism , Models, Biological , Porins/metabolism , Viral Fusion Proteins/metabolism , Viral Proteins/metabolism , Viral Proteins/physiology
19.
Virology ; 235(1): 118-28, 1997 Aug 18.
Article in English | MEDLINE | ID: mdl-9300043

ABSTRACT

The effect of the cytoplasmic tail of influenza hemagglutinin (HA) (H3 subtype) on fusion kinetics and pore growth was examined An SV40 recombinant virus was used to express wild-type (WT) HA and HA mutants containing changes in the HA cytoplasmic tail. HA and its mutants were expressed in CV-1 cells and the ability of these cells to fuse to either red blood cells (RBCs) or planar bilayer membranes was determined quantitatively. The percentage of cells expressing HA and the levels of expression were the same for WT HA or HA lacking its cytoplasmic tail (CT-), and for a mutant, MAY, in which the three HA C-terminal cysteine residues were replaced to block the addition of palmitate. When RBCs were colabeled with large and small aqueous dyes and fused to CV-1 cells expressing WT HA, transfer of the large dye was significantly slower and extent of transfer was lower than that of the small dye, indicating that pores did not expand quickly to large diameters. An absence of the HA cytoplasmic tail did not alter the time course of spread for either dye. When CV-1 cells expressing WT HA were fused to planar membranes, small pores tended to open and close repetitively ("flicker") before a pore would continue to either grow irreversibly to large conductances or grow to intermediate sizes and then contract. For HA mutants CT- and MAY, flickering was less likely to occur, but these pores did evolve in a manner identical to WT HA postflicker pores. We conclude that palmitate covalently linked to cysteine residues of the HA cytoplasmic tail is required for pore flickering, but that the tail does not play an important role in subsequent pore enlargement.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/physiology , Animals , Cell Fusion , Cell Line , Chlorocebus aethiops , Erythrocytes/immunology , Erythrocytes/physiology , Flow Cytometry , Hemagglutinin Glycoproteins, Influenza Virus/biosynthesis , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Humans , Influenza A virus/immunology , Influenza A virus/physiology , Kinetics , Lipid Bilayers , Membrane Fusion , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Simian virus 40/genetics , Transfection
20.
J Cell Biol ; 136(5): 995-1005, 1997 Mar 10.
Article in English | MEDLINE | ID: mdl-9060465

ABSTRACT

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this "stunted" fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.


Subject(s)
Erythrocyte Membrane/physiology , Glycosylphosphatidylinositols , Hemagglutinin Glycoproteins, Influenza Virus/physiology , Membrane Fusion , Animals , CHO Cells , Chlorpromazine/analogs & derivatives , Chlorpromazine/pharmacology , Cricetinae , Dibucaine/pharmacology , Fluorescent Dyes , Humans , Lipid Bilayers , Lysophosphatidylcholines , Orthomyxoviridae , Rhodamines
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