Role of hemagglutinin surface density in the initial stages of influenza virus fusion: lack of evidence for cooperativity

Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.     

Membrane fusion by single influenza hemagglutinin trimers. Kinetic evidence from image analysis of hemagglutinin-reconstituted vesicles

Influenza hemagglutinin, the receptor-binding and membrane fusion protein of the virus, is a prototypic model for studies of biological membrane fusion in general. To elucidate the minimum number of hemagglutinin trimers needed for fusion, the kinetics of fusion induced by reconstituted vesicles of hemagglutinin was studied by using single-vesicle image analysis. The surface density of hemagglutinin fusion-activity sites on the vesicles was varied, while keeping the surface density of receptor-binding activity sites constant, by co-reconstitution of the fusogenic form of hemagglutinin, HA(1,2), and the non-fusogenic form, HA(0), at various HA(1,2):(HA(1,2) + HA(0)) ratios. The rate of fusion between the hemagglutinin vesicles containing a fluorescent lipid probe, octadecylrhodamine B, and red blood cell ghost membranes was estimated from the time distribution of fusion events of single vesicles observed by fluorescence microscopy. The best fit of a log-log plot of fusion rate versus the surface density of HA(1,2) exhibited a slope of 0.85, strongly supporting the hypothesis that single hemagglutinin trimers are sufficient for fusion. When only HA(1,2) (without HA(0)) was reconstituted on vesicles, the dependence of fusion rate on the surface density of HA(1,2) was distinct from that for the HA(1,2)-HA(0) co-reconstitution. The latter result suggested interference with fusion activity by hemagglutinin-receptor binding, without having to assume a fusion mechanism involving multiple hemagglutinin trimers.     

Fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density

Influenza virus gains access to the cytoplasm of its host cell by means of a fusion event between viral and host cell membrane. Fusion is mediated by the envelope glycoprotein hemagglutinin (HA) and is triggered by low pH. To learn how many hemagglutinin trimers are necessary to cause membrane fusion, we have used two NIH 3T3 fibroblast cell lines that express HA protein at different surface densities. On the basis of quantitations of the number of HA trimers per cell and the relative surface areas of the two cell lines, the HAb-2 cells have a 1.9-fold higher plasma membrane surface density than the GP4F cells. The membrane lateral diffusion coefficient and the mobile fraction for HA is the same for both cell lines. A Scatchard analysis of the binding of glycophorin-bearing liposomes to the cells showed 1700 binding sites for the GP4F cells and 3750 binding sites for the HAb-2 cells, with effectively the same liposome-cell binding constant, about 7 x 10(10) M-1. Binding was specific for glycophorin on the liposomes and HA expressed on the cells. A competition experiment employing toxin-containing and empty liposomes allowed us to quantitate the number of liposomes that fused per cell, which was a small constant fraction of the number of bound liposomes. For the HAb-2 cells, about 1 in every 70 bound liposomes fused and for the GP4F cells about 1 in every 300 bound liposomes fused. Hence, the HAb-2 cells showed 4.4 times more fusion per bound liposome, even though the surface density of HA was only 1.9 times greater. We conclude the following: (i) One HA trimer is not sufficient to induce fusion. (ii) The HA bound to glycophorin is not the HA that induces fusion. That is, even though each HA has a binding and a fusion function, those functions are not performed by the same HA trimer.     

Measuring pKa of activation and pKi of inactivation for influenza hemagglutinin from kinetics of membrane fusion of virions and of HA expressing cells

The data for the pH dependence of lipid mixing between influenza virus (A/PR/8/34 strain) and fluorescently labeled liposomes containing gangliosides has been analyzed using a comprehensive mass action kinetic model for hemaglutinin (HA)-mediated fusion. Quantitative results obtained about the architecture of HA-mediated membrane fusion site from this analysis are in agreement with the previously reported results from analyses of data for HA-expressing cells fusing with various target membranes. Of the eight or more HAs forming a fusogenic aggregate, only two have to undergo the "essential" conformational change needed to initiate fusion. The mass action kinetic model has been extended to allow the analysis of the pKa for HA activation and pKi for HA inactivation. Inactivation and activation of HA following protonation were investigated for various experimental systems involving different strains of HA (A/PR/8/34, X:31, A/Japan). We find that the pKa for the final protonation site on each monomer of the trimer molecule is 5.6 to 5.7, irrespective of the strain. We also find that the pKi for the PR/8 strain is 4.8 to 4.9. The inactivation rate constants for HA, measured from experiments done with PR/8 virions fusing with liposomes and X:31 HA-expressing cells fusing with red blood cells, were both found to be of the order of 10(-4) s(-1). This number appears to be the minimal rate for HA's essential conformational change at low HA surface density. At high HA surface densities, we find evidence for cooperativity in the conformational change, as suggested by other studies.     

Delay time for influenza virus hemagglutinin-induced membrane fusion depends on hemagglutinin surface density.

We have studied the kinetics of low-pH-induced fusion between erythrocyte membranes and membranes containing influenza virus hemagglutinin by using assays based on the fluorescence dequenching of the lipophilic dye octadecylrhodamine. Stopped-flow mixing and fast data acquisition have been used to monitor the early stages of influenza virus fusion. We have compared this with the kinetics observed for fusion of an NIH 3T3 cell line, transformed with bovine papillomavirus, which constitutively expresses influenza virus hemagglutinin (GP4f cells). Virus and GP4f cells both display a pH-dependent time lag before the onset of fluorescence dequenching, but of an order of magnitude difference, ca. 2 s versus ca. 20 s. We have adopted two strategies to investigate whether the difference in lag time reflects the surface density of acid-activated hemagglutinin, able to undergo productive conformational change. (i) Hemagglutinin expressed on the cell surface requires proteolytic cleavage with trypsin from an inactive HAO form; we have limited the extent of proteolysis. (ii) We have used infection of CV-1 cells with a recombinant simian virus 40 bearing the influenza virus hemagglutinin gene. The surface expression of hemagglutinin is a function of time postinfection. For low-pH-induced fusion of both types of cell with erythrocytes, the lag time decreases with increasing hemagglutinin densities. Our results do not indicate a cooperative phenomenon at the level of the principal rate-determining step. We also show in the instance of virus fusion, that the magnitude of the delay time is a function of the target membrane transbilayer lipid distribution. We conclude that for a given amount of pH-activated hemagglutinin per unit area of membrane, the kinetics of fusion is determined by nonspecific physical properties of the membranes involved.     

How many trimers? Modeling influenza virus fusion yields a minimum aggregate size of six trimers, three of which are fusogenic

Conflicting reports in leading journals have indicated the minimum number of influenza hemagglutinin (HA) trimers required for fusion to be between one and eight. Interestingly, the data in these reports are either almost identical, or can be transformed to be directly comparable. Different statistical or phenomenological models, however, were used to analyze these data, resulting in the varied interpretations. In an attempt to resolve this contradiction, we use PABM, a brane calculus we recently introduced, enabling an algorithmic systems biology approach that allows the problem to be modeled in a manner following a biological logic. Since a scalable PABM executor is still under development, we sufficiently simplified the fusion model and analyzed it using the model checker, PRISM. We validated the model against older HA-expressing cell-to-cell fusion data using the same parameters with the exception of three, namely HA and sialic acid (SA) surface densities and the aggregation rate, which were expected to be different as a result of the difference in the experimental setup. Results are consistent with the interpretation that a minimum aggregate size of six HA trimers, of which three undergo a conformational change to become fusogenic, is required for fusion. Of these three, two are free, while one is bound. Finally, we determined the effects of varying the SA surface density and showed that only a limited range of densities permit fusion. Our results demonstrate the potential of modeling in providing more precise interpretations of data.     

Synchronized activation and refolding of influenza hemagglutinin in multimeric fusion machines.

At the time of fusion, membranes are packed with fusogenic proteins. Do adjacent individual proteins interact with each other in the plane of the membrane? Or does each of these proteins serve as an independent fusion machine? Here we report that the low pH-triggered transition between the initial and final conformations of a prototype fusogenic protein, influenza hemagglutinin (HA), involves a preserved interaction between individual HAs. Although the HAs of subtypes H3 and H2 show notably different degrees of activation, for both, the percentage of low pH-activated HA increased with higher surface density of HA, indicating positive cooperativity. We propose that a concerted activation of HAs, together with the resultant synchronized release of their conformational energy, is an example of a general strategy of coordination in biological design, crucial for the functioning of multiprotein fusion machines.     

Stochastic Fusion Simulations and Experiments Suggest Passive and Active Roles of Hemagglutinin during Membrane Fusion

Influenza enters the host cell cytoplasm by fusing the viral and host membrane together. Fusion is mediated by hemagglutinin (HA) trimers that undergo conformational change when acidified in the endosome. It is currently debated how many HA trimers, w, and how many conformationally changed HA trimers, q, are minimally required for fusion. Conclusions vary because there are three common approaches for determining w and q from fusion data. One approach correlates the fusion rate with the fraction of fusogenic HA trimers and leads to the conclusion that one HA trimer is required for fusion. A second approach correlates the fusion rate with the total concentration of fusogenic HA trimers and indicates that more than one HA trimer is required. A third approach applies statistical models to fusion rate data obtained at a single HA density to establish w or q and suggests that more than one HA trimer is required. In this work, all three approaches are investigated through stochastic fusion simulations and experiments to elucidate the roles of HA and its ability to bend the target membrane during fusion. We find that the apparent discrepancies among the results from the various approaches may be resolved if nonfusogenic HA participates in fusion through interactions with a fusogenic HA. Our results, based on H3 and H1 serotypes, suggest that three adjacent HA trimers and one conformationally changed HA trimer are minimally required to induce membrane fusion (w = 3 and q = 1).     

Stochastic Fusion Simulations and Experiments Suggest Passive and Active Roles of Hemagglutinin during Membrane Fusion

Influenza enters the host cell cytoplasm by fusing the viral and host membrane together. Fusion is mediated by hemagglutinin (HA) trimers that undergo conformational change when acidified in the endosome. It is currently debated how many HA trimers, w, and how many conformationally changed HA trimers, q, are minimally required for fusion. Conclusions vary because there are three common approaches for determining w and q from fusion data. One approach correlates the fusion rate with the fraction of fusogenic HA trimers and leads to the conclusion that one HA trimer is required for fusion. A second approach correlates the fusion rate with the total concentration of fusogenic HA trimers and indicates that more than one HA trimer is required. A third approach applies statistical models to fusion rate data obtained at a single HA density to establish w or q and suggests that more than one HA trimer is required. In this work, all three approaches are investigated through stochastic fusion simulations and experiments to elucidate the roles of HA and its ability to bend the target membrane during fusion. We find that the apparent discrepancies among the results from the various approaches may be resolved if nonfusogenic HA participates in fusion through interactions with a fusogenic HA. Our results, based on H3 and H1 serotypes, suggest that three adjacent HA trimers and one conformationally changed HA trimer are minimally required to induce membrane fusion (w = 3 and q = 1).     

Autocatalytic activation of influenza hemagglutinin

Enveloped viruses contain surface proteins that mediate fusion between the viral and target cell membranes following an activating stimulus. Acidic pH induces the influenza virus fusion protein hemagglutinin (HA) via irreversible refolding of a trimeric conformational state leading to exposure of hydrophobic fusion peptides on each trimer subunit. Herein, we show that cells expressing fowl plague virus HA demonstrate discrete switching behavior with respect to the HA conformational change. Partially activated states do not exist at the scale of the cell, activation of HA leads to aggregation of cell surface trimers, and newly synthesized HA refold spontaneously in the presence of previously activated HA. These observations imply a feedback mechanism involving self-catalyzed refolding of HA and thus suggest a mechanism similar to the autocatalytic refolding and aggregation of prions.     

Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. Measurement by dequenching of fluorescence

Fusion between membranes of 3T3 fibroblasts expressing hemagglutinin (HA) from the Japan strain of influenza virus and human red blood cells (RBC) was measured using an assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecylrhodamine (R18). The probe was incorporated into the membrane of intact RBC at self-quenching concentrations, and the RBCs were bound to the 3T3 cells. Fusion, which allowed movement of R18 into 3T3 cell membranes, was monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, the fluorescence increased after a delay of about 30 s at 37 degrees C, and leveled off within 2 min. In control experiments where R18 RBCs bound to 3T3 cells expressing the uncleaved precursor hemagglutinin (HA0) were incubated at 37 degrees C and low pH, no fluorescence increase was observed. This indicated that the R18 dequenching occurred as a result of HA-induced fusion of plasma membranes. Fusion showed a very steep pH dependence with a threshold at pH 5.4 and a maximum at pH 5.0, similar to HA-induced fusion seen previously using cell biological techniques. The fusion rate increased and the delay for the onset of fusion decreased as the temperature was raised above 20 degrees C. Low pH activation of the fusion process at 37 degrees C could be partially arrested by raising the pH after 2-10 s, but not after 15 s, indicating that the irreversible pH-activated conformational change of HA necessary for fusion was complete within about 15 s. Analysis of the data indicates that the pH-induced membrane fusion activity of HA is a highly cooperative event.     

The final conformation of the complete ectodomain of the HA2 subunit of influenza hemagglutinin can by itself drive low pH-dependent fusion

One of the best characterized fusion proteins, the influenza virus hemagglutinin (HA), mediates fusion between the viral envelope and the endosomal membrane during viral entry into the cell. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding toward the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? Here we explored structural properties as well as the fusogenic activity of the full sized trimeric HA2(1–185) (here called HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes in a low pH-dependent manner. Two mutations known to inhibit HA-mediated fusion strongly inhibited the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* formed small fusion pores but did not expand them. Our results confirm that the HA1 subunit responsible for receptor binding as well as the transmembrane and cytosolic domains of HA2 is not required for fusion pore opening and substantiate the hypothesis that the final form of HA2 is more important for fusion than the conformational change that generates this form.     

Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity

We tested the role of the “spring-loaded” conformational change in the fusion mechanism of the influenza hemagglutinin (HA) by assessing the effects of 10 point mutants in the region of high coiled-coil propensity, HA2 54–81. The mutants included proline substitutions at HA2 55, 71, and 80, as well as a double proline substitution at residues 55 and 71. Mutants were expressed in COS or 293T cells and assayed for cell surface expression and structural features as well as for their ability to change conformation and induce fusion at low pH. We found the following: Specific mutations affected the precise carbohydrate structure and folding of the HA trimer. All of the mutants, however, formed trimers that could be expressed at the cell surface in a form that could be proteolytically cleaved from the precursor, HA0, to the fusion-permissive form, HA1-S-S-HA2. All mutants reacted with an antibody against the major antigenic site and bound red blood cells. Seven out of ten mutants displayed a wild-type (wt) or moderately elevated pH dependence for the conformational change. V55P displayed a substantial reduction (~60– 80%) in the initial rate of lipid mixing. The other single mutants displayed efficient fusion with the same pH dependence as wt-HA. The double proline mutant V55P/ S71P displayed no fusion activity despite being well expressed at the cell surface as a proteolytically cleaved trimer that could bind red blood cells and change conformation at low pH. The impairment in fusion for both V55P and V55P/S71P was at the level of outer leaflet lipid mixing. We interpret our results in support of the hypothesis that the spring-loaded conformational change is required for fusion. An alternate model is discussed.     

Fusion Activity of Transmembrane and Cytoplasmic Domain Chimeras of the Influenza Virus Glycoprotein Hemagglutinin

The role of the sequence of transmembrane and cytoplasmic/intraviral domains of influenza virus hemagglutinin (HA, subtype H7) for HA-mediated membrane fusion was explored. To analyze the influence of the two domains on the fusogenic properties of HA, we designed HA-chimeras in which the cytoplasmic tail and/or transmembrane domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein F of Sendai virus. These chimeras, as well as constructs of HA in which the cytoplasmic tail was replaced by peptides of human neurofibromin type1 (NF1) or c-Raf-1, NF78 (residues 1441 to 1518), and Raf81 (residues 51 to 131), respectively, were expressed in CV-1 cells by using the vaccinia virus-T7 polymerase transient-expression system. Wild-type and chimeric HA were cleaved properly into two subunits and expressed as trimers. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins was studied by a lipid-mixing assay with the lipid-like fluorophore octadecyl rhodamine B chloride (R18). No profound differences in either extent or kinetics could be observed. After the pH was lowered, the above proteins also induced a flow of the aqueous fluorophore calcein from preloaded RBCs into the cytoplasm of the protein-expressing CV-1 cells, indicating that membrane fusion involves both leaflets of the lipid bilayers and leads to formation of an aqueous fusion pore. We conclude that neither HA-specific sequences in the transmembrane and cytoplasmic domains nor their length is crucial for HA-induced membrane fusion activity.     

Analysis of delay times of hemagglutinin-mediated fusion between influenza virus and cell membranes

We have studied the kinetics of low pH-induced fusion between influenza virus A/PR 8/34 and human erythrocyte membranes in suspension by using an assay based on fluorescence dequenching (FDQ) of the lipophilic dye octadecylrhodamine B chloride (R 18). As shown previously (Clague et al. 1991) the onset of FDQ is preceded by a characteristic lag time (t _lag) following pH reduction. Whereas t _lag represents only a subpopulation of fusing viruses with the shortest delay time we suggest here that a representative mean lag time µ_1ag of virus-cell fusion can be deduced from the R 18-assay. Kinetics of FDQ reflects the cumulative distribution function of lag times _lag of single fusion events with the mean value µ_lag. We show that t _lag obtained from the onset of FDQ does not always reflect the fusion behaviour of the whole population of fusing viruses. While both lag times, t _lag and µ_lag exhibit a similar temperature dependence we found a significantly different dependence of both delay times on virus inactivation by low pH-pretreatment. We conclude that the mean lag time µ_lag appears to be a more appropriate parameter describing the kinetics of virus-cell fusion. The analysis of delay times offers a new approach to test the validity of different kinetic models of HA-mediated fusion and to gain valuable information about HA-mediated fusion. The analysis confirms that the inactivation process proceeds via steps of the formation of the fusion pore. Although the increase of lag times can be explained by a depletion of fusion competent HA's, our data suggest that intermediate structures of HA along the inactivation pathway can still transform into a fusion site.Abbreviations FDQ fluorescence dequenching - HA hemagglutinin - PBS phosphate buffered saline - R18 octadecylrhodamine B chloride - t _lag lag time - µ_lag mean lag time - _lag individual delay time Correspondence to: A. Herrmann     

Kinetically differentiating influenza hemagglutinin fusion and hemifusion machines

Membrane fusion mediated by influenza virus hemagglutinin (HA) yields different phenotypes depending on the surface density of activated HAs. A key question is whether different phenotypes arise from different fusion machines or whether different numbers of identical fusion machines yield different probabilistic outcomes. If fusion were simply a less probable event than hemifusion, requiring a larger number of identical fusion machines to occur first, then two predictions can be made. First, fusion should have a shorter average delay time than hemifusion, since there are more machines. Second, fusion should have a longer execution time of lipid mixing after it begins than hemifusion, since the full event cannot be faster than the partial event. Using a new automated video microscopy technique, we simultaneously monitored many HA-expressing cells fusing with erythrocytes and identified individual cell pairs with either full or only partial redistribution of fluorescent lipids. The full lipid mixing phenotype also showed contents mixing, i.e., fusion. Kinetic screening of the digitized fluorescence data showed that the execution of lipid mixing after the onset is faster for fusion than hemifusion. We found no correlation between the delay times before the onset of lipid mixing and the final fusion phenotype. We also found that the execution time for fusion was faster than that for hemifusion. Thus, we provide the first experimental evidence for fusion and hemifusion arising from different machines.     

The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)- expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.     

Membrane fusion of Semliki Forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein.

This paper presents a kinetic analysis of low-pH-induced fusion of Semliki Forest virus (SFV) with cholesterol-containing unilamellar lipid vesicles (liposomes), consisting otherwise of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. Fusion is monitored continuously with a lipid mixing assay, involving virus bio-synthetically labeled with the fluorophore pyrene. At pH 5.55, 37 degrees C, SFV-liposome fusion occurs on the time scale of seconds. Extensive fusion (up to 60% of the virus) requires an excess of liposomes, while a low-pH preincubation of the virus alone results in inactivation of its fusion capacity. The onset of fusion after acidification of virus-liposome mixtures is preceded by a pH- and temperature-dependent lag phase. Early in this lag phase, a conformational change in the E2E1 spike glycoprotein occurs, involving formation of a trypsin-resistant E1 homotrimer, exposing a conformation-specific epitope (E1"). These changes are followed by a rapid, cholesterol-dependent binding of the virus to the liposomes (as assessed by sucrose density gradient analysis), subsequent fusion starting only after an additional delay. This sequence of events strongly suggests that the E1 homotrimeric structure represents the fusion-active conformation of the SFV spike, the actual fusion complex possibly involving a higher order oligomer of E1 trimers.     

Gating kinetics of pH-activated membrane fusion of vesicular stomatitis virus with cells: stopped-flow measurements by dequenching of octadecylrhodamine fluorescence

To identify the initial stages of membrane fusion induced by vesicular stomatitis virus, we performed stopped-flow kinetic measurement with fluorescently labeled virus attached to human erythrocyte ghosts that contained symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically using an assay based on mixing of the lipid fluorophore octadecylrhodamine. At 37 degrees C and pH values near the threshold for fusion, a lag phase of 2 s was observed. The lag time decreased steeply as the pH decreased, while the initial rate of fusion showed the reverse functional dependence on pH. The observed rapid fluorescence changes resulted from fusion of virus bound to the target, and the time lags were not due to association-dissociation reactions between virus and target. For a given pH value, the temperature dependence of the lag time was similar to that of the initial rate of fusion. The results were fitted to a multistate model similar to that resulting from ion channel gating kinetics. The model allows testing of hypotheses concerning the role of cooperativity and conformational changes in viral spike glycoprotein-mediated membrane fusion.     

Membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion

We have investigated the mechanism of cell fusion mediated by HA, the fusogenic hemagglutinin of the Influenza viral envelope. Single erythrocytes (RBCs) were attached to fibroblasts expressing the HA on their cell surface, and fusion of the paired cells was triggered by rapid acidification. The RBC membrane was stained with fluorescent lipid, and the fusion-induced escape of lipid into the fibroblast was observed by quantitative image analysis. At the same time, the formation of an aqueous connection (i.e., the fusion pore) between the two cells was monitored electrically. Within minutes after acidification, an electrical conductance between the two cells appeared abruptly as the fusion pore opened, and then increased gradually as the pore dilated. Later, fluorescent lipid diffused into the fibroblast, approaching equilibrium over the next 5-20 min. No lipid flux was seen while the pore conductance remained 0.5 nS or less. Evidently lipid flux requires a threshold pore size. Our finding suggests that the smallest and earliest fusion pores are surrounded by a ring of protein. A fusion pore expands by breaking this ring and recruiting lipid into its circumference.