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Intermediates in influenza induced membrane fusion.

Intermediates in influenza induced membrane fusion. The EMBO Journal vol.9 no. 1 3 pp.4231 - 4241, 1 990 Intermediates in influenza induced membrane fusion Toon Stegmann, takes in endosomes Judith M.Whitel and place (see Doms et al., 1989), where it is a low triggered by pH dependent conformational change Ari Helenius in HA et (Skehel al., 1982; Doms and Helenius, 1986; White Department of Cell Biology, Yale University School of and Medicine, Wilson, 1987). As a result of this conformational change 333 Cedar Street, New CT and Haven, 06510, 'Department of the so-called 'fusion peptides', or the hydrophobic N-termini Pharmacology, and the Cell Biology of Program, University of the HA2 polypeptides, are exposed. In the X-ray struc- California, San Francisco, CA USA 94143-0450, ture of the neutral pH form, these are located peptides in Communicated by P.De Camilli the subunit interface at a distance of 35 A from the viral membrane (Wilson et al., 1981). Once exposed, the fusion Our results show that the peptides of bromelain solubilized ectodomains of mechanism by which influenza HA have virus fuses with target membranes been shown to insert into the outer leaflet of a involves sequential bilayer, adop- complex changes in the ting an a-helical configuration (Harter et hemagglutinin (HA, the viral al., 1989; Brunner, fusion protein) and in the contact site 1989). Biochemical and morphological studies between virus and as well as target membrane. To render analysis of fusion mutants have individual steps amenable revealed extensive additional to study, we worked at 0°C which decreased the rate of low pH induced changes in HA structure, including the fusion and increased the efficiency. The mechanism of dissociation of the domains top of HAl (reviewed in Doms fusion at 0°C and 37°C was similar. The process began et al., 1989; Wiley and Skehel, 1987). On the basis of these with a conformational change in HA which exposed the observations, we have proposed a model for fusion in which fusion peptides but did not lead to dissociation of the tops the exposure of the fusion peptides and the opening of the of the ectodomain of the trimer. The change in the protein top domain are crucial features (see Doms and Helenius, led to immediate hydrophobic attachment of the virus 1987; Doms et al., 1989; Stegmann et al., 1989a). to the target liposomes. Attachment was followed by a In this study we the fusion analyzed reaction between lag period (4-8 min at 0°C, 0.6-2 s at 37°C) during influenza virus and liposomes (or erythrocyte ghosts) at which rearrangements occurred in the site of membrane reduced temperatures. We found that the reaction was contact between the virus and liposome. After a further dramatically slowed down at 0WC, and several kinetically series of changes the final bilayer merger took place. This and biochemically distinct steps could be detected. Most final fusion event was not pH dependent. At effi- importantly, we observed that there is a 4-8 0°C min long, low cient fusion occurred without dissociation of the pH dependent lag before top the onset of fusion during which domains of the HA trimer, suggesting that a transient a liposome- virus complex undergoes a series of complex conformation of HA is responsible for fusion at changes. Further low occur pH dependent changes after the physiological temperatures. The observations lead to a lag period, but the fusion event i.e. the itself, merger of revised model for HA mediated fusion. membranes, proved to be pH Our independent. analysis of Key words: hemagglutinin/influenza/membrane fusion the conformational in HA indicated changes that most of the observed changes previously at higher temperatures, including the opening of the top of the trimer, are not only unnecessary but in fact inhibitory to fusion. The results Introduction provide the basis for a re-evaluation of our model for HA Membrane fusion events are involved in a multitude of vital mediated membrane fusion. cellular and physiological functions. Although some progress has been made in identifying proteins which are required Results for intracellular vesicular transport (Goud et al., 1988; Malhotra et al., 1989), the only proteins demonstrated to The temperature dependence of fusion play a direct role in membrane fusion are the envelope The fusion assay we used takes of the advantage resonance proteins of animal viruses (for recent reviews see Ohki et al., transfer between two fluorescent energy lipids (N-NBD-PE 1987; Marsh and Helenius, 1989; Stegmann et al., 1989a; and N-Rh-PE) when are both in the they present target Wilschut and Hoekstra, 1990; Sowers, 1988; White, 1990). liposomes (Struck et al., Fusion of the 1981). liposome with These proteins mediate fusion between the viral membrane a virion causes dilution of the fluorescent lipids, resulting and host cell membranes and thus allow the viral genome in decreased transfer and an increase in energy fluorescence to enter the cytosol. emission from the N-NBD-PE energy donor, (Stegmann Influenza HA is the best characterized of the viral fusion et al., The membranes used were 1986). target large factors. It is a homotrimeric integral membrane protein unilamellar extrusion et liposomes prepared by (Mayer al., composed of three 84 kd With subunits; each subunit in turn is 1986). a diameter of 0.1-0.2 were in ,um, they the composed of two disulfide bonded same polypeptides, HAl and size range as the virus were particles. They composed HA2. The trimer forms a spike 135 A from of natural zwitterionic extending the and with added phospholipids (PC PE) viral membrane (Wilson et al., 1981). HA mediated While not for fusion fusion gangliosides. required et (White al., (©) Oxford Press 4231 University T.Stegmann, J.M.White and A.Helenius 1982; Stegmann et al., 1989b), the gangliosides serve as it varied within a range of 4-8 min between virus prepara- receptors for the influenza virus HA so that virus - liposome tions. The final increase in fluorescence was 12% at 37°C complexes can form prior to fusion. To allow the forma- values corresponded and 20% at 0°C (Figure 1A). These tion of virus-liposome complexes, purified virus was of and 40%, respectively, to final fusion efficiencies 24% incubated with liposomes at 0°C, at neutral pH and at a when calculated and corrected as outlined in Materials and of virus particle per liposome. Aliquots methods. The lower overall efficiency of fusion at 37°C was stoichiometry one a thermostatted probably due to the rapid, acid induced loss of viral fusion of the mixture were then injected into buffers at different et al., 1982; Sato fluorometer cuvette containing pH values. activity which is observed at 37°C (White observed A low pH dependent increase in fluorescence was et al., 1983; Stegmann et al., 1987; Junankar and Cherry, both at 37°C and at 0°C (Figure IA). To confirm that the 1986). Since such inactivation does not occur at 0°C increase in fluorescence was caused by fusion and not by (Stegmann et al., 1987), virus remains fusion active for a other modes of lipid transfer between the membranes, virus longer period of time at 0°C and consequently more virus alone was incubated at pH 5.0, 37°C and then added to is able to fuse. To obtain a more complete picture of the temperature at 5.1. Virus treated in this way remains liposomes pH dependence of fusion, we determined the initial rates of of to ganglioside-containing liposomes but capable binding fluorescence increase and the lag times at different is abolished et the fusion activity completely (Stegmann al., temperatures in the 0-37°C range. The rates of fluorescence 1987). The lack of fluorescence increase at pH 7.4 and the lack of fluorescence increase in this control (Figure IA) increase were plotted in Arrhenius plots (Figure 2A), and confirmed that the fluorescence increase at 37°C and 0°C it was found that the plot of the initial rates was linear was to virus fusion. (squared regression coefficient 0.98). The linearity indicated due -liposome that the activation energy of the rate-limiting step in fusion fusion was taking place at 0°C, That virus-liposome was the same over the entire temperature range and thus stain elec- could also be demonstrated direcfly by negative of At neutral of suggested that the mechanism fusion at these temperatures tron microscopy (Figure 1B). pH, complexes seen Virus was the same. The lag times could only be measured with virus and liposomes were (Figure panel a). lB, in Lag time was found to were from because confidence the 0-25°C range. particles readily distinguished liposomes decrease with more electron-dense After steeply increasing temperature (Figure 2B). of the spikes and their appearance. 0°C for 90 min, fusion products were incubation at pH 5.1, with were than the 'Cold fusion' erythrocyte ghosts observed (Figure iB, panel b). They larger Before analyzing the fusion event at 0°C in further detail, or were on their original virus liposomes. Spikes present it was important to determine whether fusion with natural in surface (arrows Figure 1B). membranes could also occur at reduced temperatures. To fusion started after acidification and At 37°C, immediately within 1-2 min At there do so, we investigated the fusion of influenza virus with was completed (Figure lA). 0°C, of - 7 min which no detectable fusion erythrocyte ghosts. The fusion assay used in these was a period during fusion at a rate 40 times lower than experiments employed the fluorescent fatty acid occurred, followed by of the was octadecylrhodamine (R18), which was incorporated into the at 37°C. The duration lag highly reproducible of but viral membrane at a self-quenching concentration (Hoekstra between aliquots from the same preparation virus, ..s..... " .s.X. % s, a ....... strain X-3 with Virus and were in a small volume for 15 min at neutral 1. Fusion of influenza prebound pH Fig. virus, 1, liposomes. (A) liposomes into a cuvette at In virus was at at The mixture was then 5.1, 37°C; (c) pH 7.4, 0°C. (d), pretreated pH 5, 0°C. injected (a) pH 5.1, 0°C; (b) pH bound to at for 15 and the mixture was into a cuvette at to virus for 5 min injected pH 5.1, 0°C. Liposome 37°C min, neutralized, liposomes 0°C 1:1 of membrane consisted of PC: PE: bovine brain 6:3:1 and ratio was (5 phospholipid phosphorous each). Liposomes egg egg gangliosides liM Fluorescence was measured as described in Materials and methods. Direct trace amounts of N-NBD-PE and N-Rh-PE mol% (0.6 each). (B) electron mixtures were either at or visualization of fusion at stain microscopy. Virus-liposome kept pH 7.4, 0°C (panel a) 0°C by negative after for 15 at neutral Arrows indicate on fusion Bar at for 90 min min product. incubated pH 5.1, 0°C (panel b) aggregation pH, 0°C. spikes to 100 nm. corresponds 4232 fusion intermediates Influenza et al., 1984; Stegmann et al., 1986). The labeled virus were used as membranes. was erythrocyte ghosts target Further- allowed to bind to the ghosts on ice at neutral pH, and more, the results did not depend on the particular fusion aliquots of the mixture were used. injected into cuvettes at the assay However, the slope of the line in the Arrhenius for appropriate pH and temperatures. plot virus -liposome fusion was different from that We found that fusion occurred efficiently throughout the measured with ghosts, indicating that the detailed interac- 0-37°C range. The Arrhenius plot of the rates of tions of virus with ghosts or liposomes might differ. fluorescence increase is shown in Figure 2A. Again, the results indicate that of The the mechanism fusion is similar at lag phase 0°C and the fusion rates 37°C. Overall, were higher than The existence of a lag before the onset of fusion suggested with liposomes, but the lag times were similar. Since the that preparatory processes are needed before the actual fluorescence increase was faster than with liposomes, lag merger of the two membranes takes place. To determine the times could be measured with precision even at elevated molecular basis for the lag, we analyzed the properties of temperatures. A lag of 0.6 s could be observed at virus -liposome mixtures at 0°C using a 37°C. variety of By extrapolation, a similar lag (0.6-2 s) could be calculated approaches. for fusion with liposomes at Taken the Binding 37°C. together, studies using a centrifugation assay (Stegmann results indicated that the mechanism of influenza virus fusion et al., 1987) indicated that attachment of virus to the with target membranes is similar at 0 and 37°C. The liposomes could only account for a small fraction of the lag temperature dependence was similar whether liposomes or (Figure 3); one third of the virus was already bound to the liposomes prior to acidification, and the rest was bound within the first few seconds at pH 5.1 (half time < 15 s). The of length the lag was, moreover, independent of the total virus 2 and liposome concentration over a 10-fold concentra- tion range at a constant 1:1 virus to liposome ratio. This confirmed that the rate limiting events in the lag were not determined mass by action kinetic processes such as binding of the virus to liposomes. Rather, they involved processes subsequent to the binding, i.e. processes within individual virus - liposome complexes. As expected, the rate of fluorescence increase after the 0- CD lag depended on the ratio of virus to liposomes (not shown). (D The lag time was, however, independent of virus - liposome 100 - 3.2 3.3 3.4 3.5 3.6 3.7 1 OOO/K 80 - 500 _V 40 - CO 20 - I-- co 0O 4 8 12 16 time (min) -j Fig. 3. Binding of ganglioside-containing liposomes to virus. Virus was incubated with liposomes (ratio, concentration and composition as in the legend to Figure 1) at pH 5.1, 0°C for various amounts of time and neutralized. The virus and virus-liposome complexes were then pelleted by centrifugation. Binding was determined as the amount of liposomes in the pellet relative to the total amount of liposomes, by Temperature (OC) in measuring fluorescence pellet and supernatant. Fig. 2. Temperature dependence of the initial rates of fluorescence Table I. Duration of the fusion lag phase preceding virus-liposome at increase and the duration of the lag phase. (A) Arrhenius plots of the OOC fusion of influenza virus with fluorescently labeled liposomes (open squares; at different temperatures but otherwise as in the legend to ratio Virus:liposome Lag time (min) Figure 1) or erythrocyte ghosts (closed squares; as described below). The log of the initial rate of fluorescence increase (the slope of the 1:2 7.0 of steepest part the fusion curve, after the lag phase) was plotted 1:1 6.9 the of against reciprocal absolute temperature. Results shown are the 2:1 6.7 averages of duplicates; the same preparation of virus was used in all 4:1 Virus concentration 10 7.0 experiments. yg/ml; ghost concentration 100 lOg/ml. Fluorescence was measured as described in Materials and methods. Data were fitted by linear regression, with squared regression Concentration of was while the amount of liposomes kept constant, coefficients of 0.98 for virus-liposome fusion and 0.99 for virus was varied. ratio based on membrane Liposome:virus virus-ghost fusion. (B) Lag times of virus-liposome (open squares) at 5 Results shown are for phospholipid phosphorous; liposomes AM. or fusion virus-ghost (closed squares) as described above, at different one particular of variation between within preparation virus; replicates temperatures. one was -0.4 min. preparation 4233 T.Stegmann, J.M.White and A.Helenius ratio at least in the 4:1 -1:2 range (Table I). This confirmed proceeded after the lag phase was affected by steps which that the and uniform for low This is evident in Figure 4, which duration of the lag was constant did not require pH. - every complex fusion in that had neutraliza- all virus liposome complexes. Apparently, shows that samples undergone the was 1.5-2 times faster upon re- had to undergo similar changes during the lag phase, and tion during lag than was fusion in controls which had not been these changes required a minimum time of 4-8 min. acidification to the same extent Two additional observations confirmed that the lag phase neutralized. Fusion was accelerated place early or late in the lag was determined by events taking place in virus-liposome whether the interruptions took complexes and not in the virus alone. First, preincubation period. phase of virus alone at pH 5.1 on ice for up to 10 min prior to Taken together, these results indicated that the lag mixing with liposomes did not change the duration of the encompasses low pH dependent reactions in individual started only upon addi- liposome -virus complexes. The reactions were continuous subsequent lag. Therefore, the lag of time with the and additive. They were faster when the liposomes contained tion liposomes. Secondly, the lag varied They probably represented a series of composition of the liposomes. With phosphatidylcholine/ gangliosides. of molecular at the sites of virus-liposome phosphatidylethanolamine (PC/PE) liposomes devoid rearrangements of and constituted crucial preparatory events in fusion. gangliosides, the lag was 30 min instead 4-8 min (not contact, shown). We found that this difference was not due to slower for pH dependence of fusion at 0°C binding (see below). Apparently, the effect was specific in To determine whether the lag was the only pH dependent gangliosides, as inclusion of cholesterol liposomal PE or PE and step in fusion several experiments were performed. We first membranes containing either PC and PC, on of the determined the overall pH dependence of virus -liposome gangliosides had no additional effect the duration was fusion at 0°C, the rate of fusion at different pH values lag or on the rate of fusion. The size of the liposomes (Figure SA) and the duration of the lag (Figure SB). The not critical; the same lag time was measured using vesicles for fusion was found to be 5.5 and with diameters in the 0.1-0.8 range. apparent pH threshold j.m 5.1. The threshold for fusion was indicated that the events which deter- the pH optimum was pH Further experiments mined the duration of the lag required low pH and were additive. When mixtures at 0°C were virus-liposome 1.0o neutralized at time the lag, no fusion occurred any during even after 90 min. re-acidification fusion began but C Upon 0.8 0- further The total i.e. only after a lag (Figure 4). lag time, ._ of the times before and after the sum lag neutralization, the observed in controls that had not 0.6 always equalled lag neutralized. The combined lag time did not depend on been 0 ._ the amount of time the samples were incubated at neutral 0.4 pH after neutralization, nor did it matter whether the inter- ruption occurred early or late in the lag phase (Figure 4). 0.2 These results indicated that the low pH dependent reactions which determine the length of the lag phase (and which were 0.0 for fusion) progressed steadily and additively necessary the during lag phase. pH While the duration of the was determined by lag phase low the rate with which fusion pH dependent steps, 30 - 15 - 20 - a >--fi- 10 - 0) preincubation time at pH 5. 1, O-C (min) < 1-d -0 CD 10 - a) CIO L- +- 0 4 16 4.5 8 12 20 24 5.5 6.5 time (min) pH of with after a of Fig. 5. pH dependence of fusion and lag time at 0°C. (A) Rate of Fig. 4. Fusion virus liposomes preincubation at low Virus and fluorescence increase. Duration of the lag phase. Open squares: virus-liposome complexes pH. liposomes (ratio, (B) and final concentration as in were incubated for fusion of virus and liposomes at different pH values at 0°C. Open composition Figure 1) for 15 in a concentrated at acidified to pH circles: virus and liposomes were allowed to fuse at pH 5.1, 0°C min suspension pH 7.4, 0°C, down in and reneutralized after (a) 6 min; (b) 25 min, neutralized and re-acidified to the pH indicated at 0°C. Closed 5.1, 0°C (arrow inset) in not virus and were acidified to 5.1 for 2 4 min; (c) 2 min (arrow up in inset) The sample (d) was diamonds: liposomes pH min then transferred the duration of the for this of virus), acidified and served as a control. The mixtures were (half lag phase preparation the moment the neutralized and then re-acidified to the indicated at Other to a cuvette at a final pH of 5.1, 0°C. Tracings began pH 0°C. arrow conditions as in 1. mixtures were re-acidified (large down). Figure 4234 Influenza fusion intermediates had therefore 0.1-0.2 pH units lower at 0°C than at 37°C which did not fuse after neutralization undergone increased fuse at a Virus (Doms et al., 1986). The length of the lag phase changes which allowed it to higher pH. in was dramatically with increasing pH. While it was 4-8 min at neutralized during the fusion phase as Figure 6A, pH 5. 1, it was 30 min at pH 5.3, increasing monotonously incubated at different pH values between 5.1 and neutral with pH (Figure SB). (Figure 6B). The lower the pH after re-acidification was Next, we assayed the pH dependence during different more virus was able to fuse. The rates of made, the fusion, of the Virus - were acidified measured from the steepest part of the curves after re- stages lag. liposome complexes 5.1 at for 2 to half the dura- acidification shown in 6B) are plotted in Figure to pH 0°C min (corresponding (as Figure tion of the lag phase for the virus preparation used), and SA. The for fusion was found to be broadened pH optimum then neutralized. Aliquots were then re-acidified to different occurred at values as so that considerable fusion pH high In with our pH values, and fusion was recorded. agreement as pH 6. previous results (Figure 4), we found that fusion took place Several conclusions can be drawn from these results, (i) after a further lag (Figure SB) and at a faster rate (Figure While the events leading to fusion are low pH dependent, SA) than without prior acidification. It was found, however, the final membrane merger is not. (ii) The step which and hence that the pH threshold for fusion had shifted from pH 5.5 requires the highest concentration of protons, to pH 6. This showed that while the lag was low pH depen- determines the overall pH dependence of fusion, occurs early dent throughout, the overall pH dependence of the fusion in the lag phase. This step is most likely the initial low pH reaction was determined by early events in the lag. induced conformational change in HA. (iii) All fusion These results suggested that the fusion reaction itself might competent virus-liposome complexes undergo a series of be pH independent. To obtain an estimate for the pH low during the lag phase, and the pH dependent changes dependence of the final fusion reaction, we neutralized is constant. duration of these changes (iv) Thereafter, they samples which had proceeded beyond the lag phase at pH a further series of at a undergo changes, occurring highly variable rate, including some which are not low pH 5.1 and 0°C and were actively fusing (Figure 6A). We found in contrast to our results at that, previous 37°C where fusion dependent. stopped immediately upon neutralization (Stegmann et al., 1986), fusion at 0°C continued for 30 min. The rate was - Hydrophobic virus liposome attachment 1/5 to 1/2 of the the in original rate. Upon re-acidification, To analyze what was happening the contact zone between remaining virus fused at a rate faster than the original rate we the mode of the membranes, characterized binding and reached the same as We finally level controls. These data between virus and liposomes at reduced temperatures. indicated that virus can fuse with at that influenza A virus X-47 liposomes neutral pH have previously shown rapidly once the complex has undergone all the low pH requiring attaches to liposomes, consisting only of the zwitterionic preparatory steps. At any given time after the end of the phospholipids PC and PE, in a salt resistant manner at low lag phase only a fraction of the virus has reached this state. pH and low temperature (Stegmann et al., 1987). When the In further studies, it was found that the virus population was with the strain used in this study, experiment repeated X-3 identical results were obtained. The virus and 1, were found to associate with each other with a liposomes half time of < 15 s at 5.1 at At neutral pH 0°C (Figure 7). no such took The of the pH, binding place. pH dependence C: - 8.0 n -, a) a) a) -6.0 _s 4.0 {: 20- time (min) -2 0 E 10 - 0 - 0.0 0 4 8 12 16 20 - -$ time (min) a) 7. of (n Fig. Binding zwitterionic liposomes to virus and exposure of the o 10 - fusion peptide as measured by immunoprecipitation. Open squares: binding. Virus was incubated with liposomes (5 sAM of membrane phospholipid phosphorous each; composition of the liposomes: egg PC: egg PE and trace amounts of [3H] cholesterol) at pH 5.1, 0°C 2:1, for various amounts of time and neutralized. The virus and 1 0 30 50 virus-liposome complexes were then pelleted by centrifugation and time (min) measured as the amount of liposomes in the pellet relative to binding, the total amount of was determined by liquid scintillation liposomes, Fig. 6. Effect of neutralization and re-acidification during fusion at Closed circles: amount of HA immunoprecipitated by an counting. O'C. (A) Virus-liposome complexes were neutralized during fusion at the fusion also text). A mixture of trace antibody against peptide (see arrow later 5.1 pH 5.1, O'C (b, up) and re-acidified to pH (b, arrow amounts of 35S-labeled virus and liposomes were prebound for 15 min Curve is a control at O'C throughout. (B) at neutral to and ratio as in down). (a) fusing pH 5.1, pH, subjected pH 5.1, 0°C (concentrations Virus-liposome complexes were neutralized after 25 min (arrow up), for the times and then Figure 1) indicated, neutralized and re-acidified to the indicated thereafter as in White and Wilson with pH shortly (arrow down). immunoprecipitated (1987) antibody Other conditions as in Figure 1. the fusion against peptide. 4235 T.Stegmann, J.M.White and A.Helenius was virtually identical to that of the overall fusion Non-specific control antibodies were used association HA antibodies. attachment could not be reversed by neutraliza- process. The as background controls. we tested polyclonal antipeptide antibodies to the N- tion or incubation with 1 M NaCl (not shown). Pre- First Our previous studies incubation of the virus alone at pH 5.1, 0°C followed by terminus of HA2 (the fusion peptide). recognize neutralization and addition of liposomes at neutral pH also at 25°C and 37°C have shown that these antibodies resulted in binding, but at a lower level. This indicated that exposed fusion peptides in low pH treated HA and BHA in the virus that resulted in attachment was (White and Wilson, 1987). As shown in Figure 7, the amount the change irreversible. of viral HA precipitated from virus-liposome mixtures at We that the binding is hydrophobic in nature and reached maximum values < 1 min after acidification. assume 0°C involves the fusion peptides which are Kinetically, the exposure of the antibody binding sites at 0°C that it hydrophobic under these conditions (see below). This with the attachment of the virus to irreversibly exposed coincided hydrophobic during the first few seconds of the 7). The conformational change was not mode of binding occurs liposomes (Figure point for further low neutralization, and it was not dependent on lag period, and it provides the starting reversible after lag. It is important to note of shown). An antiserum to the pH dependent changes during the the presence liposomes (not C-terminal peptide of HAl (White and Wilson, 1987) was that this binding is different from the binding of virus to also found to precipitate the HA after acidification of either liposomes containing gangliosides at neutral pH (Figure 3). interactions, virus -liposome mixtures or virus alone at 0°C (not shown). In that case, binding occurs via HA-receptor not involve the fusion of HAl is located in the stem of the trimer is not hydrophobic in nature and does The C-terminus et al., 1981). When acidified, the ganglioside-bound HA close to the fusion peptide (Wilson peptides. thus indicated that viral HA expose their fusion peptides, and attach The antipeptide antibodies molecules apparently conformational upon hydrophobic interactions. undergoes a rapid, irreversible change via additional results in the acidification at 0°C, and this change exposure and a region of the C-terminus of HAl, changes in HA of the fusion peptides The conformational Experiments with BHA confirmed which occur in both located in the stem. To determine the conformational changes furthermore, revealed that the protein at low temperatures, we used conforma- this conclusion, and HA during fusion K with kinetics that coincide antibodies which had previously been employed becomes proteinase sensitive tion specific (not shown). low induced changes in BHA (the with this change to characterize pH immunoprecipitations with a panel of soluble ectodomain of HA) and HA Further quantitative bromelain released monoclonal antibodies are summarized et 1986; Daniels conformation specific (White and Wilson, 1987; Copeland al., used in this case was HA Wilson et 1984; in Table II. The antigen purified et al., 1983; Webster et al., 1983; al., non-ionic On the basis of their The was in the presence of detergent. Green et al., 1982). analysis performed by quan- with BHA and detergent solubilized HA before In each case the amount of reactivity titative immunoprecipitation. at room or at 37°C, was and compared with and after acid treatment temperature radioactive HA precipitated counted, been classified as acid- or with conformation anti- these antibodies have previously the amount precipitated independent in HA Table II. Low pH induced conformational changes pH pH pH 5.1, 37°C Incubation 7.4, 0°C 5.1, 0°C 4 60 min 60 min 60 min 60 min 60 min min pH pH 5.1, 0°C pH 7.4, 0°C Precipitation 7.4, 0°C I II III IV V VI Antibody Epitope Neutral specific antibodies 89.9 88.5 92.7 nd nd 7.2 NI (1) HA1 192 (B) 192 101.7 93.0 92.7 nd nd 5.2 N2 (1) HA1 (B) 198 92.6 89.0 94.5 nd nd 9.2 HC-31 (2) HA1 96.4 84.2 83.9 nd nd 4.2 14-4 (3) HAI (B/D) Acid antibodies specific 5.4 20.5 nd 0.5 98.3 HA2 3.9 Al (1) 1.9 1.5 0.8 0 55.5 79.7 A2 HAl (1) 0.4 0.5 0.5 0 29.0 64.4 H26D08 HAI 98-106 (4) 0.4 4.6 20.0 21.6 10.6 47.0 88/1 (3) 100.8 96.2 103.9 88.5 42.8 77.4 Polyclonal rabbit antibody HA. in brackets denote the references et al. Daniels in The number (1) Copeland (1986) (2) Conformational changes detergent solubilized, purified Wilson et al. The letters in brackets denote as defined in Wiley et al. (1981). Antibodies et al. Webster et al. (1984). epitopes (1983) (3) (1983) (4) A2 for et H26D08 reacts with an epitope in the NI and N2 are for monomers of Al and trimers (Copeland al., 1986). specificially specific HA, induced dissociation of the of HA et The rabbit antibody sees both neutral and trimer interface that after low tops (Wilson al., 1984). appears pH at 5.1 were not completely quantitative; the results shown are for two rounds acid forms of HA. See also text. Immunoprecipitations performed pH Al did not its at low (column V) and was therefore not used at pH 5.1 (column IV). The of immunoprecipitation. Antibody recognize epitope pH of the low form with a neutral specific antibody at low pH. nature of the experiment precluded testing pH 4236 Influenza fusion intermediates neutral specific (White and Wilson, 1987; Copeland et al., (Copeland et al., 1986). With purified HA, the changes 1986; Daniels et al., Webster et Wilson recognized by were slow and 1983; al., 1983; these antibodies incomplete et al., 1984; Green et al., 1982). Their conformation (Table II). However, when virus -liposome complexes were specificity was confirmed the reversal acidified, faster changes were detected. Even the effi- by virtually complete then, of the precipitation patterns before and after low pH treat- of at remained ciency precipitation 0°C one third of that seen ment at 37°C in the presence of detergent (Table II, columns at 37°C (Figure 8A and The increase in B). precipitation I and However, after acidification at 0°C, few 88/1 with kinetics identical to the changes by occurred exposure of VI). in the antigenic properties of HA were detected by these the fusion peptide, and confirmed that an irreversible confor- antibodies. Four neutral and two of the acid mational change occurred at low pH. Interestingly, we found specific specific antibodies failed to detect alterations in that the 88/1 antibody, at concentrations of - 5 any purified detergent dig/ml, was solubilized HA after acidification and reneutralization at the only in the of antibodies listed in Table 0°C antibody panel (Table II, columns II and To determine whether the II of fusion without III). capable inhibiting inhibiting binding of antibodies detected changes during fusion, mixtures of virus, virus to liposomes. radioactive virus and liposomes (at the same concentrations The apparent lack of antigenic conversion at the top of as in the fusion cf. were experiments, Figure 1) prepared the molecule at 0°C could either mean that the changes and incubated at 0 or were then 37°C. Samples neutralized, previously described at 37°C and room temperature did not with and The results lysed detergent immunoprecipitated. occur at the low temperature, or that they occurred but were were similar to those obtained with purified HA (Table II) reversed upon reneutralization prior to immunoprecipitation. i.e the same antibodies did not detect changes (not shown). To determine whether reversible changes were occurring, most of the conformation sites for Since dependent binding immunoprecipitations with four acid specific monoclonal these antibodies have been mapped to interfaces in the top antibodies were at 5.1. as a performed directly pH First, domain of the HA trimer (binding sites B and D, see control, HA was treated at pH 5.1, 37°C to induce the Wiley et al., 1981), we conclude that major irreversible complete set of conformational changes, and precipitated at changes do not take place in the top domain during fusion at 0°C. pH 5.1, 0°C. Three of the four acid specific antibodies (A2, Apparently, the tops do not open up. 88/1 and H26D08) were able to recognize their epitopes at Of the four acid monoclonal in pH 5.1, albeit with a lower affinity (Table II, column V). specific antibodies Table 88/1 and detected Antibody Al was not able to at low II, only two, Al, changes. While the immunoprecipitate pH. for 88/1 has not been Al to HA2 When applied to samples that had acidified at 0°C, only one epitope mapped, binds and is therefore in of of the detected its located the lower half the molecule three, 88/1, epitope (Table H, column IV). In conclusion, A2 and H26D08 recognized epitopes induced by conformational at changes only elevated temperature, while 88/1's epitope was exposed by conformational changes 80 at and low high temperatures. This demonstrated that the conformational at changes 0°C were much less extensive 60 than at higher temperatures, and not simply reversed by (-) neutralization. .) 40 - We concluded that fusion at 0°C occurred without the of the of HA complete opening tops the molecule and thus without exposure of the interface of the trimer. A confor- mational change, which exposed the fusion peptide and modified the antigenic properties of the stem of HA, was, 0 15 30 45 60 however, required for fusion activity. Time (min) 100 - Discussion (V Two new into 0 major insights the mechanisms of HA 80 - mediated membrane fusion resulted from our study. First, we found that fusion is mediated by a conformational 60 - (6-i intermediate of the low pH treated HA, a form with exposed 4>i fusion peptides but without opened top domains. Second, 40 - .a we found evidence for a set of events that occur between a~ attachment of virus to the membrane target and the final 20 - Q- fusion event, suggesting complex rearrangements at the site of contact between the membranes. 0I 15 3 45 I 60 I 0 1 5 30 45 60 Conflicting results have been obtained in the concern- past the of ing ability influenza virus to fuse at low temperatures. While White et al. 8. Kinetics of of the 88/1 and the Al A (1982) and Wharton et al. (1986) reported Fig. exposure epitopes. mixture of 35S-labeled virus, egg-grown virus (open symbols) or 35S- that fusion could take at other place 0°C, workers had not labeled virus, egg-grown virus and liposomes (closed symbols) were seen it and et (Junankar Cherry, 1986; Stegmann al., 1987). incubated at or for the time pH 5.1, 0°C (squares) 37°C (diamonds) The issue is now resolved: not only is fusion possible at 0°C, then and indicated, neutralized, detergent-lysed, immunoprecipitated it is actually more efficient than at 37°C. The negative scintillation A: 88/1 also analyzed by liquid counting. antibody (see Table II) B: Al antibody (see also Table reports in the literature are the time before II). explained by lag 4237 T.Stegmann, J.M.White and A.Helenius the onset of fusion, which prevented the detection of fusion acid induced conformation of HA observed at physiological in short recordings. The linear relationship of fusion rate not temperature is fusion active. The strong correlation with temperature (when plotted in Arrhenius plots) suggests between inactivation and the secondary change argues against that the mechanism of fusion is similar at low and high the formal possibility that fusion at 0°C is caused by a minor temperatures. subpopulation of HA, undergoing both primary and secon- dary changes, and escaping detection by our antibodies. Activation and inactivation of HA Fusion, therefore, appears to be mediated by a transient form After acidification, the first event that takes place is an of the protein which has undergone the primary change but irreversible conformational change in HA (Skehel et al., not the secondary change. The published kinetics of virus 1982; Doms and Helenius, 1986; White and Wilson, 1987). inactivation at low pH indicates that the half time of func- It is now clear that this primary change, which leads to struc- tional intermediates at 37°C varies between virus strains in (Scholtissek, 1985). It is 2-4 min for fowl plague virus tural modifications mainly the stem, is followed by a (White et al., 1982) and 30 s for X-31. Apparently, the secondary conformational change at elevated temperatures. A/Japan strain is not inactivated by low pH (Ellens et al., As a result of the primary change, the fusion peptides move 1990; Puri et al., 1990). out of their pockets in the subunit interface. With these What biological significance could a built-in deactivation peptides exposed, the HA becomes hydrophobic, and the process have? One possibility is that HA needs to be rendered virus binds hydrophobically to target membranes. Also as sensitive to after its function in a result of the primary change, the trypsin cleavage site proteases having performed virus entry. After the primary conformational change, the (between HAl residues 27 and 28), located in the loop half protein is still resistant to a variety of proteases. After the way up the stem, is exposed (Wharton et al., 1986). During secondary change, it aggregates (Junankar and Cherry, 1986) the secondary change the top domains of the trimer dissociate from each other (White and Wilson, 1987). This change and becomes quite protease sensitive. This may be impor- in its from results in the opening of the top domains and an additional tant preventing recycling the endosomes to the in such as the surface and thus its detection on the cell surface by the increase protease sensitivity trypsin cleavage HAl 224-225 in the subunit interface in the domain immune surveillance of the host. Our studies have shown site top (Wharton et al., 1986). Studies with the bromelain that the HA of the incoming virus is, indeed, rapidly in of solubilized ectodomain ofHA (BHA) indicate that the secon- degraded lysosomes CHO cells (Martin and Helenius, is slower than the 1991). dary change considerably primary change and The of the (White Wilson, 1987). antigenic properties top domain are altered at this time, and the interactions Events durng the lag between subunits of the trimer are weakened Doms and The primary conformational change in HA occurs within (see 15 s after acidification. of it is Helenius, 1988). Regardless temperature, we monitored the at 0 and a followed salt resistant attachment of the When changes 37°C using instantaneously by of conformation antibodies Table virus to target membranes (Figure 7). The attachment is most panel specific (see II), and we found that likely mediated by the insertion of the exposed fusion binding assays protease digestion, only the occurs at Electron ofHA into the Recent studies primary change 0°C. microscopic peptides target bilayer. labeling of unstained virus confirm this. with photo-activatable lipid analogues indicate that the fusion analysis vitrified, samples No detectable in overall HA are of BHA HA insert as changes morphology peptides (isolated ectodomains) when virus is to low at a-helices into the outer leaflet of observed exposed pH 0°C (Stegmann amphiphilic liposomal while conformational dissociation is membranes Harter et 1989). et al., 1987), gross (Brunner, 1989; al., this and other The next detectable event is fusion between the target readily demonstrated by morphological after incubation et al., membrane and the viral membrane. It occurs after a techniques 37°C (White 1982; lag et the is suffi- which between 0.6 s and 30 min Stegmann al., 1987). Thus, primary change period ranges depending cient for HA's fusion and HA trimers can mediate on temperature, pH and lipid composition. At low activity, fusion without of the where the between and 30 opening tops. temperature, lag ranges min, of The result raises many questions. The most important our results show that the lag involves a series changes which we will consider concerns the in the virus -target membrane complex. lag phase has question, later, mechanism of fusion without trimer dissociation. Others previously been described for fusion of HA-expressing concern the function of the conformational fibroblasts with red cell ghosts (Morris et al., 1989; Sarkar secondary change. it affect the fusion kinetic a What role does it have? How does et measurements, al., 1989). Using stopped-flow In the of our we conclude that was also for another low activity? light present data, lag recently observed pH triggered the conformational is not vesicular stomatitis virus et secondary change only unnecessary virus, (Clague al., 1990) but most for fusion. Most strains of influenza that it be a feature in virus fusion. likely inhibitory suggesting may general A virus rapidly lose fusion activity when incubated at low The reason it has not been observed before why routinely pH in the absence of target membranes (White et al., 1982; is that it is very short at physiological temperature. Sato et Junankar Stegmann et al., 1987; al., 1983; and Our studies show that the events the occurring during lag Cherry, 1986). are continuous, additive and irreversible. Throughout the the virus loses its to interact the must be but the Concomitantly, ability lag, pH low, pH requirement does not with the membrane et remain constant. The step that requires the lowest pH in the hydrophobically target (Stegmann al., At the conformational does entire fusion thus the overall 1987). 0°C, secondary change reaction, determining pH not occur as we have (Stegmann the conformational change (Table I), previously shown dependence, is probably primary et al., 1987) and inactivation does not take place. On the in HA. During the lag, the pH required keeps rising. Since of time is also basis these observations, it seems very likely that the final the lag determined by the lipid composition, and 4238 fusion intermediates Influenza recently been described (Spruce et al., 1989). It since it is initiated only upon addition of target membranes pores has is possible that fusion occurs at the center of the HA complex to acidified virus, we can assume that the key reactions take sites between the two membranes. is where the initial pore opens up. place in the contact and this did not detect changes in HA beyond Our antibody probes the initial primary alteration during or after the lag at 0°C. model for fusion A revised suspect that the lag is not dependent on major We therefore the basis for a revised fusion model Our results provide intramolecular changes in HA. To explain the lag, we favor five operationally distinct steps, as which encompasses the possibility that it reflects the time needed for lateral move- The first is the primary confor- shown in Figure 9 (B -F). ment and reorganization of the HA molecules in the contact 9b). The fusion peptides are mational change in HA (Figure zone. Such rearrangement could, for instance, be required change in the top exposed without a major, permanent for the formation of a multimeric fusion complex. Several the molecule domain. [Minor alterations throughout independent reports have suggested that HA mediated fusion since the change in including the tops are likely, however, may require more than one trimeric spike (Doms and the stem is dramatic and point mutations in the top quite Helenius, 1986; Sarkar et al., 1989; Morris et al., 1989; are known to affect the threshold pH of fusion domain et al., 1990), and rosette shaped complexes of HA Ellens by Wiley and Skehel, 1987)]. The exposure of (reviewed in acidified virus (Doms and Helenius, have been observed leads to attachment of some the fusion peptides hydrophobic 1986). HA molecules to the external bilayer leaflet of the of the Since the ectodomains of HA trimers do target membrane. The fusion phase dissociate, it is difficult to envisage how the fusion fusion phase, during which further not The lag is followed by a are located close to the viral membrane) can - interface must be occurring, peptides (which changes in the virus liposome membrane. To achieve contact the HA in of the lipid bilayers. The rate of reach the target culminating the merger tilt contact with the target membrane as on temperature, pH and trimers may upon fusion during this phase depended This would at least one of the shown in Figure 9c. bring the composition of the target membrane. The main difference trimer into contact with the target fusion peptides of each with the lag phase was that only some of the changes were The other fusion peptide(s) membrane and allow insertion. dependent; fusion continued for some time even if low pH interact with the viral membrane as has of the trimer may was neutralized. Thus, while the reactions that the sample et al., 1982). Attachment complex are low pH depen- been proposed previously (Skehel lead up to the fusion competent external leaflet of the of the fusion peptides to the bilayer the final fusion event itself is not. dent, lead to a salt resistant membrane would stable, Given the limited information available on the role of the target intermediates that we could complex, which is one of the membrane lipids in the fusion reaction, it is difficult to readily see at low temperature. envisage what exactly occurs during the final fusion step. the HA Without major additional conformational changes, Freeze fracture studies indicate a local point mechanism of attachment between the molecules located in the site et al., 1988), and the initial formation of small fusion (Burger it' 1.I Formaltion <fufsionl A. Neutral f'ortii of 11A pH contiple r ( (I B. of fusiion ExposLure I.o. \1mgej o n ntiumins plepLtidsls F of in Ih'ssociation elmluni s C . <ttachenlli1t to targezat Hydrophlobica J~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~r....: .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ .. i t _ _)~~ i.~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ in HA and the molecular in the site of fusion. When combined with the model for the conformational changes rearrangements Fig. 9. Schematic that fusion in five (A) Neutral form of HA. (B) The primary conformational change results from studies, our results suggest proceeds steps. previous membrane via the fusion (D The rearrangement of HA trimers in the site of contact in HA. The attachment to the target exposed peptides. (C) after which occurs. After fusion at 37°C, the HA undergoes secondary the membranes. A of variable length bilayer merger (F) between (E) period are dissociated. which the top domains conformational changes during 4239 T.Stegmann, J.M.White and A.Helenius were carried out under continuous in a membranes may next undergo lateral rearrangement to form stinring, thermostatted cuvette holder in 2 ml (final volume) of 135 mM NaCl, 15 mM sodium citrate, 10 mM a fusion complex. On the basis of electron microscopic data MES, 5 mM HEPES set to various pH values by HCI or NaOH. (Doms and Helenius, 1986), we picture it to be a rosette- Temperatures were maintained within 0.1 'C and pH within 0.05 pH units. like structure with three or more HA trimers (Figure 9d). The increase in fluorescence, resulting from dilution of the fluorophores The final fusion reaction may now take place (Figure 9e) into the viral membrane upon fusion, was recorded continuously at excita- tion and emission wavelengths of 465 and 530 nm, respectively. A 515 nm resulting in lipid mixing and the opening of a narrow fusion long-pass filter was placed between cuvette and emission monochromator channel. The HA molecules may finally undergo the secon- (Stegmann et al., 1985). For the R18 assay, virus was labeled with R18 dary conformational change (Figure 9f) which-in the as described earlier (Stegmann et al., 1986) and injected into a cuvette cellular context of virus entry-could prepare the HA for containing erythrocyte ghosts and a buffer as described above. Fluorescence rapid degradation. dequenching resulting from probe dilution was monitored continuously at excitation and emission wavelengths of 560 and 590 nm, respectively. An SLM-8000 fluorometer was used for all measurements. For calibra- Materials and methods tion of the fluorescence scale, the initial residual fluorescence of the liposomes or the labeled virus was set to zero and the fluorescence at infinite probe Chemicals dilution at 100%. The latter value was obtained after addition of TX-100 N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (N-Rh-PE) (0.5% v/v) to the liposomes and the fluorescence intensity was then corrected and N-(7-nitro-2,1,3-benzoxadiazol4yl)phosphatidylethanolamine (N-NBD- for sample dilution and in the case of the resonance energy transfer assay PE), egg phosphatidylethanolamine (PE) and egg phosphatidylcholine (PC) also for the effect of TX-100 on the quantum yield of N-NBD-PE (Struck from Avanti Polar Lipids (Birmingham, AL) were used without further et al., 1981). Initial rates of fluorescence increase were measured as the purification. Cholesterol, bovine brain gangliosides (type E), TPCK-treated slope of the fusion curve, immediately after the lag phase. For calculations trypsin, HEPES [4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid] and of the final extent of fusion, it was assumed that viral envelopes and liposomes sepharose coupled Ricinus communis agglutinin were purchased from Sigma are of equal size and that, as was found previously (Stegmann et al., 1985, (St Louis, MO), octadecyl rhodamine B chloride (R18) from Molecular 1989b), at a one to one ratio of virus to liposomes one virus particle fuses acid Probes (Junction City, OR), 2-(4-morpholino)-ethanesulfonic (MES) with one liposome. For fusion drawings, curves were manually traced from and from Fisher (Fairlawn, NJ). Goat anti-mouse IgG was Tris-HCI the original stripchart recordings using a digitizer pad (Kurta, Phoenix, AR) purchased from TAGO (Burlingame, CA), scintillation fluid (Opti-fluor) and a drawing program (Canvas, Deneba Software, Miami, FL). In either from Packard (Drowners Grove, IL), and [la,2a(n)-3H]cholesterol assay, at these concentrations of the fluorescent probes, the fluorescence [35S]methionine (Trans-label) from Amersham (Arlington Heights, IL). increases linearly with probe dilution (Hoekstra et al., 1984; Struck et al., Heat-killed Staphylococcus aureus (Zysorbin) was obtained from Zymed 1981). Experiments involving low pH preincubation were performed in Laboratories Inc. (San Francisco, CA) and purified by two washes in 20 mM concentrated suspension (100-150 The pH was adjusted by the addi- MES, 30 mM Tris, 100 mM NaCl, 0.5% TX-100, pelleting the bacteria 1A). tion of 1 M HEPES pH 7.8 or 1 M sodium citrate pH 4.0. Temperature at 1500 g for 4 min. was maintained within 0.5'C in these experiments. Virus purified HA, erythrocyte ghosts and liposomes For binding experiments, virus was added to liposomes, containing trace The X-3 1 recombinant strain of influenza virus was propagated from a single amounts of [ H]cholesteryloleate, at 1 a 1: (phospholipid ratio phosphate) plaque (C-22; Doms et al., 1986) in the allantoic cavity of embryonated in buffers at temperatures and pH as described above for fusion experiments. eggs, purified, handled and stored essentially as described before (Stegmann After incubation and if necessary, neutralization by the 1 M addition of et al., 1985). Viral phospholipid was extracted according to Folch et al. HEPES, the mixture was centrifuged at 0'C for 20 min at 16 000 g. In (1957) after which phosphate was determined according to Bottcher et al. the absence of liposomes, - 90% of the virus pellets under these conditions. (1961). For the production of radioactive virus and HA, confluent monolayers of MDCK cells were infected with virus at high m.o.i. (10-30) for 1 h Immunoprecipitations at 37'C in Dulbecco's Modified Eagles Minimal Essential Medium For immunoprecipitations, immune complexes were preformed by incubating (DMEM). The monolayers were then supplemented with DMEM containing 50 of a 10% (v/v) slurry of washed, killed in 20 mM S.aureus bacteria I.I 10% fetal calf serum and incubated at 37'C for 3 h. The monolayers were MES, 30 mM Tris, 100 mM NAC1 (MNT), pH 7.4 containing 0.1% then washed and the medium was replaced with methionine-free DMEM, TX-100 with td 5 of a 2 mg/ml solution of goat 1 anti-mouse IgG for h to which 1 mCi of [35S]methionine was added per 150 cm2 flask. Virus at 0'C with continuous agitation. Next, aliquots of mouse monoclonal production was allowed to continue for 6 h at 37'C. TPCK-treated trypsin antibodies from different sources (White and Wilson, 1987; Copeland et al., was than added (5 and the culture was incubated for another 8 h 1986; Daniels et al., 1983; Webster et al., 1983; Wilson et al., 1984; Green /Ag/ml) at 37'C. For radioactive virus, the medium was harvested, spun twice at et al., 1982), were added to the mixture and incubated for another hour 2000 g for 5 min and the was loaded on a discontinuous sucrose supematant with continuous agitation. Mixtures were washed with MNT containing 0.5% gradient (3 ml 60% sucrose, 20 ml 30% sucrose, in 145 mM NaCl, 2.5 mM TX-100, pelleted for 4 min at 1500 g, and to resuspended added the purified for 90 min. The HEPES) in an SW 28 rotor and spun at 25 000 r.p.m. HA or virus solution, either in MNT (plus 0.1% TX-100 and 0.25% BSA) interface between the sucrose layers was collected and used as a source or in 135 mM NaCI, 15 mM sodium citrate, 10 mM MES, 5 mM HEPES, of radioactive virus. For HA, virus infected MDCK monolayers were labeled 0.1% TX, 0.25% BSA pH 7.4 or 5.1) and incubated for 1 h with vigorous as described above, trypsin activated as above, and then the cells were agitation. Complexes were then washed three times by pelleting as above collected by centifugation at 2000 g, lysed with 0.5% TX-100, 0.5 M KCI, and resuspending 1 in ml of buffer as described above, after which pellets 20 mM MES, 30 mM pH 7.4,25 soybean trypsin inhibitor Tris-HCI were counted in a liquid scintillation counter or loaded on gels. For Ag/ml and phenylmethylsulfonylfluoride. Nuclei were pelleted at 12 000 g for experiments involving virus, trace virus were mixed amounts of 35S-labeled 10 min, the supernatant was loaded onto a 1 ml column containing with egg-grown virus and liposomes prior to acidification, neutralization R. agglutinin coupled to Sepharose and the column was washed communis and the addition of TX-100 to 0.1 % final, at concentrations comparable with 40 ml of 145 mM NaCl, 2.5 mM HEPES pH 7.4. HA was eluted with those used in the fusion experiments. off the column with 0.2 M D(+) galactose, 145 mM NaCl, 2.5 mM HEPES pH 7.4 and aliquots were quickly frozen in liquid nitrogen and stored at Electron microscopy -70°C. For electron microscopic evaluation of fusion, liposomes and virus were Large unilamellar liposomes were prepared by repeated low-pressure extru- incubated at neutral pH, 0'C for 15 min. For some samples, the pH was sion of multilamellar liposomes through defined pore polycarbonate filters, then lowered to 5.1, 0'C. Samples were applied to Formvar/carbon coated 0.2 in diameter, according to Mayer et al. (1986). Multilamellar copper grids 1 ttm which had been freshly % glow-discharged, stained with liposomes were frozen and thawed three to five times before extrusion. After phosphotungsten acid, pH 7.4 and viewed in a Philips 301 electron extrusion, residual multilameller liposomes were removed by centrifuga- microscope, operating at 80 kV. tion at 16 000 g for 20 min and the radioactivity in pellet and supernatant was determined. Phospholipid phosphate was determined according to Bctt- cher et al. (1961). Erythrocyte ghosts were prepared as in Steck and Kant Acknowledgements (1974) with minor modifications (Stegmann et al., 1986). The authors wish to thank Dr J.J.Skehel and Dr R.G.Webster for the use Fusion and binding experiments of their antibodies, Drs J.M.Delfino, F.Richards, J.Brunner, and D.Wiley For the resonance energy transfer assay, 0.6 mol % of N-NBD-PE and N- P.Zagouras for stimulating discussions, D.Mason and for H.Hoover-Litty Rh-PE was incorporated into liposomes (Struck et al., 1981). Measurements technical assistance, Dr P.Webster for help with the electron microscopy, 4240 fusion Influenza intermediates Dr Webster,R.G., Brown,L.E. and Jackson,D.C. (1983) Virology, R.Lerner for the use of his antibodies and K.Martin for critical reading 126, 587-599. of the manuscript. The work presented in this paper was made possible and by grants from the National Institutes of Health Al 22470, Al 18599 and Wharton,S.A., Skehel,J.J. Wiley, D.C. (1986) Virology, 149, 27-35. White,J.M. (1990) Annu. Rev. GM 38346. Physiol., 52, 675-697. J. Cell White,J. and Wilson,I.A. (1987) Biol., 105, 2887-2896. White,J., Kartenbeck,J. and Helenius,A. (1982) EMBO J., 1, 217-222. Wiley,D.C. and Skehel,J.J. (1987) Annu. Rev. Biochem., 56, 365-394. References Wiley,D.C., Wilson,I.A. and Skehel,J.J. (1981) Nature, 289, 373-378. Wilschut,J. and Hoekstra,D. (1990) Membrane Fusion: Cellular Mechanisms Bottcher,C.J.F., Van Gent,C.M. and Fries,C. (1961) Anal. Chim. Acta, and Biotechnological Applications. Marcel Dekker, New York, in press. 24, 203-204. Wilson,I.A., Skehel,J.J. and Wiley,D.C. (1981) Nature, 289, 366-375. Brunner,J. (1989) FEBS Lett., 257, 369-372. Wilson,I.A., Niman,H.L., Houghten,R.A., Cherenson,A.R., Burger,K.N., Knoll,G. and Verkleij,A.J. (1988) Biochim. Biophys. Acta., Connolly,M.L. and Lerner,R.A. (1984) Cell, 37, 767-778. 89-101. 939, Clague,M.J., Schoch,C., Zech,L. and Blumenthal,R. (1990) Biochemistry, Received on revised on October July 20, 1990; 8, 1990 29, 1303-1308. Copeland,C.S., Doms,R.W., Bolzau,E.M., Webster,R.G. and Helenius,A. J. Cell (1986) Biol., 103, 1179-1191. Daniels,R.S., Douglas,A.R., Skehel,J.J. and Wiley,D.C. (1983) J. Gen. Virol., 64, 1657-1662. Doms,R.W. and Helenius,A. (1986) J. Virol., 60, 833-839. Doms,R.W. and Helenius,A. (1987) In Ohki,S., Flanagan,T.D., Doyle,D., Hui,S.W. and Helenius,A. (eds), Molecular Mechanisms ofMembrane Fusion. Plenum Press, New York, pp. 385-398. Doms,R.W., Gething,M.J., Henneberry,J., White,J. and Helenius,A. (1986) J. Virol., 57, 603-613. Doms,R.W., Stegmann,T. and Helenius,A. (1989) In Notkins,A.L. and Oldstone,M.B.A. (eds), Concepts in Viral Pathogenesis, Springer-Verlag, New York., Vol III, pp. 114-120. Ellens,H., Bentz,J., Mason,D., Zhang,F. and White,J.M. (1990) Biochemistry, 29, 9697 -9707. Folch,J., Lees,M. and Sloane Stanley,G.H. (1957) J. Biol. Chem., 226, 497-509. Goud,B., Salminen,A., Walworth,N.C. and Novick,P.J. (1988) Cell, 53, 753 -768. Green,N., Alexander,H., Olson,A., Alexander,S., Shinnick,T.M., Sutcliffe,J.G. and Lerner,R.A. (1982) Cell, 28, 477-487. Harter,C., James,P., Bachi,T., Semenza,B. and Brunner,J. (1989) J. Biol. Chem., 264, 6459-6464. Hoekstra,D., de Boer,T., Klappe,K. and Wilschut,J. (1984) Biochemistry, 23, 5675-5681. Junankar,P.R. and Cherry,R.J. (1986) Biochim. Biophys. Acta., 854, 198-206. Malhotra,V., Orci,L., Glick,B.S., Block,J.E. and Rothmann,J.E. (1989) Cell, 54, 221-227. Marsh,M. and Helenius,A. (1989) Adv. Virus Res., 36, 107-151. Martin,K. and Helenius,A.J. (1991) Virology, in press. Mayer,L.D., Hope,M.J. and Cullis,P.R. (1986) Biochim. Biophys. Acta., 858, 161-168. Morris,S.J., Sarkar,D.P., White,J.M. and Blumenthal,R. (1989) J. Biol Chem., 264, 3972-3978. and Ohki,S., Doyle,D., Flanagan,T.D., Hui,S.W. Mayhew,E. (eds) (1987) Molecular Mechanisms Membrane Fusion. Plenum New York. of Press, White,J.M. and Puri,A., Booy,F., Doms,R., Blumenthal,R. (1990) J. Virol., in press. Sarkar,D.P., Morris,S.J., and Eidelman,O., Zimmerberg,J. Blumenthal,R. (1989) J. Cell Biol., 113-122. 109, Sato,S.B., Kawasaki,K. and Proc. Natl. Acad. Sci. Ohnishi,S.I. (1983) USA, 80, 3153-3157. Vaccine 215-218. Scholtissek,C. (1985) (supplement), 3, Skehel,J.J., Bayley,P.M., Brown,E.B., Martin,S.R., Waterfield,M.D., White,J.M., Wilson,I.A. and Wiley,D.C. (1982) Proc. Natl. Acad. Sci. 968-972. USA, 79, Cell York. Sowers,A.E. (1988) Fusion, Plenum Press, New White,J.M. and 342. Spruce,A.E., Iwata,A., Almers,W. (1989) Nature, 555-558. and Kant,J.M. Methods 172-180. Steck,T.L. (1974) Enzymol., 31, and Stegmann,T., Hoekstra,D., Scherphof,G. Wilschut,J. (1985) Biochemistry. 24, 3107-3113. and J. Biol. Stegmann,T., Hoekstra,D., Scherphof,G. Wilschut,J. (1986) 10966-10969. Chem., 261, and J. Biol. Stegmann,T., Booy,F.P. Wilschut,J. (1987) Chem., 262, 17744-17749. and Annu. Rev. Stegmann,T., Doms,R.S. Helenius,A. (1989a) Biophys. Biophys, Chem., 18, 187-211. and 1698 - 1704. Stegmann,T., Nir.S. Wilschut,J. (1989b) Biochemistry, 28, and Struck,D.K., Hoekstra,D. Pagano,R.E. (1981) Biochemistry, 20, 4093-4099. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Intermediates in influenza induced membrane fusion.

Stegmann, T.; White, J. M.; Helenius, A.
The EMBO Journal , Volume 9 (13) – Dec 1, 1990
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Abstract

The EMBO Journal vol.9 no. 1 3 pp.4231 - 4241, 1 990 Intermediates in influenza induced membrane fusion Toon Stegmann, takes in endosomes Judith M.Whitel and place (see Doms et al., 1989), where it is a low triggered by pH dependent conformational change Ari Helenius in HA et (Skehel al., 1982; Doms and Helenius, 1986; White Department of Cell Biology, Yale University School of and Medicine, Wilson, 1987). As a result of this conformational change 333 Cedar Street, New CT and Haven, 06510, 'Department of the so-called 'fusion peptides', or the hydrophobic N-termini Pharmacology, and the Cell Biology of Program, University of the HA2 polypeptides, are exposed. In the X-ray struc- California, San Francisco, CA USA 94143-0450, ture of the neutral pH form, these are located peptides in Communicated by P.De Camilli the subunit interface at a distance of 35 A from the viral membrane (Wilson et al., 1981). Once exposed, the fusion Our results show that the peptides of bromelain solubilized ectodomains of mechanism by which influenza HA have virus fuses with target membranes been shown to insert into the outer leaflet of a involves sequential bilayer, adop- complex changes in the ting an a-helical configuration (Harter et hemagglutinin (HA, the viral al., 1989; Brunner, fusion protein) and in the contact site 1989). Biochemical and morphological studies between virus and as well as target membrane. To render analysis of fusion mutants have individual steps amenable revealed extensive additional to study, we worked at 0°C which decreased the rate of low pH induced changes in HA structure, including the fusion and increased the efficiency. The mechanism of dissociation of the domains top of HAl (reviewed in Doms fusion at 0°C and 37°C was similar. The process began et al., 1989; Wiley and Skehel, 1987). On the basis of these with a conformational change in HA which exposed the observations, we have proposed a model for fusion in which fusion peptides but did not lead to dissociation of the tops the exposure of the fusion peptides and the opening of the of the ectodomain of the trimer. The change in the protein top domain are crucial features (see Doms and Helenius, led to immediate hydrophobic attachment of the virus 1987; Doms et al., 1989; Stegmann et al., 1989a). to the target liposomes. Attachment was followed by a In this study we the fusion analyzed reaction between lag period (4-8 min at 0°C, 0.6-2 s at 37°C) during influenza virus and liposomes (or erythrocyte ghosts) at which rearrangements occurred in the site of membrane reduced temperatures. We found that the reaction was contact between the virus and liposome. After a further dramatically slowed down at 0WC, and several kinetically series of changes the final bilayer merger took place. This and biochemically distinct steps could be detected. Most final fusion event was not pH dependent. At effi- importantly, we observed that there is a 4-8 0°C min long, low cient fusion occurred without dissociation of the pH dependent lag before top the onset of fusion during which domains of the HA trimer, suggesting that a transient a liposome- virus complex undergoes a series of complex conformation of HA is responsible for fusion at changes. Further low occur pH dependent changes after the physiological temperatures. The observations lead to a lag period, but the fusion event i.e. the itself, merger of revised model for HA mediated fusion. membranes, proved to be pH Our independent. analysis of Key words: hemagglutinin/influenza/membrane fusion the conformational in HA indicated changes that most of the observed changes previously at higher temperatures, including the opening of the top of the trimer, are not only unnecessary but in fact inhibitory to fusion. The results Introduction provide the basis for a re-evaluation of our model for HA Membrane fusion events are involved in a multitude of vital mediated membrane fusion. cellular and physiological functions. Although some progress has been made in identifying proteins which are required Results for intracellular vesicular transport (Goud et al., 1988; Malhotra et al., 1989), the only proteins demonstrated to The temperature dependence of fusion play a direct role in membrane fusion are the envelope The fusion assay we used takes of the advantage resonance proteins of animal viruses (for recent reviews see Ohki et al., transfer between two fluorescent energy lipids (N-NBD-PE 1987; Marsh and Helenius, 1989; Stegmann et al., 1989a; and N-Rh-PE) when are both in the they present target Wilschut and Hoekstra, 1990; Sowers, 1988; White, 1990). liposomes (Struck et al., Fusion of the 1981). liposome with These proteins mediate fusion between the viral membrane a virion causes dilution of the fluorescent lipids, resulting and host cell membranes and thus allow the viral genome in decreased transfer and an increase in energy fluorescence to enter the cytosol. emission from the N-NBD-PE energy donor, (Stegmann Influenza HA is the best characterized of the viral fusion et al., The membranes used were 1986). target large factors. It is a homotrimeric integral membrane protein unilamellar extrusion et liposomes prepared by (Mayer al., composed of three 84 kd With subunits; each subunit in turn is 1986). a diameter of 0.1-0.2 were in ,um, they the composed of two disulfide bonded same polypeptides, HAl and size range as the virus were particles. They composed HA2. The trimer forms a spike 135 A from of natural zwitterionic extending the and with added phospholipids (PC PE) viral membrane (Wilson et al., 1981). HA mediated While not for fusion fusion gangliosides. required et (White al., (©) Oxford Press 4231 University T.Stegmann, J.M.White and A.Helenius 1982; Stegmann et al., 1989b), the gangliosides serve as it varied within a range of 4-8 min between virus prepara- receptors for the influenza virus HA so that virus - liposome tions. The final increase in fluorescence was 12% at 37°C complexes can form prior to fusion. To allow the forma- values corresponded and 20% at 0°C (Figure 1A). These tion of virus-liposome complexes, purified virus was of and 40%, respectively, to final fusion efficiencies 24% incubated with liposomes at 0°C, at neutral pH and at a when calculated and corrected as outlined in Materials and of virus particle per liposome. Aliquots methods. The lower overall efficiency of fusion at 37°C was stoichiometry one a thermostatted probably due to the rapid, acid induced loss of viral fusion of the mixture were then injected into buffers at different et al., 1982; Sato fluorometer cuvette containing pH values. activity which is observed at 37°C (White observed A low pH dependent increase in fluorescence was et al., 1983; Stegmann et al., 1987; Junankar and Cherry, both at 37°C and at 0°C (Figure IA). To confirm that the 1986). Since such inactivation does not occur at 0°C increase in fluorescence was caused by fusion and not by (Stegmann et al., 1987), virus remains fusion active for a other modes of lipid transfer between the membranes, virus longer period of time at 0°C and consequently more virus alone was incubated at pH 5.0, 37°C and then added to is able to fuse. To obtain a more complete picture of the temperature at 5.1. Virus treated in this way remains liposomes pH dependence of fusion, we determined the initial rates of of to ganglioside-containing liposomes but capable binding fluorescence increase and the lag times at different is abolished et the fusion activity completely (Stegmann al., temperatures in the 0-37°C range. The rates of fluorescence 1987). The lack of fluorescence increase at pH 7.4 and the lack of fluorescence increase in this control (Figure IA) increase were plotted in Arrhenius plots (Figure 2A), and confirmed that the fluorescence increase at 37°C and 0°C it was found that the plot of the initial rates was linear was to virus fusion. (squared regression coefficient 0.98). The linearity indicated due -liposome that the activation energy of the rate-limiting step in fusion fusion was taking place at 0°C, That virus-liposome was the same over the entire temperature range and thus stain elec- could also be demonstrated direcfly by negative of At neutral of suggested that the mechanism fusion at these temperatures tron microscopy (Figure 1B). pH, complexes seen Virus was the same. The lag times could only be measured with virus and liposomes were (Figure panel a). lB, in Lag time was found to were from because confidence the 0-25°C range. particles readily distinguished liposomes decrease with more electron-dense After steeply increasing temperature (Figure 2B). of the spikes and their appearance. 0°C for 90 min, fusion products were incubation at pH 5.1, with were than the 'Cold fusion' erythrocyte ghosts observed (Figure iB, panel b). They larger Before analyzing the fusion event at 0°C in further detail, or were on their original virus liposomes. Spikes present it was important to determine whether fusion with natural in surface (arrows Figure 1B). membranes could also occur at reduced temperatures. To fusion started after acidification and At 37°C, immediately within 1-2 min At there do so, we investigated the fusion of influenza virus with was completed (Figure lA). 0°C, of - 7 min which no detectable fusion erythrocyte ghosts. The fusion assay used in these was a period during fusion at a rate 40 times lower than experiments employed the fluorescent fatty acid occurred, followed by of the was octadecylrhodamine (R18), which was incorporated into the at 37°C. The duration lag highly reproducible of but viral membrane at a self-quenching concentration (Hoekstra between aliquots from the same preparation virus, ..s..... " .s.X. % s, a ....... strain X-3 with Virus and were in a small volume for 15 min at neutral 1. Fusion of influenza prebound pH Fig. virus, 1, liposomes. (A) liposomes into a cuvette at In virus was at at The mixture was then 5.1, 37°C; (c) pH 7.4, 0°C. (d), pretreated pH 5, 0°C. injected (a) pH 5.1, 0°C; (b) pH bound to at for 15 and the mixture was into a cuvette at to virus for 5 min injected pH 5.1, 0°C. Liposome 37°C min, neutralized, liposomes 0°C 1:1 of membrane consisted of PC: PE: bovine brain 6:3:1 and ratio was (5 phospholipid phosphorous each). Liposomes egg egg gangliosides liM Fluorescence was measured as described in Materials and methods. Direct trace amounts of N-NBD-PE and N-Rh-PE mol% (0.6 each). (B) electron mixtures were either at or visualization of fusion at stain microscopy. Virus-liposome kept pH 7.4, 0°C (panel a) 0°C by negative after for 15 at neutral Arrows indicate on fusion Bar at for 90 min min product. incubated pH 5.1, 0°C (panel b) aggregation pH, 0°C. spikes to 100 nm. corresponds 4232 fusion intermediates Influenza et al., 1984; Stegmann et al., 1986). The labeled virus were used as membranes. was erythrocyte ghosts target Further- allowed to bind to the ghosts on ice at neutral pH, and more, the results did not depend on the particular fusion aliquots of the mixture were used. injected into cuvettes at the assay However, the slope of the line in the Arrhenius for appropriate pH and temperatures. plot virus -liposome fusion was different from that We found that fusion occurred efficiently throughout the measured with ghosts, indicating that the detailed interac- 0-37°C range. The Arrhenius plot of the rates of tions of virus with ghosts or liposomes might differ. fluorescence increase is shown in Figure 2A. Again, the results indicate that of The the mechanism fusion is similar at lag phase 0°C and the fusion rates 37°C. Overall, were higher than The existence of a lag before the onset of fusion suggested with liposomes, but the lag times were similar. Since the that preparatory processes are needed before the actual fluorescence increase was faster than with liposomes, lag merger of the two membranes takes place. To determine the times could be measured with precision even at elevated molecular basis for the lag, we analyzed the properties of temperatures. A lag of 0.6 s could be observed at virus -liposome mixtures at 0°C using a 37°C. variety of By extrapolation, a similar lag (0.6-2 s) could be calculated approaches. for fusion with liposomes at Taken the Binding 37°C. together, studies using a centrifugation assay (Stegmann results indicated that the mechanism of influenza virus fusion et al., 1987) indicated that attachment of virus to the with target membranes is similar at 0 and 37°C. The liposomes could only account for a small fraction of the lag temperature dependence was similar whether liposomes or (Figure 3); one third of the virus was already bound to the liposomes prior to acidification, and the rest was bound within the first few seconds at pH 5.1 (half time < 15 s). The of length the lag was, moreover, independent of the total virus 2 and liposome concentration over a 10-fold concentra- tion range at a constant 1:1 virus to liposome ratio. This confirmed that the rate limiting events in the lag were not determined mass by action kinetic processes such as binding of the virus to liposomes. Rather, they involved processes subsequent to the binding, i.e. processes within individual virus - liposome complexes. As expected, the rate of fluorescence increase after the 0- CD lag depended on the ratio of virus to liposomes (not shown). (D The lag time was, however, independent of virus - liposome 100 - 3.2 3.3 3.4 3.5 3.6 3.7 1 OOO/K 80 - 500 _V 40 - CO 20 - I-- co 0O 4 8 12 16 time (min) -j Fig. 3. Binding of ganglioside-containing liposomes to virus. Virus was incubated with liposomes (ratio, concentration and composition as in the legend to Figure 1) at pH 5.1, 0°C for various amounts of time and neutralized. The virus and virus-liposome complexes were then pelleted by centrifugation. Binding was determined as the amount of liposomes in the pellet relative to the total amount of liposomes, by Temperature (OC) in measuring fluorescence pellet and supernatant. Fig. 2. Temperature dependence of the initial rates of fluorescence Table I. Duration of the fusion lag phase preceding virus-liposome at increase and the duration of the lag phase. (A) Arrhenius plots of the OOC fusion of influenza virus with fluorescently labeled liposomes (open squares; at different temperatures but otherwise as in the legend to ratio Virus:liposome Lag time (min) Figure 1) or erythrocyte ghosts (closed squares; as described below). The log of the initial rate of fluorescence increase (the slope of the 1:2 7.0 of steepest part the fusion curve, after the lag phase) was plotted 1:1 6.9 the of against reciprocal absolute temperature. Results shown are the 2:1 6.7 averages of duplicates; the same preparation of virus was used in all 4:1 Virus concentration 10 7.0 experiments. yg/ml; ghost concentration 100 lOg/ml. Fluorescence was measured as described in Materials and methods. Data were fitted by linear regression, with squared regression Concentration of was while the amount of liposomes kept constant, coefficients of 0.98 for virus-liposome fusion and 0.99 for virus was varied. ratio based on membrane Liposome:virus virus-ghost fusion. (B) Lag times of virus-liposome (open squares) at 5 Results shown are for phospholipid phosphorous; liposomes AM. or fusion virus-ghost (closed squares) as described above, at different one particular of variation between within preparation virus; replicates temperatures. one was -0.4 min. preparation 4233 T.Stegmann, J.M.White and A.Helenius ratio at least in the 4:1 -1:2 range (Table I). This confirmed proceeded after the lag phase was affected by steps which that the and uniform for low This is evident in Figure 4, which duration of the lag was constant did not require pH. - every complex fusion in that had neutraliza- all virus liposome complexes. Apparently, shows that samples undergone the was 1.5-2 times faster upon re- had to undergo similar changes during the lag phase, and tion during lag than was fusion in controls which had not been these changes required a minimum time of 4-8 min. acidification to the same extent Two additional observations confirmed that the lag phase neutralized. Fusion was accelerated place early or late in the lag was determined by events taking place in virus-liposome whether the interruptions took complexes and not in the virus alone. First, preincubation period. phase of virus alone at pH 5.1 on ice for up to 10 min prior to Taken together, these results indicated that the lag mixing with liposomes did not change the duration of the encompasses low pH dependent reactions in individual started only upon addi- liposome -virus complexes. The reactions were continuous subsequent lag. Therefore, the lag of time with the and additive. They were faster when the liposomes contained tion liposomes. Secondly, the lag varied They probably represented a series of composition of the liposomes. With phosphatidylcholine/ gangliosides. of molecular at the sites of virus-liposome phosphatidylethanolamine (PC/PE) liposomes devoid rearrangements of and constituted crucial preparatory events in fusion. gangliosides, the lag was 30 min instead 4-8 min (not contact, shown). We found that this difference was not due to slower for pH dependence of fusion at 0°C binding (see below). Apparently, the effect was specific in To determine whether the lag was the only pH dependent gangliosides, as inclusion of cholesterol liposomal PE or PE and step in fusion several experiments were performed. We first membranes containing either PC and PC, on of the determined the overall pH dependence of virus -liposome gangliosides had no additional effect the duration was fusion at 0°C, the rate of fusion at different pH values lag or on the rate of fusion. The size of the liposomes (Figure SA) and the duration of the lag (Figure SB). The not critical; the same lag time was measured using vesicles for fusion was found to be 5.5 and with diameters in the 0.1-0.8 range. apparent pH threshold j.m 5.1. The threshold for fusion was indicated that the events which deter- the pH optimum was pH Further experiments mined the duration of the lag required low pH and were additive. When mixtures at 0°C were virus-liposome 1.0o neutralized at time the lag, no fusion occurred any during even after 90 min. re-acidification fusion began but C Upon 0.8 0- further The total i.e. only after a lag (Figure 4). lag time, ._ of the times before and after the sum lag neutralization, the observed in controls that had not 0.6 always equalled lag neutralized. The combined lag time did not depend on been 0 ._ the amount of time the samples were incubated at neutral 0.4 pH after neutralization, nor did it matter whether the inter- ruption occurred early or late in the lag phase (Figure 4). 0.2 These results indicated that the low pH dependent reactions which determine the length of the lag phase (and which were 0.0 for fusion) progressed steadily and additively necessary the during lag phase. pH While the duration of the was determined by lag phase low the rate with which fusion pH dependent steps, 30 - 15 - 20 - a >--fi- 10 - 0) preincubation time at pH 5. 1, O-C (min) < 1-d -0 CD 10 - a) CIO L- +- 0 4 16 4.5 8 12 20 24 5.5 6.5 time (min) pH of with after a of Fig. 5. pH dependence of fusion and lag time at 0°C. (A) Rate of Fig. 4. Fusion virus liposomes preincubation at low Virus and fluorescence increase. Duration of the lag phase. Open squares: virus-liposome complexes pH. liposomes (ratio, (B) and final concentration as in were incubated for fusion of virus and liposomes at different pH values at 0°C. Open composition Figure 1) for 15 in a concentrated at acidified to pH circles: virus and liposomes were allowed to fuse at pH 5.1, 0°C min suspension pH 7.4, 0°C, down in and reneutralized after (a) 6 min; (b) 25 min, neutralized and re-acidified to the pH indicated at 0°C. Closed 5.1, 0°C (arrow inset) in not virus and were acidified to 5.1 for 2 4 min; (c) 2 min (arrow up in inset) The sample (d) was diamonds: liposomes pH min then transferred the duration of the for this of virus), acidified and served as a control. The mixtures were (half lag phase preparation the moment the neutralized and then re-acidified to the indicated at Other to a cuvette at a final pH of 5.1, 0°C. Tracings began pH 0°C. arrow conditions as in 1. mixtures were re-acidified (large down). Figure 4234 Influenza fusion intermediates had therefore 0.1-0.2 pH units lower at 0°C than at 37°C which did not fuse after neutralization undergone increased fuse at a Virus (Doms et al., 1986). The length of the lag phase changes which allowed it to higher pH. in was dramatically with increasing pH. While it was 4-8 min at neutralized during the fusion phase as Figure 6A, pH 5. 1, it was 30 min at pH 5.3, increasing monotonously incubated at different pH values between 5.1 and neutral with pH (Figure SB). (Figure 6B). The lower the pH after re-acidification was Next, we assayed the pH dependence during different more virus was able to fuse. The rates of made, the fusion, of the Virus - were acidified measured from the steepest part of the curves after re- stages lag. liposome complexes 5.1 at for 2 to half the dura- acidification shown in 6B) are plotted in Figure to pH 0°C min (corresponding (as Figure tion of the lag phase for the virus preparation used), and SA. The for fusion was found to be broadened pH optimum then neutralized. Aliquots were then re-acidified to different occurred at values as so that considerable fusion pH high In with our pH values, and fusion was recorded. agreement as pH 6. previous results (Figure 4), we found that fusion took place Several conclusions can be drawn from these results, (i) after a further lag (Figure SB) and at a faster rate (Figure While the events leading to fusion are low pH dependent, SA) than without prior acidification. It was found, however, the final membrane merger is not. (ii) The step which and hence that the pH threshold for fusion had shifted from pH 5.5 requires the highest concentration of protons, to pH 6. This showed that while the lag was low pH depen- determines the overall pH dependence of fusion, occurs early dent throughout, the overall pH dependence of the fusion in the lag phase. This step is most likely the initial low pH reaction was determined by early events in the lag. induced conformational change in HA. (iii) All fusion These results suggested that the fusion reaction itself might competent virus-liposome complexes undergo a series of be pH independent. To obtain an estimate for the pH low during the lag phase, and the pH dependent changes dependence of the final fusion reaction, we neutralized is constant. duration of these changes (iv) Thereafter, they samples which had proceeded beyond the lag phase at pH a further series of at a undergo changes, occurring highly variable rate, including some which are not low pH 5.1 and 0°C and were actively fusing (Figure 6A). We found in contrast to our results at that, previous 37°C where fusion dependent. stopped immediately upon neutralization (Stegmann et al., 1986), fusion at 0°C continued for 30 min. The rate was - Hydrophobic virus liposome attachment 1/5 to 1/2 of the the in original rate. Upon re-acidification, To analyze what was happening the contact zone between remaining virus fused at a rate faster than the original rate we the mode of the membranes, characterized binding and reached the same as We finally level controls. These data between virus and liposomes at reduced temperatures. indicated that virus can fuse with at that influenza A virus X-47 liposomes neutral pH have previously shown rapidly once the complex has undergone all the low pH requiring attaches to liposomes, consisting only of the zwitterionic preparatory steps. At any given time after the end of the phospholipids PC and PE, in a salt resistant manner at low lag phase only a fraction of the virus has reached this state. pH and low temperature (Stegmann et al., 1987). When the In further studies, it was found that the virus population was with the strain used in this study, experiment repeated X-3 identical results were obtained. The virus and 1, were found to associate with each other with a liposomes half time of < 15 s at 5.1 at At neutral pH 0°C (Figure 7). no such took The of the pH, binding place. pH dependence C: - 8.0 n -, a) a) a) -6.0 _s 4.0 {: 20- time (min) -2 0 E 10 - 0 - 0.0 0 4 8 12 16 20 - -$ time (min) a) 7. of (n Fig. Binding zwitterionic liposomes to virus and exposure of the o 10 - fusion peptide as measured by immunoprecipitation. Open squares: binding. Virus was incubated with liposomes (5 sAM of membrane phospholipid phosphorous each; composition of the liposomes: egg PC: egg PE and trace amounts of [3H] cholesterol) at pH 5.1, 0°C 2:1, for various amounts of time and neutralized. The virus and 1 0 30 50 virus-liposome complexes were then pelleted by centrifugation and time (min) measured as the amount of liposomes in the pellet relative to binding, the total amount of was determined by liquid scintillation liposomes, Fig. 6. Effect of neutralization and re-acidification during fusion at Closed circles: amount of HA immunoprecipitated by an counting. O'C. (A) Virus-liposome complexes were neutralized during fusion at the fusion also text). A mixture of trace antibody against peptide (see arrow later 5.1 pH 5.1, O'C (b, up) and re-acidified to pH (b, arrow amounts of 35S-labeled virus and liposomes were prebound for 15 min Curve is a control at O'C throughout. (B) at neutral to and ratio as in down). (a) fusing pH 5.1, pH, subjected pH 5.1, 0°C (concentrations Virus-liposome complexes were neutralized after 25 min (arrow up), for the times and then Figure 1) indicated, neutralized and re-acidified to the indicated thereafter as in White and Wilson with pH shortly (arrow down). immunoprecipitated (1987) antibody Other conditions as in Figure 1. the fusion against peptide. 4235 T.Stegmann, J.M.White and A.Helenius was virtually identical to that of the overall fusion Non-specific control antibodies were used association HA antibodies. attachment could not be reversed by neutraliza- process. The as background controls. we tested polyclonal antipeptide antibodies to the N- tion or incubation with 1 M NaCl (not shown). Pre- First Our previous studies incubation of the virus alone at pH 5.1, 0°C followed by terminus of HA2 (the fusion peptide). recognize neutralization and addition of liposomes at neutral pH also at 25°C and 37°C have shown that these antibodies resulted in binding, but at a lower level. This indicated that exposed fusion peptides in low pH treated HA and BHA in the virus that resulted in attachment was (White and Wilson, 1987). As shown in Figure 7, the amount the change irreversible. of viral HA precipitated from virus-liposome mixtures at We that the binding is hydrophobic in nature and reached maximum values < 1 min after acidification. assume 0°C involves the fusion peptides which are Kinetically, the exposure of the antibody binding sites at 0°C that it hydrophobic under these conditions (see below). This with the attachment of the virus to irreversibly exposed coincided hydrophobic during the first few seconds of the 7). The conformational change was not mode of binding occurs liposomes (Figure point for further low neutralization, and it was not dependent on lag period, and it provides the starting reversible after lag. It is important to note of shown). An antiserum to the pH dependent changes during the the presence liposomes (not C-terminal peptide of HAl (White and Wilson, 1987) was that this binding is different from the binding of virus to also found to precipitate the HA after acidification of either liposomes containing gangliosides at neutral pH (Figure 3). interactions, virus -liposome mixtures or virus alone at 0°C (not shown). In that case, binding occurs via HA-receptor not involve the fusion of HAl is located in the stem of the trimer is not hydrophobic in nature and does The C-terminus et al., 1981). When acidified, the ganglioside-bound HA close to the fusion peptide (Wilson peptides. thus indicated that viral HA expose their fusion peptides, and attach The antipeptide antibodies molecules apparently conformational upon hydrophobic interactions. undergoes a rapid, irreversible change via additional results in the acidification at 0°C, and this change exposure and a region of the C-terminus of HAl, changes in HA of the fusion peptides The conformational Experiments with BHA confirmed which occur in both located in the stem. To determine the conformational changes furthermore, revealed that the protein at low temperatures, we used conforma- this conclusion, and HA during fusion K with kinetics that coincide antibodies which had previously been employed becomes proteinase sensitive tion specific (not shown). low induced changes in BHA (the with this change to characterize pH immunoprecipitations with a panel of soluble ectodomain of HA) and HA Further quantitative bromelain released monoclonal antibodies are summarized et 1986; Daniels conformation specific (White and Wilson, 1987; Copeland al., used in this case was HA Wilson et 1984; in Table II. The antigen purified et al., 1983; Webster et al., 1983; al., non-ionic On the basis of their The was in the presence of detergent. Green et al., 1982). analysis performed by quan- with BHA and detergent solubilized HA before In each case the amount of reactivity titative immunoprecipitation. at room or at 37°C, was and compared with and after acid treatment temperature radioactive HA precipitated counted, been classified as acid- or with conformation anti- these antibodies have previously the amount precipitated independent in HA Table II. Low pH induced conformational changes pH pH pH 5.1, 37°C Incubation 7.4, 0°C 5.1, 0°C 4 60 min 60 min 60 min 60 min 60 min min pH pH 5.1, 0°C pH 7.4, 0°C Precipitation 7.4, 0°C I II III IV V VI Antibody Epitope Neutral specific antibodies 89.9 88.5 92.7 nd nd 7.2 NI (1) HA1 192 (B) 192 101.7 93.0 92.7 nd nd 5.2 N2 (1) HA1 (B) 198 92.6 89.0 94.5 nd nd 9.2 HC-31 (2) HA1 96.4 84.2 83.9 nd nd 4.2 14-4 (3) HAI (B/D) Acid antibodies specific 5.4 20.5 nd 0.5 98.3 HA2 3.9 Al (1) 1.9 1.5 0.8 0 55.5 79.7 A2 HAl (1) 0.4 0.5 0.5 0 29.0 64.4 H26D08 HAI 98-106 (4) 0.4 4.6 20.0 21.6 10.6 47.0 88/1 (3) 100.8 96.2 103.9 88.5 42.8 77.4 Polyclonal rabbit antibody HA. in brackets denote the references et al. Daniels in The number (1) Copeland (1986) (2) Conformational changes detergent solubilized, purified Wilson et al. The letters in brackets denote as defined in Wiley et al. (1981). Antibodies et al. Webster et al. (1984). epitopes (1983) (3) (1983) (4) A2 for et H26D08 reacts with an epitope in the NI and N2 are for monomers of Al and trimers (Copeland al., 1986). specificially specific HA, induced dissociation of the of HA et The rabbit antibody sees both neutral and trimer interface that after low tops (Wilson al., 1984). appears pH at 5.1 were not completely quantitative; the results shown are for two rounds acid forms of HA. See also text. Immunoprecipitations performed pH Al did not its at low (column V) and was therefore not used at pH 5.1 (column IV). The of immunoprecipitation. Antibody recognize epitope pH of the low form with a neutral specific antibody at low pH. nature of the experiment precluded testing pH 4236 Influenza fusion intermediates neutral specific (White and Wilson, 1987; Copeland et al., (Copeland et al., 1986). With purified HA, the changes 1986; Daniels et al., Webster et Wilson recognized by were slow and 1983; al., 1983; these antibodies incomplete et al., 1984; Green et al., 1982). Their conformation (Table II). However, when virus -liposome complexes were specificity was confirmed the reversal acidified, faster changes were detected. Even the effi- by virtually complete then, of the precipitation patterns before and after low pH treat- of at remained ciency precipitation 0°C one third of that seen ment at 37°C in the presence of detergent (Table II, columns at 37°C (Figure 8A and The increase in B). precipitation I and However, after acidification at 0°C, few 88/1 with kinetics identical to the changes by occurred exposure of VI). in the antigenic properties of HA were detected by these the fusion peptide, and confirmed that an irreversible confor- antibodies. Four neutral and two of the acid mational change occurred at low pH. Interestingly, we found specific specific antibodies failed to detect alterations in that the 88/1 antibody, at concentrations of - 5 any purified detergent dig/ml, was solubilized HA after acidification and reneutralization at the only in the of antibodies listed in Table 0°C antibody panel (Table II, columns II and To determine whether the II of fusion without III). capable inhibiting inhibiting binding of antibodies detected changes during fusion, mixtures of virus, virus to liposomes. radioactive virus and liposomes (at the same concentrations The apparent lack of antigenic conversion at the top of as in the fusion cf. were experiments, Figure 1) prepared the molecule at 0°C could either mean that the changes and incubated at 0 or were then 37°C. Samples neutralized, previously described at 37°C and room temperature did not with and The results lysed detergent immunoprecipitated. occur at the low temperature, or that they occurred but were were similar to those obtained with purified HA (Table II) reversed upon reneutralization prior to immunoprecipitation. i.e the same antibodies did not detect changes (not shown). To determine whether reversible changes were occurring, most of the conformation sites for Since dependent binding immunoprecipitations with four acid specific monoclonal these antibodies have been mapped to interfaces in the top antibodies were at 5.1. as a performed directly pH First, domain of the HA trimer (binding sites B and D, see control, HA was treated at pH 5.1, 37°C to induce the Wiley et al., 1981), we conclude that major irreversible complete set of conformational changes, and precipitated at changes do not take place in the top domain during fusion at 0°C. pH 5.1, 0°C. Three of the four acid specific antibodies (A2, Apparently, the tops do not open up. 88/1 and H26D08) were able to recognize their epitopes at Of the four acid monoclonal in pH 5.1, albeit with a lower affinity (Table II, column V). specific antibodies Table 88/1 and detected Antibody Al was not able to at low II, only two, Al, changes. While the immunoprecipitate pH. for 88/1 has not been Al to HA2 When applied to samples that had acidified at 0°C, only one epitope mapped, binds and is therefore in of of the detected its located the lower half the molecule three, 88/1, epitope (Table H, column IV). In conclusion, A2 and H26D08 recognized epitopes induced by conformational at changes only elevated temperature, while 88/1's epitope was exposed by conformational changes 80 at and low high temperatures. This demonstrated that the conformational at changes 0°C were much less extensive 60 than at higher temperatures, and not simply reversed by (-) neutralization. .) 40 - We concluded that fusion at 0°C occurred without the of the of HA complete opening tops the molecule and thus without exposure of the interface of the trimer. A confor- mational change, which exposed the fusion peptide and modified the antigenic properties of the stem of HA, was, 0 15 30 45 60 however, required for fusion activity. Time (min) 100 - Discussion (V Two new into 0 major insights the mechanisms of HA 80 - mediated membrane fusion resulted from our study. First, we found that fusion is mediated by a conformational 60 - (6-i intermediate of the low pH treated HA, a form with exposed 4>i fusion peptides but without opened top domains. Second, 40 - .a we found evidence for a set of events that occur between a~ attachment of virus to the membrane target and the final 20 - Q- fusion event, suggesting complex rearrangements at the site of contact between the membranes. 0I 15 3 45 I 60 I 0 1 5 30 45 60 Conflicting results have been obtained in the concern- past the of ing ability influenza virus to fuse at low temperatures. While White et al. 8. Kinetics of of the 88/1 and the Al A (1982) and Wharton et al. (1986) reported Fig. exposure epitopes. mixture of 35S-labeled virus, egg-grown virus (open symbols) or 35S- that fusion could take at other place 0°C, workers had not labeled virus, egg-grown virus and liposomes (closed symbols) were seen it and et (Junankar Cherry, 1986; Stegmann al., 1987). incubated at or for the time pH 5.1, 0°C (squares) 37°C (diamonds) The issue is now resolved: not only is fusion possible at 0°C, then and indicated, neutralized, detergent-lysed, immunoprecipitated it is actually more efficient than at 37°C. The negative scintillation A: 88/1 also analyzed by liquid counting. antibody (see Table II) B: Al antibody (see also Table reports in the literature are the time before II). explained by lag 4237 T.Stegmann, J.M.White and A.Helenius the onset of fusion, which prevented the detection of fusion acid induced conformation of HA observed at physiological in short recordings. The linear relationship of fusion rate not temperature is fusion active. The strong correlation with temperature (when plotted in Arrhenius plots) suggests between inactivation and the secondary change argues against that the mechanism of fusion is similar at low and high the formal possibility that fusion at 0°C is caused by a minor temperatures. subpopulation of HA, undergoing both primary and secon- dary changes, and escaping detection by our antibodies. Activation and inactivation of HA Fusion, therefore, appears to be mediated by a transient form After acidification, the first event that takes place is an of the protein which has undergone the primary change but irreversible conformational change in HA (Skehel et al., not the secondary change. The published kinetics of virus 1982; Doms and Helenius, 1986; White and Wilson, 1987). inactivation at low pH indicates that the half time of func- It is now clear that this primary change, which leads to struc- tional intermediates at 37°C varies between virus strains in (Scholtissek, 1985). It is 2-4 min for fowl plague virus tural modifications mainly the stem, is followed by a (White et al., 1982) and 30 s for X-31. Apparently, the secondary conformational change at elevated temperatures. A/Japan strain is not inactivated by low pH (Ellens et al., As a result of the primary change, the fusion peptides move 1990; Puri et al., 1990). out of their pockets in the subunit interface. With these What biological significance could a built-in deactivation peptides exposed, the HA becomes hydrophobic, and the process have? One possibility is that HA needs to be rendered virus binds hydrophobically to target membranes. Also as sensitive to after its function in a result of the primary change, the trypsin cleavage site proteases having performed virus entry. After the primary conformational change, the (between HAl residues 27 and 28), located in the loop half protein is still resistant to a variety of proteases. After the way up the stem, is exposed (Wharton et al., 1986). During secondary change, it aggregates (Junankar and Cherry, 1986) the secondary change the top domains of the trimer dissociate from each other (White and Wilson, 1987). This change and becomes quite protease sensitive. This may be impor- in its from results in the opening of the top domains and an additional tant preventing recycling the endosomes to the in such as the surface and thus its detection on the cell surface by the increase protease sensitivity trypsin cleavage HAl 224-225 in the subunit interface in the domain immune surveillance of the host. Our studies have shown site top (Wharton et al., 1986). Studies with the bromelain that the HA of the incoming virus is, indeed, rapidly in of solubilized ectodomain ofHA (BHA) indicate that the secon- degraded lysosomes CHO cells (Martin and Helenius, is slower than the 1991). dary change considerably primary change and The of the (White Wilson, 1987). antigenic properties top domain are altered at this time, and the interactions Events durng the lag between subunits of the trimer are weakened Doms and The primary conformational change in HA occurs within (see 15 s after acidification. of it is Helenius, 1988). Regardless temperature, we monitored the at 0 and a followed salt resistant attachment of the When changes 37°C using instantaneously by of conformation antibodies Table virus to target membranes (Figure 7). The attachment is most panel specific (see II), and we found that likely mediated by the insertion of the exposed fusion binding assays protease digestion, only the occurs at Electron ofHA into the Recent studies primary change 0°C. microscopic peptides target bilayer. labeling of unstained virus confirm this. with photo-activatable lipid analogues indicate that the fusion analysis vitrified, samples No detectable in overall HA are of BHA HA insert as changes morphology peptides (isolated ectodomains) when virus is to low at a-helices into the outer leaflet of observed exposed pH 0°C (Stegmann amphiphilic liposomal while conformational dissociation is membranes Harter et 1989). et al., 1987), gross (Brunner, 1989; al., this and other The next detectable event is fusion between the target readily demonstrated by morphological after incubation et al., membrane and the viral membrane. It occurs after a techniques 37°C (White 1982; lag et the is suffi- which between 0.6 s and 30 min Stegmann al., 1987). Thus, primary change period ranges depending cient for HA's fusion and HA trimers can mediate on temperature, pH and lipid composition. At low activity, fusion without of the where the between and 30 opening tops. temperature, lag ranges min, of The result raises many questions. The most important our results show that the lag involves a series changes which we will consider concerns the in the virus -target membrane complex. lag phase has question, later, mechanism of fusion without trimer dissociation. Others previously been described for fusion of HA-expressing concern the function of the conformational fibroblasts with red cell ghosts (Morris et al., 1989; Sarkar secondary change. it affect the fusion kinetic a What role does it have? How does et measurements, al., 1989). Using stopped-flow In the of our we conclude that was also for another low activity? light present data, lag recently observed pH triggered the conformational is not vesicular stomatitis virus et secondary change only unnecessary virus, (Clague al., 1990) but most for fusion. Most strains of influenza that it be a feature in virus fusion. likely inhibitory suggesting may general A virus rapidly lose fusion activity when incubated at low The reason it has not been observed before why routinely pH in the absence of target membranes (White et al., 1982; is that it is very short at physiological temperature. Sato et Junankar Stegmann et al., 1987; al., 1983; and Our studies show that the events the occurring during lag Cherry, 1986). are continuous, additive and irreversible. Throughout the the virus loses its to interact the must be but the Concomitantly, ability lag, pH low, pH requirement does not with the membrane et remain constant. The step that requires the lowest pH in the hydrophobically target (Stegmann al., At the conformational does entire fusion thus the overall 1987). 0°C, secondary change reaction, determining pH not occur as we have (Stegmann the conformational change (Table I), previously shown dependence, is probably primary et al., 1987) and inactivation does not take place. On the in HA. During the lag, the pH required keeps rising. Since of time is also basis these observations, it seems very likely that the final the lag determined by the lipid composition, and 4238 fusion intermediates Influenza recently been described (Spruce et al., 1989). It since it is initiated only upon addition of target membranes pores has is possible that fusion occurs at the center of the HA complex to acidified virus, we can assume that the key reactions take sites between the two membranes. is where the initial pore opens up. place in the contact and this did not detect changes in HA beyond Our antibody probes the initial primary alteration during or after the lag at 0°C. model for fusion A revised suspect that the lag is not dependent on major We therefore the basis for a revised fusion model Our results provide intramolecular changes in HA. To explain the lag, we favor five operationally distinct steps, as which encompasses the possibility that it reflects the time needed for lateral move- The first is the primary confor- shown in Figure 9 (B -F). ment and reorganization of the HA molecules in the contact 9b). The fusion peptides are mational change in HA (Figure zone. Such rearrangement could, for instance, be required change in the top exposed without a major, permanent for the formation of a multimeric fusion complex. Several the molecule domain. [Minor alterations throughout independent reports have suggested that HA mediated fusion since the change in including the tops are likely, however, may require more than one trimeric spike (Doms and the stem is dramatic and point mutations in the top quite Helenius, 1986; Sarkar et al., 1989; Morris et al., 1989; are known to affect the threshold pH of fusion domain et al., 1990), and rosette shaped complexes of HA Ellens by Wiley and Skehel, 1987)]. The exposure of (reviewed in acidified virus (Doms and Helenius, have been observed leads to attachment of some the fusion peptides hydrophobic 1986). HA molecules to the external bilayer leaflet of the of the Since the ectodomains of HA trimers do target membrane. The fusion phase dissociate, it is difficult to envisage how the fusion fusion phase, during which further not The lag is followed by a are located close to the viral membrane) can - interface must be occurring, peptides (which changes in the virus liposome membrane. To achieve contact the HA in of the lipid bilayers. The rate of reach the target culminating the merger tilt contact with the target membrane as on temperature, pH and trimers may upon fusion during this phase depended This would at least one of the shown in Figure 9c. bring the composition of the target membrane. The main difference trimer into contact with the target fusion peptides of each with the lag phase was that only some of the changes were The other fusion peptide(s) membrane and allow insertion. dependent; fusion continued for some time even if low pH interact with the viral membrane as has of the trimer may was neutralized. Thus, while the reactions that the sample et al., 1982). Attachment complex are low pH depen- been proposed previously (Skehel lead up to the fusion competent external leaflet of the of the fusion peptides to the bilayer the final fusion event itself is not. dent, lead to a salt resistant membrane would stable, Given the limited information available on the role of the target intermediates that we could complex, which is one of the membrane lipids in the fusion reaction, it is difficult to readily see at low temperature. envisage what exactly occurs during the final fusion step. the HA Without major additional conformational changes, Freeze fracture studies indicate a local point mechanism of attachment between the molecules located in the site et al., 1988), and the initial formation of small fusion (Burger it' 1.I Formaltion <fufsionl A. Neutral f'ortii of 11A pH contiple r ( (I B. of fusiion ExposLure I.o. \1mgej o n ntiumins plepLtidsls F of in Ih'ssociation elmluni s C . <ttachenlli1t to targezat Hydrophlobica J~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~r....: .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ .. i t _ _)~~ i.~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ in HA and the molecular in the site of fusion. When combined with the model for the conformational changes rearrangements Fig. 9. Schematic that fusion in five (A) Neutral form of HA. (B) The primary conformational change results from studies, our results suggest proceeds steps. previous membrane via the fusion (D The rearrangement of HA trimers in the site of contact in HA. The attachment to the target exposed peptides. (C) after which occurs. After fusion at 37°C, the HA undergoes secondary the membranes. A of variable length bilayer merger (F) between (E) period are dissociated. which the top domains conformational changes during 4239 T.Stegmann, J.M.White and A.Helenius were carried out under continuous in a membranes may next undergo lateral rearrangement to form stinring, thermostatted cuvette holder in 2 ml (final volume) of 135 mM NaCl, 15 mM sodium citrate, 10 mM a fusion complex. On the basis of electron microscopic data MES, 5 mM HEPES set to various pH values by HCI or NaOH. (Doms and Helenius, 1986), we picture it to be a rosette- Temperatures were maintained within 0.1 'C and pH within 0.05 pH units. like structure with three or more HA trimers (Figure 9d). The increase in fluorescence, resulting from dilution of the fluorophores The final fusion reaction may now take place (Figure 9e) into the viral membrane upon fusion, was recorded continuously at excita- tion and emission wavelengths of 465 and 530 nm, respectively. A 515 nm resulting in lipid mixing and the opening of a narrow fusion long-pass filter was placed between cuvette and emission monochromator channel. The HA molecules may finally undergo the secon- (Stegmann et al., 1985). For the R18 assay, virus was labeled with R18 dary conformational change (Figure 9f) which-in the as described earlier (Stegmann et al., 1986) and injected into a cuvette cellular context of virus entry-could prepare the HA for containing erythrocyte ghosts and a buffer as described above. Fluorescence rapid degradation. dequenching resulting from probe dilution was monitored continuously at excitation and emission wavelengths of 560 and 590 nm, respectively. An SLM-8000 fluorometer was used for all measurements. For calibra- Materials and methods tion of the fluorescence scale, the initial residual fluorescence of the liposomes or the labeled virus was set to zero and the fluorescence at infinite probe Chemicals dilution at 100%. The latter value was obtained after addition of TX-100 N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (N-Rh-PE) (0.5% v/v) to the liposomes and the fluorescence intensity was then corrected and N-(7-nitro-2,1,3-benzoxadiazol4yl)phosphatidylethanolamine (N-NBD- for sample dilution and in the case of the resonance energy transfer assay PE), egg phosphatidylethanolamine (PE) and egg phosphatidylcholine (PC) also for the effect of TX-100 on the quantum yield of N-NBD-PE (Struck from Avanti Polar Lipids (Birmingham, AL) were used without further et al., 1981). Initial rates of fluorescence increase were measured as the purification. Cholesterol, bovine brain gangliosides (type E), TPCK-treated slope of the fusion curve, immediately after the lag phase. For calculations trypsin, HEPES [4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid] and of the final extent of fusion, it was assumed that viral envelopes and liposomes sepharose coupled Ricinus communis agglutinin were purchased from Sigma are of equal size and that, as was found previously (Stegmann et al., 1985, (St Louis, MO), octadecyl rhodamine B chloride (R18) from Molecular 1989b), at a one to one ratio of virus to liposomes one virus particle fuses acid Probes (Junction City, OR), 2-(4-morpholino)-ethanesulfonic (MES) with one liposome. For fusion drawings, curves were manually traced from and from Fisher (Fairlawn, NJ). Goat anti-mouse IgG was Tris-HCI the original stripchart recordings using a digitizer pad (Kurta, Phoenix, AR) purchased from TAGO (Burlingame, CA), scintillation fluid (Opti-fluor) and a drawing program (Canvas, Deneba Software, Miami, FL). In either from Packard (Drowners Grove, IL), and [la,2a(n)-3H]cholesterol assay, at these concentrations of the fluorescent probes, the fluorescence [35S]methionine (Trans-label) from Amersham (Arlington Heights, IL). increases linearly with probe dilution (Hoekstra et al., 1984; Struck et al., Heat-killed Staphylococcus aureus (Zysorbin) was obtained from Zymed 1981). Experiments involving low pH preincubation were performed in Laboratories Inc. (San Francisco, CA) and purified by two washes in 20 mM concentrated suspension (100-150 The pH was adjusted by the addi- MES, 30 mM Tris, 100 mM NaCl, 0.5% TX-100, pelleting the bacteria 1A). tion of 1 M HEPES pH 7.8 or 1 M sodium citrate pH 4.0. Temperature at 1500 g for 4 min. was maintained within 0.5'C in these experiments. Virus purified HA, erythrocyte ghosts and liposomes For binding experiments, virus was added to liposomes, containing trace The X-3 1 recombinant strain of influenza virus was propagated from a single amounts of [ H]cholesteryloleate, at 1 a 1: (phospholipid ratio phosphate) plaque (C-22; Doms et al., 1986) in the allantoic cavity of embryonated in buffers at temperatures and pH as described above for fusion experiments. eggs, purified, handled and stored essentially as described before (Stegmann After incubation and if necessary, neutralization by the 1 M addition of et al., 1985). Viral phospholipid was extracted according to Folch et al. HEPES, the mixture was centrifuged at 0'C for 20 min at 16 000 g. In (1957) after which phosphate was determined according to Bottcher et al. the absence of liposomes, - 90% of the virus pellets under these conditions. (1961). For the production of radioactive virus and HA, confluent monolayers of MDCK cells were infected with virus at high m.o.i. (10-30) for 1 h Immunoprecipitations at 37'C in Dulbecco's Modified Eagles Minimal Essential Medium For immunoprecipitations, immune complexes were preformed by incubating (DMEM). The monolayers were then supplemented with DMEM containing 50 of a 10% (v/v) slurry of washed, killed in 20 mM S.aureus bacteria I.I 10% fetal calf serum and incubated at 37'C for 3 h. The monolayers were MES, 30 mM Tris, 100 mM NAC1 (MNT), pH 7.4 containing 0.1% then washed and the medium was replaced with methionine-free DMEM, TX-100 with td 5 of a 2 mg/ml solution of goat 1 anti-mouse IgG for h to which 1 mCi of [35S]methionine was added per 150 cm2 flask. Virus at 0'C with continuous agitation. Next, aliquots of mouse monoclonal production was allowed to continue for 6 h at 37'C. TPCK-treated trypsin antibodies from different sources (White and Wilson, 1987; Copeland et al., was than added (5 and the culture was incubated for another 8 h 1986; Daniels et al., 1983; Webster et al., 1983; Wilson et al., 1984; Green /Ag/ml) at 37'C. For radioactive virus, the medium was harvested, spun twice at et al., 1982), were added to the mixture and incubated for another hour 2000 g for 5 min and the was loaded on a discontinuous sucrose supematant with continuous agitation. Mixtures were washed with MNT containing 0.5% gradient (3 ml 60% sucrose, 20 ml 30% sucrose, in 145 mM NaCl, 2.5 mM TX-100, pelleted for 4 min at 1500 g, and to resuspended added the purified for 90 min. The HEPES) in an SW 28 rotor and spun at 25 000 r.p.m. HA or virus solution, either in MNT (plus 0.1% TX-100 and 0.25% BSA) interface between the sucrose layers was collected and used as a source or in 135 mM NaCI, 15 mM sodium citrate, 10 mM MES, 5 mM HEPES, of radioactive virus. For HA, virus infected MDCK monolayers were labeled 0.1% TX, 0.25% BSA pH 7.4 or 5.1) and incubated for 1 h with vigorous as described above, trypsin activated as above, and then the cells were agitation. Complexes were then washed three times by pelleting as above collected by centifugation at 2000 g, lysed with 0.5% TX-100, 0.5 M KCI, and resuspending 1 in ml of buffer as described above, after which pellets 20 mM MES, 30 mM pH 7.4,25 soybean trypsin inhibitor Tris-HCI were counted in a liquid scintillation counter or loaded on gels. For Ag/ml and phenylmethylsulfonylfluoride. Nuclei were pelleted at 12 000 g for experiments involving virus, trace virus were mixed amounts of 35S-labeled 10 min, the supernatant was loaded onto a 1 ml column containing with egg-grown virus and liposomes prior to acidification, neutralization R. agglutinin coupled to Sepharose and the column was washed communis and the addition of TX-100 to 0.1 % final, at concentrations comparable with 40 ml of 145 mM NaCl, 2.5 mM HEPES pH 7.4. HA was eluted with those used in the fusion experiments. off the column with 0.2 M D(+) galactose, 145 mM NaCl, 2.5 mM HEPES pH 7.4 and aliquots were quickly frozen in liquid nitrogen and stored at Electron microscopy -70°C. For electron microscopic evaluation of fusion, liposomes and virus were Large unilamellar liposomes were prepared by repeated low-pressure extru- incubated at neutral pH, 0'C for 15 min. For some samples, the pH was sion of multilamellar liposomes through defined pore polycarbonate filters, then lowered to 5.1, 0'C. Samples were applied to Formvar/carbon coated 0.2 in diameter, according to Mayer et al. (1986). Multilamellar copper grids 1 ttm which had been freshly % glow-discharged, stained with liposomes were frozen and thawed three to five times before extrusion. After phosphotungsten acid, pH 7.4 and viewed in a Philips 301 electron extrusion, residual multilameller liposomes were removed by centrifuga- microscope, operating at 80 kV. tion at 16 000 g for 20 min and the radioactivity in pellet and supernatant was determined. Phospholipid phosphate was determined according to Bctt- cher et al. (1961). Erythrocyte ghosts were prepared as in Steck and Kant Acknowledgements (1974) with minor modifications (Stegmann et al., 1986). The authors wish to thank Dr J.J.Skehel and Dr R.G.Webster for the use Fusion and binding experiments of their antibodies, Drs J.M.Delfino, F.Richards, J.Brunner, and D.Wiley For the resonance energy transfer assay, 0.6 mol % of N-NBD-PE and N- P.Zagouras for stimulating discussions, D.Mason and for H.Hoover-Litty Rh-PE was incorporated into liposomes (Struck et al., 1981). Measurements technical assistance, Dr P.Webster for help with the electron microscopy, 4240 fusion Influenza intermediates Dr Webster,R.G., Brown,L.E. and Jackson,D.C. (1983) Virology, R.Lerner for the use of his antibodies and K.Martin for critical reading 126, 587-599. of the manuscript. The work presented in this paper was made possible and by grants from the National Institutes of Health Al 22470, Al 18599 and Wharton,S.A., Skehel,J.J. Wiley, D.C. (1986) Virology, 149, 27-35. White,J.M. (1990) Annu. Rev. GM 38346. Physiol., 52, 675-697. J. Cell White,J. and Wilson,I.A. (1987) Biol., 105, 2887-2896. White,J., Kartenbeck,J. and Helenius,A. (1982) EMBO J., 1, 217-222. Wiley,D.C. and Skehel,J.J. (1987) Annu. Rev. Biochem., 56, 365-394. References Wiley,D.C., Wilson,I.A. and Skehel,J.J. (1981) Nature, 289, 373-378. Wilschut,J. and Hoekstra,D. (1990) Membrane Fusion: Cellular Mechanisms Bottcher,C.J.F., Van Gent,C.M. and Fries,C. (1961) Anal. Chim. Acta, and Biotechnological Applications. Marcel Dekker, New York, in press. 24, 203-204. Wilson,I.A., Skehel,J.J. and Wiley,D.C. (1981) Nature, 289, 366-375. Brunner,J. (1989) FEBS Lett., 257, 369-372. Wilson,I.A., Niman,H.L., Houghten,R.A., Cherenson,A.R., Burger,K.N., Knoll,G. and Verkleij,A.J. (1988) Biochim. Biophys. Acta., Connolly,M.L. and Lerner,R.A. (1984) Cell, 37, 767-778. 89-101. 939, Clague,M.J., Schoch,C., Zech,L. and Blumenthal,R. (1990) Biochemistry, Received on revised on October July 20, 1990; 8, 1990 29, 1303-1308. Copeland,C.S., Doms,R.W., Bolzau,E.M., Webster,R.G. and Helenius,A. J. Cell (1986) Biol., 103, 1179-1191. Daniels,R.S., Douglas,A.R., Skehel,J.J. and Wiley,D.C. (1983) J. Gen. Virol., 64, 1657-1662. Doms,R.W. and Helenius,A. (1986) J. Virol., 60, 833-839. Doms,R.W. and Helenius,A. (1987) In Ohki,S., Flanagan,T.D., Doyle,D., Hui,S.W. and Helenius,A. (eds), Molecular Mechanisms ofMembrane Fusion. Plenum Press, New York, pp. 385-398. Doms,R.W., Gething,M.J., Henneberry,J., White,J. and Helenius,A. (1986) J. Virol., 57, 603-613. Doms,R.W., Stegmann,T. and Helenius,A. (1989) In Notkins,A.L. and Oldstone,M.B.A. (eds), Concepts in Viral Pathogenesis, Springer-Verlag, New York., Vol III, pp. 114-120. Ellens,H., Bentz,J., Mason,D., Zhang,F. and White,J.M. (1990) Biochemistry, 29, 9697 -9707. Folch,J., Lees,M. and Sloane Stanley,G.H. (1957) J. Biol. Chem., 226, 497-509. Goud,B., Salminen,A., Walworth,N.C. and Novick,P.J. (1988) Cell, 53, 753 -768. Green,N., Alexander,H., Olson,A., Alexander,S., Shinnick,T.M., Sutcliffe,J.G. and Lerner,R.A. (1982) Cell, 28, 477-487. Harter,C., James,P., Bachi,T., Semenza,B. and Brunner,J. (1989) J. Biol. Chem., 264, 6459-6464. Hoekstra,D., de Boer,T., Klappe,K. and Wilschut,J. (1984) Biochemistry, 23, 5675-5681. Junankar,P.R. and Cherry,R.J. (1986) Biochim. Biophys. Acta., 854, 198-206. Malhotra,V., Orci,L., Glick,B.S., Block,J.E. and Rothmann,J.E. (1989) Cell, 54, 221-227. Marsh,M. and Helenius,A. (1989) Adv. Virus Res., 36, 107-151. Martin,K. and Helenius,A.J. (1991) Virology, in press. Mayer,L.D., Hope,M.J. and Cullis,P.R. (1986) Biochim. Biophys. Acta., 858, 161-168. Morris,S.J., Sarkar,D.P., White,J.M. and Blumenthal,R. (1989) J. Biol Chem., 264, 3972-3978. and Ohki,S., Doyle,D., Flanagan,T.D., Hui,S.W. Mayhew,E. (eds) (1987) Molecular Mechanisms Membrane Fusion. Plenum New York. of Press, White,J.M. and Puri,A., Booy,F., Doms,R., Blumenthal,R. (1990) J. Virol., in press. Sarkar,D.P., Morris,S.J., and Eidelman,O., Zimmerberg,J. Blumenthal,R. (1989) J. Cell Biol., 113-122. 109, Sato,S.B., Kawasaki,K. and Proc. Natl. Acad. Sci. Ohnishi,S.I. (1983) USA, 80, 3153-3157. Vaccine 215-218. Scholtissek,C. (1985) (supplement), 3, Skehel,J.J., Bayley,P.M., Brown,E.B., Martin,S.R., Waterfield,M.D., White,J.M., Wilson,I.A. and Wiley,D.C. (1982) Proc. Natl. Acad. Sci. 968-972. USA, 79, Cell York. Sowers,A.E. (1988) Fusion, Plenum Press, New White,J.M. and 342. Spruce,A.E., Iwata,A., Almers,W. (1989) Nature, 555-558. and Kant,J.M. Methods 172-180. Steck,T.L. (1974) Enzymol., 31, and Stegmann,T., Hoekstra,D., Scherphof,G. Wilschut,J. (1985) Biochemistry. 24, 3107-3113. and J. Biol. Stegmann,T., Hoekstra,D., Scherphof,G. Wilschut,J. (1986) 10966-10969. Chem., 261, and J. Biol. Stegmann,T., Booy,F.P. Wilschut,J. (1987) Chem., 262, 17744-17749. and Annu. Rev. Stegmann,T., Doms,R.S. Helenius,A. (1989a) Biophys. Biophys, Chem., 18, 187-211. and 1698 - 1704. Stegmann,T., Nir.S. Wilschut,J. (1989b) Biochemistry, 28, and Struck,D.K., Hoekstra,D. Pagano,R.E. (1981) Biochemistry, 20, 4093-4099.

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The EMBO Journal – Springer Journals

Published: Dec 1, 1990

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