Virus Structures
Transcript of Part 2: Viral Membrane Fusion
00:00:03.24 Hello, I'm Stephen Harrison from the Harvard Medical 00:00:07.02 School, Children's Hospital Boston, and the Howard 00:00:09.18 Hughes Medical Institute. Welcome to Part 2 of this series 00:00:15.17 on virus structures. This part is about viral membrane 00:00:19.08 fusion, the process by which enveloped viruses get into 00:00:23.20 cells. As those of you who watched Part 1 will know, 00:00:29.23 enveloped viruses, those with lipid bilayer membranes, 00:00:33.10 acquire their membrane by budding out through the 00:00:37.13 surface or into an internal compartment of the host cell. 00:00:44.22 And likewise, they penetrate cells that they are about to 00:00:49.04 infect by fusion, a reverse of the budding process, by 00:00:53.13 fusion of viral and cellular membranes. Different viruses 00:00:58.18 have different triggers or sensors, if you wish, to initiate 00:01:05.15 the fusion process. Influenza virus, which enters through 00:01:10.14 endosomes, depends on the low pH of the endosome to 00:01:13.22 initiate fusion. Viruses such as HIV can fuse at the cell 00:01:18.21 surface, and they depend on the sensing the receptor, 00:01:24.18 which triggers conformational changes of its own and in, 00:01:30.15 in the case of HIV, a co-receptor as well. What is 00:01:33.17 membrane fusion? Membrane fusion is, in the simplest 00:01:38.05 sense, making one bilayer out of two. But it's a relatively 00:01:43.15 complicated process in practice, although it's 00:01:47.15 thermodynamically downhill, that is, the fused structure is 00:01:52.17 ultimately stabler than the two separate structures, but 00:01:57.16 there's a substantial kinetic barrier, and it's overcoming 00:02:01.02 that kinetic barrier that is the role of the viral fusion 00:02:06.15 proteins, or of cellular fusion proteins. So an intermediate 00:02:14.20 in the fusion process is generally accepted to be a 00:02:19.20 structure in which the apposed monolayers, the apposed 00:02:26.15 leaflets, of the two bilayers have merged, but not yet the 00:02:31.08 distal ones, and that's called a "hemifusion" structure, or 00:02:34.22 a "hemifusion stalk." And while there's some debates 00:02:38.17 about the detailed organization of the hemifusion 00:02:41.23 intermediate, it's clear from a number of studies that that is 00:02:47.17 an important step on route to fusion. Indeed, the barrier 00:02:52.10 between two bilayers and the hemifusion structure is one 00:02:58.26 of the major kinetic barriers in this process of fusion, and 00:03:04.04 there is probably a kinetic barrier between hemifusion and 00:03:08.04 the ultimate merging of the distal leaflets that lead to the 00:03:13.12 formation of a fusion pore. In the case of viral proteins, 00:03:19.25 there's a sequence of events that's reasonably 00:03:23.12 stereotypical, it turns out, even though the molecular 00:03:29.17 machinery driving this series of events may look very 00:03:34.27 different. That is, the fusion proteins of different viruses, 00:03:39.18 although from the point of view of their protein 00:03:41.17 architecture may be very different, the underlying process 00:03:47.04 that they catalyze (and there's a real sense in which this a 00:03:50.26 catalysis, since as I said it's thermodynamically downhill, 00:03:55.05 but with a high kinetic barrier)... the sequence of events 00:03:58.11 that they catalyze is reasonably stereotypical in all cases. 00:04:04.24 And so, before events begin, the fusion protein is in some 00:04:14.02 conformation, and this is a purely schematic 00:04:16.10 representation, and there are two bilayers: the bilayer of the membrane 00:04:24.02 in the virus, and the bilayer of the membrane of the cell to 00:04:29.07 which the virus is attached. Some event, proton-binding 00:04:37.08 or receptor-binding, induces or fixes a conformational 00:04:41.26 change in the fusion protein that leads to the formation of 00:04:48.13 an extended intermediate in which a hydrophobic 00:04:51.17 element, either an N-terminal peptide or a loop in the 00:04:56.02 middle of an extended part of the protein structure, 00:05:00.00 interacts with the target cell membrane. And that 00:05:03.17 extended intermediate, which is transient, then collapses 00:05:08.18 into a structure that is ultimately a stable structure for the 00:05:15.05 fusion protein, and drags the two membranes together. As 00:05:19.14 I suggested, there are probably kinetic barriers from the 00:05:22.23 point of view of the lipid bilayer itself, both between the 00:05:25.28 two bilayers state and the hemifusion state, and between 00:05:29.20 the hemifusion state and the final formation of a fusion 00:05:33.07 pore, and it is the role of fusion protein to lower that 00:05:38.06 kinetic barrier, as suggested by these dashed, red lines. 00:05:45.12 We'll talk almost entirely about the fusion protein of 00:05:50.19 influenza virus, the so-called hemagglutinin. It's a member 00:05:57.03 of a class of viral fusion proteins, all of which have the 00:06:00.17 following properties, and it's sometimes because they 00:06:04.04 were the earliest ones characterized in molecular 00:06:07.06 structural terms, have come to be called "Class I" viral 00:06:11.02 fusion proteins. These proteins are synthesized as a 00:06:15.00 precursor, which is cleaved, usually, en route to the cell 00:06:23.23 surface, by a protease in the late compartments of the 00:06:31.16 secretory pathway (furin, for example) into an N-terminal 00:06:38.03 element, which is usually a receptor-binding domain (some 00:06:43.07 viruses have proteins like this, but have a separate 00:06:46.27 receptor-binding protein) and a fusion modular, the C- 00:06:52.21 terminal half in general, which is anchored by a C-terminal 00:06:56.11 transmembrane segment in the viral membrane. Examples 00:07:00.09 of this sort of protein (they are all trimeric assemblies of 00:07:06.14 this sort of organization) are influenza, HIV, and the 00:07:13.00 filoviruses such as Ebola. In the case of influenza, where 00:07:19.28 the protein hemagglutinin sticks off of the surface of the 00:07:24.13 virus... along with another protein, which is an enzyme, 00:07:29.26 called neuraminidase, and we won't talk about that today. 00:07:33.07 The hemagglutinin is a trimeric structure, as I suggested, 00:07:38.06 with three functions. It binds the virus to its receptor, the 00:07:43.07 receptor is sialic acid on glycolipids or glycoproteins on 00:07:47.25 the surface of the target cell. It has structures on the 00:07:54.03 outside that can vary without compromising its two other 00:08:00.19 essential functions, so that the virus can evolve to 00:08:05.01 escape neutralization by the immune system of its hosts. 00:08:11.18 And finally, it is, as I've suggested, the protein that 00:08:15.02 catalyzes the membrane fusion process when suitably 00:08:19.02 triggered by proton-binding. So as I've said, it's 00:08:23.00 synthesized as a precursor. This diagram is overly 00:08:26.28 complicated, but all that matters for today is that, at the N 00:08:32.28 terminus of the so-called HA2... the precursor is called 00:08:38.22 HA0, and the two fragments are known as HA1 00:08:42.26 (hemagglutinin 1) and HA2. At the N terminus of HA2 is a 00:08:50.05 hydrophobic peptide exposed, if you wish (it's actually not 00:08:55.12 exposed in the structure, but made N-terminal rather 00:08:59.24 internal by the cleavage process), that interacts with the 00:09:06.19 target cell membrane and is known as the fusion peptide. 00:09:10.20 And then there is a transmembrane segment very near the 00:09:13.06 C terminus that anchors the protein in the viral membrane. 00:09:18.27 So, the representation here shows you the overall 00:09:23.23 structure of the hemagglutinin. This particular 00:09:29.20 representation is based on x-ray crystallography and does 00:09:33.15 not show the transmembrane segment or the very short 00:09:38.05 segment of about 11 residues that extends into the interior 00:09:43.12 of the virus particle or, before budding, into the cytosol of 00:09:47.07 the cell. As you see, most of the HA1 part, which would 00:09:54.03 be, let us say, red (and the HA2 part would be green of 00:10:00.28 one of the subunits), most of the HA1 part folds into a 00:10:05.27 globular domain at the top of the molecule. It contains the 00:10:11.01 site for binding sialic acid. HA2 forms a stalk that projects 00:10:17.18 it outward from the surface of the virus. The sialic acid- 00:10:25.03 binding site here (there's one on each of the three 00:10:28.19 subunits) faces outward; it's the one very conserved 00:10:34.22 feature of an otherwise antigenically variable surface that 00:10:41.00 the molecule presents to the outside world. Here's a 00:10:44.26 slightly more readable representation, both of the 00:10:51.02 monomer on the left, and of the trimeric, spike-like 00:10:58.05 hemagglutinin on the right. Let's look at the monomer. As I 00:11:04.02 said, the HA1 part is largely out at the surface with its 00:11:08.28 sialic acid-binding site, the HA2 part forms the stalk of the 00:11:15.12 molecule. The N terminus of HA2, remember that's the 00:11:20.19 fusion peptide, is here, tucked in along the threefold axis 00:11:26.16 of the trimer. And so the fusion peptide is hidden and 00:11:32.23 can't interact with hydrophobic targets in the structure of 00:11:40.27 the protein as we see it here, but as you'll see, once 00:11:45.01 exposed to low pH, once protons bind, a major 00:11:49.23 underfolding occurs that allows this fusion peptide to 00:11:54.02 emerge and interact with a target membrane. So here's 00:11:57.29 the low pH-triggered conformational change, and one 00:12:01.02 way of describing it from the point of view of the 00:12:03.21 monomer, is that the HA2 part turns itself inside out. That 00:12:11.19 is, the part of the HA2 (and perhaps it's easier to see in 00:12:16.01 this representation with colored segments)... the part of 00:12:20.05 HA2 that's on the outside in the trimer, which is red and 00:12:25.15 then merging into blue, is on the inside after the 00:12:30.19 conformational change, and the part that's on the inside 00:12:34.09 (green and yellow) turns around and comes up the 00:12:38.01 outside. This structure is most simply described as a trimer 00:12:47.06 of hairpin conformations. There's a fair amount of twisting 00:12:51.27 and turning at the turnaround of the hairpin, but 00:12:54.23 fundamentally, you can think of this as three polypeptide 00:12:58.10 chains that begin up here (with the purple arrow which 00:13:03.09 represents the fusion peptide, it's not represented here 00:13:06.28 since it's based on a crystal structure), comes down, and 00:13:10.29 then turns around and comes right back up to the 00:13:13.29 transmembrane segment, which would follow the yellow 00:13:17.24 arrow. So the hemagglutinin then undergoes two 00:13:27.25 irreversible changes in the course of its maturation and 00:13:33.07 exposure to low pH, because indeed the conformational 00:13:37.28 change I just showed you is irreversible. If you then n 00:13:41.12 eutralize, you don't go backwards. And that's because of 00:13:45.01 the first irreversible change, which is the cleavage of a 00:13:47.28 peptide bond. That now means that the structure we see, 00:13:53.05 which is very stable if you keep it at pH 7 (soluble flu 00:13:56.27 hemagglutinin can hang around for months or years stably 00:14:00.23 in the laboratory), but if you expose it to low pH very, very 00:14:05.05 rapidly, it rearranges as shown and that rearrangement 00:14:08.19 doesn't go backwards, and it doesn't go backwards 00:14:11.13 because there's no way of reknitting that peptide bond, 00:14:15.11 since this structure is actually not the lowest free energy 00:14:19.19 state, it's just there's a very high barrier here that's 00:14:22.11 lowered when protons bind. And it is that second change 00:14:31.16 and the free energy recovered from that second 00:14:36.00 conformational change that is coupled to the process of 00:14:40.18 membrane fusion. And so the fusion mechanism can be 00:14:46.15 thought of as cleaving the precursor, or priming this fusion 00:14:53.15 machinery; localizing the virus to the cell by receptor 00:15:02.09 binding, ultimately by uptake into the endosome; and the 00:15:09.02 triggering of refolding, in the case of flu, by low pH, in the 00:15:13.08 case of other viruses, let us say, by a receptor or co- 00:15:16.11 receptor binding, that leads to this stereotypical sequence 00:15:20.06 of events: Exposure of the fusion peptide (that's that 00:15:24.23 extended intermediate), insertion of the fusion peptide into 00:15:28.16 the target membrane, and a folding back of the protein 00:15:33.01 that brings together the target and viral membranes. And it 00:15:36.05 is that folding back that overcomes the first of the kinetic 00:15:40.06 barriers. There is a substantial kinetic barrier to squeezing 00:15:44.15 two membranes any closer together than about 10 or 15 00:15:47.12 angstroms. That is why liposomes, let's say, in solution are 00:15:52.25 stable, although once fused, they are even more stable. 00:15:59.26 But a liposome preparation doesn't spontaneously fuse 00:16:04.08 because of that kinetic barrier to bringing two bilayers 00:16:08.22 close together. And it is that process that is at least one of 00:16:15.03 the crucial ways in which these proteins facilitate 00:16:19.10 membrane fusion, and they do so by recovering free 00:16:23.13 energy in this fold-back process because the primed state 00:16:29.13 is, in one way or another, metastable. So the fusion of 00:16:40.00 membranes by influenza virus can be through of, then, as 00:16:45.13 a triggering process (we don't show actually the sialic acid 00:16:49.17 attachment here)... but a triggering process that leads to 00:16:55.03 dissociation of the HA1 domains at the top. There 00:17:00.13 happens to be a disulfide bond down here that keeps 00:17:03.07 HA1 from actually floating away, but some experiments 00:17:07.11 done already 10 or 15 years ago (actually, more than that, 00:17:11.20 nearly 20 years ago, now that I think of it), showed that if 00:17:16.02 you knit the tops together, then this process can't occur, 00:17:19.27 so we know that this dissociation of the tops from the 00:17:25.01 stalk occurs, and that allows the stalk, the HA2 stalk, to 00:17:32.11 unfold and refold, so to speak. That is, allows the fusion 00:17:37.12 peptide to flip up, associate with the target bilayer, and 00:17:47.21 then, along with the rest of the protein, collapse together 00:17:52.17 to squeeze the two bilayers together, leading to 00:17:56.24 membrane fusion. I said that the description of the post- 00:18:09.08 fusion conformation of the flu hemagglutinin of HA2 00:18:13.26 corresponds to a trimer of hairpin-like structures, and it 00:18:19.28 turns out that for large numbers of these so-called Class I 00:18:24.24 viral fusion proteins, that simple analogy is true. Indeed, in 00:18:29.05 the case of HIV and SIV, the hairpin is particularly simple. 00:18:34.06 It's just a helix coming down, a loop turning around, and a 00:18:38.18 helix coming up, and so the membrane fusion process is 00:18:46.17 nicely represented in this animation from Gaël McGill 00:18:52.11 based on the structure of the post-fusion state of the HIV 00:18:57.04 and SIV conformational proteins, which you can see 00:19:00.10 going from the extended intermediate at the beginning, to 00:19:05.09 a fused state at the end. So we can then ask, in the case 00:19:14.03 of flu hemagglutinin, which makes a post-fusion structure 00:19:19.01 that's also a trimer of hairpins (although as it happens, as 00:19:21.26 you saw, the outer layer isn't a simple α-helix, the 00:19:25.21 structure is a bit more complicated, but it's still 00:19:28.12 fundamentally just coming one way and going back the 00:19:32.24 other way), how many trimers are needed to make such a 00:19:35.24 fusion structure, and indeed, how long does the process 00:19:38.29 take? And so in some experiments that our laboratory 00:19:44.12 undertook with the collaboration of Antoine van Oijen, 00:19:49.08 through the work of a graduate student named Dan 00:19:52.11 Floyd, we sought to use contemporary techniques in 00:19:57.27 single-molecule fluorescence microscopy to try to carry 00:20:03.16 out measurements of fusion, looking at individual virus 00:20:07.22 particles. Because it was clear that the only way we could 00:20:10.29 begin to answer the questions I was just raising about 00:20:14.15 timescale and about numbers of hemagglutinins needed 00:20:20.29 could only be answered in that way. And the experimental 00:20:25.05 setup that Dan Floyd devised is shown schematically 00:20:31.02 here. A lipid bilayer supported on a thin layer of a dextran 00:20:38.11 polymer is doped with a bit of ganglioside, lipids that have 00:20:48.26 sialic acid on their head group and therefore are receptors 00:20:55.15 for flu hemagglutinin. An influenza virus that has been 00:21:04.09 exposed to two different fluorescent dyes, that has taken 00:21:07.17 up two different fluorescent dyes, is allowed to bind to this 00:21:11.12 surface. The green dye is a hydrophobic dye that inserts 00:21:16.03 into the membrane. The red dye is a more soluble dye that 00:21:19.26 can be soaked into the virus particle, and then the 00:21:21.24 excess washed out, and the virus used in the experiment 00:21:25.11 before any of it leaks back out. And so those two dyes 00:21:29.06 report, on the one hand, mixture of lipids in the two 00:21:33.27 membranes, and hence the hemifusion step, and 00:21:37.08 formation of an aqueous channel between the virus and 00:21:45.06 the solute in the swollen dextran polymer layer, that shows 00:21:54.26 the formation of a full fusion pore. And finally, there's a 00:21:59.05 fluorescein pH sensor to tell us... in the bilayer, fluorescein 00:22:04.01 is bleached when the pH drops below about pH 6, and 00:22:07.27 that tells us when, in the experiment you're about to see, 00:22:12.28 the pH in the region of the virus particle fell below a 00:22:16.15 critical value. And so, here's the kind of measurement that 00:22:22.01 is done, and you'll see here the recording both of the 00:22:32.04 signal from the pH sensor; the signal from the green dye 00:22:37.06 that's in the bilayer, the hydrophobic dye; and the signal 00:22:42.24 from the red fluorophore that is inside the virus particle. 00:22:48.04 And what happens when the pH drops is that, with a 00:22:51.10 certain time delay, there is suddenly a rise and then a 00:22:55.22 rapid fall of the fluorescence from the hydrophobic 00:23:03.00 fluorophore. That's because there's enough of it in the 00:23:08.04 membrane that the signal is quenched. This represents 00:23:12.27 de-quenching as the two membranes begin to merge, as 00:23:16.23 the hemifusion event occurs, and then the fluorophore 00:23:21.24 diffuses away in the target membrane. Then with a further 00:23:25.08 time delay, there is mixing of the content of the virus with 00:23:31.02 the aqueous substrate in the dextran layer underneath the 00:23:37.24 bilayer, and one sees loss of fluorescence from the dye 00:23:44.09 that was inside the virus particle as it diffuses away. And 00:23:49.10 so if one does lots of these measurements, and they can 00:23:54.08 be done in parallel because in a suitable microscope, as 00:23:58.06 you see here, there are lots of particles in a field, then one 00:24:02.12 can get a histogram of the times to hemifusion, that is, 00:24:07.02 lipid mixing, and the times to pore formation. We can do 00:24:11.03 this as a function of a variety of parameters, including the 00:24:14.23 final pH of the buffer that was flowed into the little 00:24:23.11 chamber in the microscope, and other parameters of the 00:24:26.28 experiment. Analyzing this kind of experiment, in which... 00:24:33.03 and I guess I should go back to explain that, as you see, 00:24:36.22 hemifusion always involves a rise and then a fall, and 00:24:42.05 when you have a kinetic event that has a delay of that 00:24:45.19 kind, and so then if we're looking at the time to 00:24:48.16 hemifusion, there is a certain delay that has a distribution 00:24:55.03 from particle to particle that looks like this, then you know 00:24:58.25 that there are multiple kinetic steps, whereas if there's a 00:25:01.15 simple, single kinetic step, you would just see a single 00:25:04.23 exponential decay, as you indeed do if you, on a particle- 00:25:12.10 by-particle basis, plot the time between hemifusion and 00:25:17.09 fusion. So to fit the kinetics of the hemifusion event, we 00:25:31.13 chose a relatively simply kinetic scheme with two 00:25:34.10 parameters, in which there might be "N" sequential steps, 00:25:46.09 rate-limiting steps, each with a similar rate constant "k," or 00:25:50.19 "N" independent parallel steps. And they turn out under 00:25:54.14 suitable conditions to have essentially the same sort of 00:25:57.17 functional behavior. And as a result, if we fit these 00:26:02.13 histograms that I showed you in the previous slide, we 00:26:06.22 find that the best fit involves an N of 3 and a rate 00:26:13.17 constant appropriate for the times involved: These 00:26:17.04 experiments were done at room temperature of about 20 00:26:21.08 seconds or so, as a kind of mean time to hemifusion 00:26:25.15 under the conditions of this experiment. Whereas the 00:26:29.29 pore-forming event was a single kinetic step from the 00:26:36.24 hemifusion state to pore formation. Now what's the 00:26:40.28 interpretation of this sort of kinetic analysis? Well, as I 00:26:46.16 said, there were several possibilities. One might be that 00:26:49.27 there are N sequential steps, three. Another might be that 00:26:54.09 there are three parallel steps. By looking at the pH 00:26:57.29 dependence, as indicated here, we found that N was 00:27:03.06 essentially independent of the final pH. And it seemed to 00:27:08.22 us unlikely that one could have N distinct sequential 00:27:12.14 steps that would vary identically with pH, whereas the 00:27:17.02 same is much more likely to be true of N parallel steps. 00:27:22.12 And so we've interpreted N as representing the number of 00:27:26.21 hemagglutinin molecules, the number of hemagglutinin 00:27:28.29 trimers, needed to form a successful fusion pore. That 00:27:35.13 number, of course, might be two in some cases and four 00:27:38.13 in others, but on average over the large number of events 00:27:43.20 analyzed, the number comes out to just about three. In 00:27:48.25 other words, that the free energy recovered from three 00:27:54.08 hemagglutinin conformational changes appears to be 00:27:57.22 sufficient to drive the process that I was showing you in 00:28:01.26 the previous slide. So, which of the various steps in this 00:28:10.06 sort of scheme are we looking at is the rate-limiting step in 00:28:17.06 this sort of analysis? From the pH dependence, and I 00:28:23.15 won't go into the details, we believe that there's an initial 00:28:29.20 rapid equilibrium between a protonated and unprotonated 00:28:35.03 state, but that as soon as this extended intermediate 00:28:39.23 forms, then the process is essentially irreversible. And 00:28:45.21 indeed, as a result of looking at some variance of the 00:28:53.28 hemagglutinin, we're pretty convinced that it is this step 00:28:58.17 that in the measurements I just showed you we're looking 00:29:01.20 at. So there happens to be a very conserved interaction 00:29:07.22 just where the fusion peptide tucks into the trimeric stalk. 00:29:16.08 And mutations here, either in a completely conserved 00:29:22.13 aspartic acid or a completely conserved glycine residue 00:29:26.22 that stabilize the tucking in, mutations here accelerate 00:29:31.04 fusion. And so, we take that as evidence that it is this 00:29:39.06 step that we're looking at. Now, in practice, on the 00:29:45.05 surface of the virus, there are very tightly packed 00:29:50.24 hemagglutinin molecules. There are two of them 00:29:53.13 superposed on this electron micrograph. And so it is also 00:29:59.11 plausible that three of these humagglutinins clustered in 00:30:06.09 one region might well be the minimum needed to catalyze 00:30:13.29 this fusion process. Also, because of the tight packing, it's 00:30:19.12 very unlikely that in the surface of the virus the proteins 00:30:24.03 can move around very much, and so again, the process 00:30:27.26 is presumably carried out by a local set of interactions at 00:30:33.26 an attachment point between the viral membrane and the 00:30:36.29 cell surface. So, these sorts of measurements obviously 00:30:43.09 are just the beginning at trying to understand the details of 00:30:48.21 this sort of process, but here, from now just about 50 00:30:52.28 years ago is an electron micrograph of influenza virus in 00:31:00.15 what would now be called an endosome, to indicate to 00:31:04.11 you, to give you a bit of perspective, and to suggest to 00:31:06.21 you that, from this sort of information, at a stage when one 00:31:13.28 didn't even know what the molecules on the surface of 00:31:17.01 the virus might be, we're now at a stage... we're at the 00:31:20.15 level of dissecting the kinetics of the events and, hence, 00:31:25.19 trying to understand sensitive points for neutralization by 00:31:29.17 antibodies, for example. We can actually get at the 00:31:37.19 molecular details of the process that would lead to the 00:31:45.07 release of the nucleoproteins from inside the particle (you 00:31:51.27 can actually see some of them in cross section here 00:31:55.03 probably) into the cytoplasm through fusion of the lipid 00:31:59.07 bilayer of the virus with the lipid bilayer of the endosome. 00:32:04.26 I've mentioned Dan Floyd and Antoine van Oijen, I should 00:32:08.07 mention also Tijana Ivanovic and John Skehel as 00:32:12.20 collaborators in the measurements I've been showing you, 00:32:15.26 illustrating how one can use structure and biophysical 00:32:21.03 measurements to dissect the fusion mechanism. And I 00:32:25.29 should add further credit to Gaël McGill, whose animation 00:32:30.16 of the HIV fusion mechanism is particularly helpful in trying 00:32:35.02 to understand what we believe these fusion proteins are 00:32:39.00 doing. Thank you very much. 00:32:43.03
