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