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Virus Structures

Transcript of Part 3: Non-Enveloped Virus Entry

00:00:03.22		Hello, I'm Stephen Harrison of Harvard Medical School,
00:00:07.26		Children's Hospital, and the Howard Hughes Medical
00:00:10.09		Institute. Welcome to Part 3 of this series of lectures on
00:00:15.13		virus structure. In this part, we'll talk about the structure of
00:00:20.07		a non-enveloped virus particle and its implications for the
00:00:26.12		mechanism by which this sort of virus particle gets into
00:00:29.26		cells. As you'll recall, viruses come in two major flavors:
00:00:37.27		enveloped viruses with lipid bilayers, like influenza virus
00:00:42.01		that was the subject of Part 2; and non-enveloped
00:00:47.05		viruses, viruses that have a tightly fitting protein coat to
00:00:52.29		protect the nucleic acid, but no lipid bilayer, such as
00:00:58.15		rotovirus, which we'll be talking about largely today.
00:01:02.21		Rotoviruses are the cause of childhood diarrhea, they're a
00:01:08.04		virus that grows in the small intestine, and is the major
00:01:12.05		source of infantile dehydrating diarrhea, which is
00:01:17.12		particularly serious in developing countries. There's a
00:01:20.29		recent introduction of a vaccine that may ameliorate the
00:01:26.09		spread of this virus, which has been, in recent years,
00:01:29.09		responsible for as many as half a million childhood deaths
00:01:34.24		each year. The virus particle is shown here in an electron
00:01:43.27		micrograph. You'll notice that the contrast appears to be
00:01:50.19		much less than in the micrograph that I show of influenza
00:01:55.18		virus because this is a micrograph taken with a
00:02:02.05		cryopreserved specimen with no stain, rather than a
00:02:05.19		micrograph contrasted with negative stain. We're going to
00:02:09.23		be talking a little bit in the course of looking at the
00:02:13.15		structure of rotovirus about how one uses electron
00:02:18.13		cryomicroscopy to get three-dimensional structures of
00:02:23.24		large macromolecular assemblies such as this one. I also
00:02:28.17		point out on this slide that enveloped viruses enter cells,
00:02:36.11		penetrate cells, by membrane fusion, as we discussed in
00:02:39.22		great detail in the last part, and non-enveloped viruses
00:02:46.01		need to get in by some sort of perforation process, since
00:02:50.07		they don't have a membrane of their own. We want to
00:02:53.29		talk a little bit about what we know about the mechanism
00:02:56.26		of this process in the second half of today's talk. So
00:03:02.15		here's an introduction to the rotovirus particle. It's
00:03:05.06		sometimes called a triple-layered particle because it has
00:03:08.14		three protein layers, an inner blue layer, an intermediate
00:03:13.10		green layer, and an outer yellow and red layer, composed
00:03:18.29		of proteins known as viral protein 2, rather unimaginative
00:03:23.02		nomenclature, VP2, viral protein 6 (the green one), and
00:03:28.18		viral protein 7 and 4 (the yellow and red). The red protein
00:03:33.20		is cleaved in a step that we're going to talk about a little
00:03:36.28		bit, to two fragments known as VP8 and VP5, and those
00:03:43.09		of you who have followed the previous part may begin to
00:03:48.22		see similarities as we go forward between this sort of
00:03:52.20		cleavage, and the kind of cleavage that activates the
00:03:56.06		viral fusion proteins, such as flu hemagglutinin. The
00:04:00.27		particle, by the way, is about 800 angstroms in diameter,
00:04:06.24		so it's almost as large as the 1000 angstrom diameter
00:04:11.25		influenza virus particle. It packages a double-strand RNA
00:04:16.10		genome. In this animation, I show you that the virus
00:04:22.04		particle, as it originally leaves a cell, does not have these
00:04:29.13		extended spikes, but a cleavage, that cleavage from VP4
00:04:35.10		to VP5 and 8, erects the spikes. The spike protein is quite
00:04:40.26		an unusual structure, we're going to talk about it. It's
00:04:43.26		anchored in the inner layer by the yellow outer layer
00:04:50.07		protein. It's the job of the outer layer to deliver the inner
00:05:01.02		particle into the cytoplasm. The inner particle never
00:05:05.15		uncoats, it has a polymerase and a capping enzyme.
00:05:11.11		There are 11 segments of double-strand RNA wound
00:05:15.15		inside, and the polymerase can transcribe that RNA, the
00:05:22.20		capping enzyme can cap it, and the message is extruded
00:05:26.15		from this so-called double-layer particle, or "DLP." Now
00:05:33.00		this animation is not pure fantasy, it's based on detailed
00:05:36.28		structural data: x-ray crystal structures of various proteins
00:05:42.05		and their fragments; an x-ray crystal structure of the intact
00:05:46.00		DLP; and, as we'll talk about in a little bit more detail, a
00:05:53.01		three-dimensional image reconstruction, or several
00:05:55.05		different three-dimensional image reconstructions, from
00:05:58.00		electron cryomicroscopy, or "cryoEM" for short. This slide
00:06:08.24		shows you that recent advances in electron
00:06:13.12		cryomicroscopy mean that we can now obtain density maps
00:06:20.12		representing the structure with essentially the same detail
00:06:25.20		that we've been able to get from x-ray crystallography of
00:06:28.16		large assemblies hitherto. And so, here's a comparison of
00:06:37.01		the x-ray crystal structure, or the density map obtained
00:06:41.07		from that analysis for the double-layered particle, one
00:06:45.02		particular little bit of it, and the similar density map from
00:06:51.27		cryoEM. This was done in collaboration with a colleague
00:06:56.12		at Brandeis named Niko Grigorieff, and these two
00:07:01.08		members, Xing Zhang and Ethan Settembre, of our
00:07:05.20		laboratories. Now, the process by which this sort of
00:07:13.10		analysis is carried out depends on being able to suspend
00:07:20.13		the particles you wish to analyze in a very thin film of
00:07:25.21		vitreous ice, ice or a solution suspension frozen so rapidly
00:07:32.10		that the ice doesn't form crystalline ice, and hence
00:07:34.29		doesn't expand in volume and distort the solute. In the
00:07:42.05		electron microscope, one is seeing a projection of each
00:07:46.14		particle, and by combining data from literally thousands of
00:07:53.09		such images, it's possible to obtain the sort of
00:07:57.29		reconstructed view that I showed you. The single
00:08:01.05		particles are randomly oriented in the vitreous ice, so one
00:08:04.19		has ever conceivable view, and mathematical algorithms
00:08:07.24		had been worked out (you might be familiar with some of
00:08:11.05		them from CAT scans) to determine the relative orientation
00:08:17.28		of all those views, and to combine the data into a three-
00:08:26.27		dimensional picture of the object in question. This
00:08:29.18		obviously depends on the fact that all of the particles are
00:08:33.13		identical. And so cryoEM images of biological structures
00:08:38.12		are possible when those particles are very uniform. But as
00:08:42.16		I've emphasized, the images themselves are very noisy
00:08:47.03		because of the very low electron dose that's required to
00:08:50.12		avoid specimen damage. If you were to try to get a less
00:08:55.02		noisy image, one with higher signal to noise, then you'd fry
00:08:59.24		the specimen. And so, you depend on the fact that the
00:09:04.03		particles are very uniform, and in the case of virus
00:09:07.08		particles, the additional huge advantage of their high
00:09:10.05		symmetry, to make it possible to reach the molecular
00:09:13.05		resolution that I've suggested. So what we're going to
00:09:18.29		focus on for today are the outer layer proteins and their
00:09:24.15		role in delivering the double-layered particle into the cell.
00:09:29.19		Remember that the outer layer proteins are VP4 (which
00:09:33.12		gets cleaved when this erected conformation is
00:09:38.25		established, to VP8 and VP5) and the yellow protein, as I
00:09:43.29		called it, VP7, which locks everything in place. Now VP4,
00:09:51.11		the spike protein, is actually, as you probably noticed, a
00:09:55.06		very curious structure indeed. I've colored the VP8 part in
00:10:04.25		magenta and the three different (it's a trimer, as you'll see
00:10:08.29		in a minute) VP5 parts in various shades of sort of red and
00:10:15.01		orange. And of course the first thing you probably noticed,
00:10:19.22		even with the first image I showed you, was this thing
00:10:23.07		looks like a dimer. There are two lobes sticking up and
00:10:27.27		two ears sticking off of them, and yet I've just said to you
00:10:31.11		it's a trimer, what's going on? Well, turns out that this is an
00:10:35.01		extremely unusual sort of asymmetric arrangement of three
00:10:41.09		proteins. The bottom part, we call it the foot, as you'll see,
00:10:47.06		is perfectly trimeric. The outer projecting spike has a very
00:10:56.08		nice twofold axis. And one is adapted to the other, by this
00:11:03.26		sort of diagonal cantilever. So if you now look, you'll see
00:11:09.14		that the beginning of each protein subunit (remember that
00:11:16.04		VP8 is magenta, and I'm about to show you it's the N-
00:11:20.24		terminal part) is down here, and there are three of them.
00:11:26.21		Polypeptide chain comes up, one of them "quits," and the
00:11:30.10		other two come on up and form these ears. Polypeptide
00:11:34.17		chain then resumes (the cleavage is between VP8 and
00:11:39.13		VP5) as this bean-shaped domain of VP5, and continues
00:11:45.18		on back down. The third bean-shaped domain is here,
00:11:50.27		and so we've lost one ear, it's almost certainly been
00:11:57.02		cleaved by a cleavage here and a cleavage here, as I'll
00:12:00.13		explain in a minute. And that third bean-shaped domain
00:12:05.04		forms this diagonal cantilever that supports the twofold
00:12:10.14		clustered spike of the remaining two. Now already this is a
00:12:16.05		pretty unusual conformation, but I'm about to show you
00:12:21.15		some other gyrations that this protein appears to be able
00:12:25.18		to go through. So as I said, the tryptic cleavage that
00:12:31.09		separates VP8 and VP5... and that probably occurs in the
00:12:37.25		gut as the virus emerges by lysis of the intestinal cells that
00:12:43.17		it has infected, or emerges by some other secretory
00:12:51.27		method, the actual emergence is a bit unclear. The tryptic
00:13:02.00		cleavage then allows the rearrangement of this spike.
00:13:07.27		The three protein subunits on the outer parts of them are
00:13:12.26		probably more disordered if the protein has not been
00:13:20.22		cleaved, and if you look in the electron microscope, you
00:13:23.13		don't see any ordered parts of the outer assembly. And
00:13:28.01		since I've said you have to average many images in order
00:13:31.17		to get a decent three-dimensional representation, then if
00:13:38.23		the outer part is disordered, the averaging will basically
00:13:42.05		blur out all of those elements, and all you'll see is the
00:13:47.23		ordered foot. So if we compare now these spike regions,
00:13:55.08		which in the electron micrographs, or in the three-
00:13:57.15		dimensional reconstruction from electron micrographs, are
00:14:00.21		not quite as well ordered as the parts I showed you,
00:14:03.23		because of some slight flexibility as these structures stick
00:14:10.01		out from the virus. But we can sit them very well with
00:14:13.29		known x-ray structures. If we compare them, see that an
00:14:17.05		x-ray structure of the beam-shaped domain shows a nice
00:14:24.00		dimer like this. It has some hydrophobic loops at the tips
00:14:28.15		that we're going to talk about, and then a separate x-ray
00:14:31.12		structure of this region yields a lectin-like domain that
00:14:39.16		binds sialic acid, which is a receptor for rhesus rotovirus,
00:14:45.07		from which these proteins in our experiments were
00:14:49.00		derived. Now, we therefore believe that the protein,
00:14:58.08		which is synthesized as a monomer, combines with the
00:15:03.18		double-layered particle, three of them for each of the
00:15:07.21		positions on which it sits (you may have noticed that there
00:15:10.04		were 60 spikes sticking out, corresponding to the
00:15:15.20		icosahedral symmetry), and inserts and is locked in by
00:15:21.28		VP7, but is flexible until tryptic cleavage occurs, in which
00:15:28.12		case, one of the VP8s is excised, and the rest of the
00:15:35.15		structure reorganizes to the unusual-looking spike that
00:15:43.05		I've shown you. Now, as if this weren't odd enough, if you
00:15:50.14		make a piece of the protein that's a bit longer than the
00:15:54.24		one that gave that dimer I showed you, you get this
00:15:59.20		trimeric structure, in which a segment of the protein that
00:16:06.09		was missing from the dimeric construct is present, and it
00:16:10.07		has formed a beautiful, trimeric, coiled coil, and the
00:16:15.28		hydrophobic loops of the bean-shaped domain are now
00:16:18.25		pointing, if you wish, down rather than up. In other words,
00:16:23.19		it's as if the structure has gone from this sort of
00:16:28.17		arrangement to this one, and those of you who were
00:16:34.08		following some of the conformational changes of
00:16:39.04		enveloped virus fusion proteins may find that familiar,
00:16:43.22		since it's essentially the same kind of conformational
00:16:48.04		change we've seen there. And just anticipating, we
00:16:51.29		believe that indeed it is that conformational change that,
00:16:55.22		in this case, drives not fusion (there's no membrane on
00:16:58.19		the virus) but drives the membrane disruption that will get
00:17:04.29		the double-layer particle into the cytoplasm. And so, our
00:17:11.24		scheme based on this information so far is that this spike-
00:17:17.27		like structure (and the tryptic cleavage is essential for viral
00:17:22.06		infectivity)... this spike-like structure attaches through
00:17:28.06		sialic acid, some suitable trigger (and we actually don't yet
00:17:34.17		know what that is) allows the two VP8s that are attached
00:17:44.06		to separate enough that these bean-shaped domains can
00:17:50.14		insert or interact with the target cell membrane through
00:17:56.00		those hydrophobic loops, and then this umbrella-like
00:18:01.18		folding back leads, in a way that we hope to understand
00:18:05.19		but don't fully yet, to a disruption event that can allow the
00:18:11.02		double-layer particle to translocate into the cytosol. Now,
00:18:17.27		this list basically tells you what we're thinking. An
00:18:23.21		extended intermediate forms, hydrophobic loops contact
00:18:27.08		the membrane, the protein folds back to the umbrella-like
00:18:30.10		conformation, and there's a coupling of the fold back and
00:18:34.08		the membrane, to perforate the bilayer. To test these
00:18:40.05		notions, we can take advantage of an extremely
00:18:43.22		interesting property of double-strand RNA viruses like
00:18:47.08		rotovirus, which I like to call functional recoating. If you'd
00:18:52.19		like to study the entry of a virus by genetic methods, by
00:18:59.11		trying to making mutations that would impair entry, you
00:19:03.03		have a problem on your hands, because how are you
00:19:07.09		going to get enough of an entry-incompetent virus to
00:19:10.25		study in the first place? You can get around that problem
00:19:15.22		with these viruses in quite a clever way. If you take
00:19:20.16		double-layered particles prepared by removing the outer
00:19:25.12		shell, VP7 and VP4 (or VP8 plus VP5), from infectious
00:19:34.26		virus particles, the double-layer particle's no longer
00:19:38.21		infectious because it can't get in. But if you recoat it with
00:19:45.02		recombinant proteins, recombinant VP4 and recombinant
00:19:50.04		VP7, and then treat with trypsin to cleave and activate
00:19:54.27		the VP4, you get perfectly infectious particles. This is a
00:20:00.07		process worked out (mimicking some experiments
00:20:06.26		originally done by Kartik Chandran and Max Nibert on
00:20:10.10		rheovirus) by Shane Trask and Phil Dormitzer. As a result,
00:20:16.05		we can use this trick to do two things: First, we can
00:20:19.13		mutate the hydrophobic loops and ask does the virus get
00:20:23.08		in or infect, and if it doesn't, what sort of properties does it
00:20:28.04		have? And second, as you'll see, we can use this
00:20:33.28		scheme actually to watch virus particles getting into a
00:20:37.07		cell. And so, some experiments carried out by Irene Kim, a
00:20:42.11		graduate student, showed that if you mutate the
00:20:45.12		hydrophobic residues, you lose infectivity, the virus
00:20:50.28		becomes engulfed in the cell, it's taken up by a process
00:20:55.07		I'm about to show you, but never infects. And you can
00:20:59.22		also show that is never disrupts a membrane, because
00:21:03.06		the virus can allow a toxin called α-sarcin to get into the
00:21:09.00		cell, sort of sweeps it in, so to speak, if it is actually
00:21:13.26		infectious, if it actually perforate some sort of endosomal
00:21:18.21		membrane, but the hydrophobic loop mutations not only
00:21:26.21		block infectivity, but block the capacity of the virus to
00:21:30.17		sweep this toxin into the cell along with it. And so, those
00:21:36.11		experiments are strongly consistent with the view that the
00:21:47.16		hydrophobic loops matter, but in order to understand more
00:21:52.12		of this process, we've really got to understand more about
00:21:56.02		the compartment that the virus arrives in when taken up
00:22:00.28		in a cell, about the kinetics of those events, and about
00:22:06.00		other characteristics, so that we can try to figure out how
00:22:10.03		to design experiments to look at these subsequent steps.
00:22:15.27		In order to describe the experiments that we've devised to
00:22:23.24		try to do this, I should mention the outer shell protein VP7,
00:22:28.03		the other partner here, which, as you see from these
00:22:32.01		images and from the animation early in this lecture, locks
00:22:40.08		the spike protein, which we believe to be the membrane
00:22:44.12		perforator, so to speak, into the particle. VP7 is a trimeric
00:22:50.11		protein, and it's held together by calcium, there are two
00:22:53.15		calcium ions that the x-ray crystal structure showed us
00:22:59.23		hold the protein subunits together. There are negatively
00:23:06.15		charged residues interacting with those calcium ions so
00:23:11.08		that the ligation of calcium is critical for the stability of this
00:23:15.20		trimer. As a result, we believe that some sort of calcium
00:23:20.27		withdrawal may be part of a triggering signal, but we have
00:23:26.10		as yet to define that more precisely. So, in order to try to
00:23:34.24		understand what's going on, we've taken advantage of
00:23:39.01		the recoating experiment in a different way. We found, or
00:23:45.23		Aliaa Abdelhakim in the laboratory has found, that one
00:23:50.27		can label with a fluorescent dye each of the components
00:23:58.15		of the particle before recoating, that is, the double-layered
00:24:03.17		particle, VP7, and VP4, with distinct fluorescent dyes, and
00:24:09.17		therefore follow not only the binding and entry of the
00:24:17.04		particle into the cell, but can detect the point at which the
00:24:21.11		double-layer particle is released into the cytosol, as I'll
00:24:25.02		show you. So here's an experiment with VP7 pseudo-
00:24:31.10		colored in red, the fluorophore on VP7, and the double-
00:24:35.14		layer particle in green. The experiment is done by looking
00:24:40.10		at the very thin edge of an epithelial cell in culture, so that
00:24:45.06		you can look at the top surface without much aberration,
00:24:48.12		and as you'll see, the particle in the circle will suddenly
00:24:53.27		release and fly around in the cytoplasm. If you look
00:24:59.27		closely, you can probably see a trace of red remaining in
00:25:04.01		the circle. This experiment in which the three components
00:25:12.11		had been labeled separately is a little easier to follow. I'll
00:25:16.23		go back and show you that this particle suddenly
00:25:22.29		releases, and if you look at the circles, you'll see that
00:25:26.13		there's still some purple, that is, VP4 and VP7 (or rather,
00:25:32.02		VP5/VP8 plus VP7), at the site of entry, and we stopped
00:25:41.01		the frame as the double-layer particle was diffusing
00:25:44.04		around rapidly in the cell after release. A summary of what
00:25:49.25		we think these images are telling us might be roughly as
00:25:53.23		follows. One has the particle that is activated by trypsin,
00:25:58.11		that is probably even before it emerges from one host and
00:26:04.13		enters another. This is a fecal-oral transmission virus and
00:26:09.01		probably is already exposed to trypsin in the gut of the
00:26:14.02		initial infected individual. It binds to the surface of the cell
00:26:20.08		it's going to infect, in the case of the virus infecting a
00:26:27.00		person or a monkey, since this is a rhesus rotovirus, then
00:26:32.13		it will bind to a small intestinal epithelial cell. It's taken up
00:26:38.15		by a process I'm going to tell you a little bit more about,
00:26:43.09		that does not depend on familiar endocytic routes like
00:26:47.12		clathrin-mediated uptake. Something triggers the
00:26:52.26		conformational changes we've seen, so the membrane
00:26:55.19		perforates, the particle escapes and can start to make
00:27:00.10		RNA. And so, what can we learn from this sort of
00:27:08.04		experiment about this sequence of events? We're just in
00:27:13.21		the process of being able to try to do that, these
00:27:16.09		experiments aren't even yet published, but I think that
00:27:22.29		what I'd like to end on then is a series of questions that I
00:27:27.27		think this kind of experiment can answer. And I hope that
00:27:32.21		they will illustrate to you that, by combining structural data
00:27:37.09		with the kinds of, in this case, single virus particle imaging
00:27:41.27		on living cells that takes advantage of contemporary
00:27:46.20		fluorescence microscopy, we can begin to answer these
00:27:51.05		sorts of questions. So the first question is about the
00:27:55.03		engulfment. Is it simply what I like to call an
00:27:58.14		"autoendosome," that is, does the virus just wrap itself up
00:28:01.22		in membrane? There are various reasons why we think
00:28:04.02		that's at least a reasonable possibility. One is that the
00:28:07.22		process is quite rapid and that it doesn't depend on any of
00:28:11.06		the known cellular pathways. Within a minute or two, the
00:28:15.15		virus particle is sequestered from agents like EDTA,
00:28:23.15		which would take it apart by pulling calcium off, or
00:28:27.11		antibodies that might bind to it. A second question is
00:28:33.14		about the membrane disruption that then occurs. Is the
00:28:38.04		membrane disruption by VP5, this folding back that I
00:28:43.25		showed you, purely mechanochemical, if you wish, as in
00:28:47.26		fusion, or are there other more, in you wish, detergent-like
00:28:54.18		qualities of that interaction? Does it indeed do what I
00:29:02.02		pictured in the previous slide, is there an extended
00:29:05.02		intermediate? Does it remain anchored in the particle
00:29:08.22		when it undergoes this whole process? What triggers it,
00:29:13.09		and when does VP7 come off? But I think you can
00:29:16.12		imagine we can begin by combining the powers of
00:29:22.14		structure-based mutation and design of constrained or
00:29:29.21		altered proteins with recoating and with the direct
00:29:34.14		visualization, particle by particle, of this entry process, to
00:29:38.15		answer some of these questions. And so in conclusion, let
00:29:43.01		me simply acknowledge the large number of people who
00:29:46.09		have participated in this work. I should acknowledge both
00:29:52.17		people in my own laboratory, as well as in collaborating
00:29:55.14		laboratories. Let me just name the specific collaborators
00:30:00.02		with whom the work has been carried out: Phil Dormitzer,
00:30:04.22		who began as a member of our laboratory, and I then
00:30:07.16		have collaborated with him extensively since he
00:30:12.10		established an independent research program; Niko
00:30:16.05		Grigorieff at Brandeis, one of the pioneers of the sorts of
00:30:21.01		cryoEM analysis that I described briefly to you; Tom
00:30:26.00		Kirchhausen, whose work on live-cell imaging and whose
00:30:30.17		development of technologies has allowed us to do the
00:30:34.07		experiments I just described; and Dick Bellamy at the
00:30:37.18		University of Auckland, who first got me interested in
00:30:40.23		rotovirus in the first place. And thank you.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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