Stephen Harrison (Harvard) Part 2: Viral membrane fusion

Hello, I’m Stephen Harrison from the Harvard Medical School, Children’s Hospital Boston, and the Howard Hughes Medical Institute. Welcome to Part 2 of this series on virus arrangements. This part is about viral layer fusion, the process by which enclose viruses get into cells. As those of you who watched Part 1 are aware of the fact, enclose viruses, those with lipid bilayer sheaths, acquire their membrane by budding out through the surface or into an internal chamber of the multitude cadre. And likewise, they infiltrate cadres that they are about to infect by synthesi, a overturn of the budding process, by synthesi of viral and cellular membranes. Different viruses have different triggers or sensors, if you are interested in, to initiate the synthesi process. Influenza virus, which registers through endosomes, depends on the low-toned pH of the endosome to initiate fusion. Viruses such as HIV can fuse at the cadre surface, and they depend on the sensing the receptor, which triggers conformational alterations “of ones own” and in, in the case of HIV, a co-receptor as well.What is layer fusion? Membrane fusion is, in the simplest sense, making one bilayer out of two. But it’s a relatively complicated process in practice, although it’s thermodynamically downhill, that is, the fused organize is ultimately stabler than the two separate organizations, but there’s a substantial kinetic roadblock, and it’s overcoming that kinetic obstruction that is the role of the viral fusion proteins, or of cellular synthesi proteins. So an intermediate in the synthesi process is generally accepted to be a structure in which the apposed monolayers, the apposed booklets, of the two bilayers have incorporated, but have still not been the distal ones, and that’s called a “hemifusion” structure, or a “hemifusion stalk.” And while there’s some debates about the detailed organization of the hemifusion intermediate, it’s clear from a number of studies that that is an important step on route to fusion.Indeed, the barrier between two bilayers and the hemifusion formation is one of its most important kinetic hindrances in this process of synthesi, and there is probably a kinetic obstacle between hemifusion and the ultimate unite of the distal leaflets that lead to the formation of a fusion hole. In situations of viral proteins, there’s a sequence of occasions that’s reasonably stereotypical, it is about to change, even if they are the molecular apparatu driving this series of events may examine very different. That is, the synthesi proteins of different viruses, although from the viewpoints of their protein building may be very different, the underlying process that they catalyze( and there’s a real ability in which this a catalysis, since as I said it’s thermodynamically downhill, but with a high kinetic impediment )… The sequence of contests that they catalyze is reasonably stereotypical in all cases. And so, before events begin, the synthesi protein is in some conformation, and this is a strictly schematic representation, and there are two bilayers: the bilayer of the sheath in the virus, and the bilayer of the membrane of the cell to which the virus is attached. Some event, proton-binding or receptor-binding, persuasions or deposits a conformational change in the synthesi protein that leads to the formation of an extended intermediate in which a hydrophobic element, either an N-terminal peptide or a loop in the middle of an extended part of the protein design, interacts with the target cell membrane.And that extended intermediate, which is transient, then collapses into a formation that is ultimately a stable organize for the synthesi protein, and draws the two tissues together. As I showed, there are probably kinetic obstacles from the viewpoints of the lipid bilayer itself, both between the two bilayers state and the hemifusion state, and between the hemifusion commonwealth and the final shaping of a fusion pore, and it is the role of fusion protein to lower that kinetic obstruction, as suggested by these scooted, red lines.We’ll talk almost entirely about the fusion protein of influenza virus, the so-called hemagglutinin. It’s a member of a class of viral synthesi proteins, all of which have the following qualities, and it’s sometimes because they were the earliest ones characterized in molecular structural expressions, have come to be called “Class I” viral fusion proteins. These proteins are synthesized as a precursor, which is cleaved, usually, en route to the cell surface, by a protease in the late lockers of the secretory pathway( furin, for example) into an N-terminal element, which is generally a receptor-binding domain( some viruses have proteins like this, but have a separate receptor-binding protein) and a fusion modular, the C- terminal half in general, which is anchored by a C-terminal transmembrane segment in the viral membrane.Examples of this sort of protein( they are all trimeric forums of this sort of organization) are influenza, HIV, and the filoviruses such as Ebola. In the case of influenza, where the protein hemagglutinin stays off of the surface of the virus … along with another protein, which is an enzyme, announced neuraminidase, and we won’t talk about that today. The hemagglutinin is a trimeric design, as I intimated, with three runs. It fixes the virus to its receptor, the receptor is sialic battery-acid on glycolipids or glycoproteins on the surface of the target cell. It has formations on the outside that can vary without compromising its two other critical capacities, so that the virus can progress to escape neutralization by the immune system of its emcees. And ultimately, it is, as I’ve suggested, the protein that catalyzes the membrane synthesi process when appropriately triggered by proton-binding. So as I’ve said, it’s synthesized as a precursor. This sketch is overly involved, but all that matters for today is that, at the N terminus of the so-called HA2 … the precursor is called HA0, and the two scraps are known as HA1( hemagglutinin 1) and HA2. At the N terminus of HA2 is a hydrophobic peptide exposed, if you are interested in( it’s actually not exposed in the structure, but constituted N-terminal preferably internal by the cleavage process ), that interacts with the target cell membrane and is known as the fusion peptide.And then there is a transmembrane segment very near the C terminus that fixes the protein in the viral layer. So, the representation now goes to show the overall organize of the hemagglutinin. This particular representation is based on x-ray crystallography and does not show the transmembrane segment or the very short segment of about 11 residues that extends into the interior of the virus corpuscle or, before budding, into the cytosol of the cadre. As you accompany, the majority of members of the HA1 part, which “wouldve been”, let us say, red( and the HA2 part would be green of one of the subunits ), most of the HA1 part folds into a spherical subject among the priorities of the molecule. It contains the site for binding sialic acid. HA2 sorts a branch that projects it outward from the surface area of the virus. The sialic acid- binding site here( there’s one on each of the three subunits) faces outward; it’s the one extremely conserved facet of an otherwise antigenically variable skin-deep that the molecule presents to the outside world. Here’s a slightly more intelligible illustration, both of the monomer on the left, and of the trimeric, spike-like hemagglutinin on the right.Let’s look at the monomer. As I said, the HA1 part is largely out at the surface with its sialic acid-binding site, the HA2 part ways the stalk of the molecule. The N terminus of HA2, remember that’s the synthesi peptide, is here, folded in along the threefold axis of the trimer. And so the fusion peptide is obstructed and can’t interact with hydrophobic targets in such structures of the protein as we see it now, but as you’ll witnes, once exposed to low-spirited pH, formerly protons bind, a major underfolding appears that allows this fusion peptide to surface and interact with a target sheath. So here’s the low-grade pH-triggered conformational convert, and one mode of describing him from the viewpoints of the monomer, is that the HA2 part turns itself inside out. That is, the part of the HA2( and perhaps it’s easier to see in this representation with colored segments )… the part of HA2 that’s on the outside in the trimer, which is red and then merging into blue, is on the inside after the conformational alter, and the division that’s on the inside( dark-green and yellow) turns around and comes up the outside.This structure is most simply described as a trimer of hairpin conformations. There’s a fair sum of distort and turning at the turnaround of the hairpin, but basically, you can think of this as three polypeptide series that was started up here( with the violet arrow which represents the fusion peptide, it’s not represented here since it’s based on a crystal organize ), comes down, and then turns around and comes right back up to the transmembrane segment, which would be applied the yellowed arrow. So the hemagglutinin then experiences two irreversible changes in the course of its maturation and showing to low pH, because indeed the conformational modification I just testified you is irreversible. If you then n eutralize, you don’t go backwards.And that’s because of the first irreversible change, which is the cleavage of a peptide ligament. That now means that the structure we read, which is very stable if you keep it at pH 7( soluble flu hemagglutinin can hang around for months or times stably in the lab ), but if you expose it to low pH awfully, very rapidly, it rearranges as establish and that rearrangement doesn’t go backwards, and it doesn’t go backwards because there’s no way of reknitting that peptide bond, since this structure is actually not the lowest free energy state, it’s just there’s a very high barrier here that’s lowered when protons bind.And it is that second change and the free energy recovered from that second conformational modify that is coupled to the process of membrane fusion. And so the fusion mechanism can be thought of as cleaving the precursor, or primary this fusion system; localizing the virus to the cell by receptor attaching, ultimately by uptake into the endosome; and the triggering of refolding, in the case of flu, by low pH, in the instances of other viruses, let us say, by a receptor or co- receptor binding, that leads to this stereotypical cycle of affairs: Exposure of the fusion peptide( that’s that extended intermediate ), insertion of the fusion peptide into the target membrane, and a folding back of the protein that brings together the target and viral membranes.And it is that folding back that overcomes the first of the kinetic railings. There is a substantial kinetic barrier to squeezing two membranes any closer together than about 10 or 15 angstroms. That is why liposomes, let’s say, in solution are stable, although once fused, they are even more stable. But a liposome cooking doesn’t spontaneously fuse because of that kinetic hindrance to bringing two bilayers close together. And it is that process that is at least one of the crucial natures in which these proteins promote layer fusion, and they do so by recovering free energy in this fold-back process because the primed regime is, in one way or another, metastable. So the synthesi of sheaths by influenza virus can be through of, then, as a triggering process( we don’t demo actually the sialic acid attachment now )… but a triggering process that leads to dissociation of the HA1 domains at the top.There happens to be a disulfide bond down now that impedes HA1 from actually swimming apart, but some ventures done already 10 or 15 years ago( actually, more than that, nearly 20 years ago , now that I think of it ), showed that if you join the pinnacles together, then this process can’t occur, so we know that this dissociation of the crowns from the stem arises, and that allows the stalk, the HA2 stalk, to unfold and refold, so to speak. That is, allows the fusion peptide to flip up, associate with the target bilayer, and then, along with the rest of the protein, crumble together to constrict the two bilayers together, leading to membrane fusion. I said that the description of the post- fusion conformation of the influenza hemagglutinin of HA2 corresponds to a trimer of hairpin-like structures, and it turns out that for large quantities of these so-called Class I viral fusion proteins, that simple analogy is true.Indeed, in the case of HIV and SIV, the hairpin is particularly simple. It’s just a helix coming down, a curve turned back, and a helix come through here, and so the sheath synthesi process is neatly represented in this animation from Gal McGill based on the structure of the post-fusion state of the HIV and SIV conformational proteins, which you can see going from the extended intermediate at the beginning, to a fused country at the end. So we can then ask, in the instances of flu hemagglutinin, which makes a post-fusion structure that’s also a trimer of hairpins( although as it happens, as you checked, the outer layer isn’t a simple -helix, the structure is a bit more complicated, but it’s still fundamentally time coming one way and going back the other way ), how many trimers are needed to make such a synthesi structure, and undoubtedly, how long does the process make? And so in some experimentations that our laboratory undertake with the collaboration of Antoine van Oijen, through the activities of a grad student mentioned Dan Floyd, we sought to use contemporary proficiencies in single-molecule fluorescence microscopy to try to carry out measurements of fusion, looking at individual virus particles.Because it was clear that the only way we could begin to answer the questions I was just farm about timescale and about numbers of hemagglutinins needed could only be answered in that way. And the experimental setup that Dan Floyd bequeathed is shown schematically here. A lipid bilayer corroborated on a thin stratum of a dextran polymer is drugged with a little of ganglioside, lipids that have sialic acid on their leader group and therefore are receptors for influenza hemagglutinin. An influenza virus that has been exposed to two different fluorescent pigments, that has taken up two different fluorescent colours, is allowed to bind to this surface. The green color is a hydrophobic pigment that inserts into the membrane. The wine color is a more soluble pigment that can be soaked into the virus corpuscle, and then the excess washed out, and the virus used in the venture before any of it discloses back out. And so those two stains report, on the one paw, combination of lipids in the two sheaths, and hence the hemifusion gradation, and formation of an aqueous path between the virus and the solute in the swell dextran polymer layer, that shows the formation of a full fusion pore.And eventually, there’s a fluorescein pH sensor to tell us … in the bilayer, fluorescein is bleached when the pH drops below about pH 6, and that tells us when, in the venture you’re about to see, the pH in the region of the virus speck descended below a critical value. And so, here’s the kind of measurement that is done, and you’ll see here the recording both of the signal from the pH sensor; the signal from the green color that’s in the bilayer, the hydrophobic colour; and the signal from the pink fluorophore that is inside the virus particle.And what happens when the pH plunges is that, with a certain time delay, there is suddenly a rise and then a rapid fall of the fluorescence from the hydrophobic fluorophore. That’s because there’s enough of it in the sheath that the signal is quenched. This represents de-quenching as the two layers begin to merge, as the hemifusion occasion results, and then the fluorophore diffuses apart in the target membrane. Then with a further time delay, there is mixing of the content of the virus with the aqueous substrate in the dextran seam underneath the bilayer, and one hears loss of fluorescence from the dye that was inside the virus particle as it disperses away.And so if one does lots of these measurements, and they can be done in parallel because in a suitable microscope, as you see here, there are lots of particles in an area, then one can get a histogram of the times to hemifusion, that is, lipid mixing, and the times to pore shaping. We can do this as a function of various categories of parameters, including the final pH of the buffer that was spurted into the little chamber in the microscope, and other parameters of the venture. Analyzing this kind of experiment, in which … and I guess I should go back to explain that, as you find, hemifusion always involves a rise and then a autumn, and when you have a kinetic occasion that has a retard of that nature, and so then if we’re looking at the time to hemifusion, there is a certain delay that has a distribution from speck to particle that looks like this, then you know that there are multiple kinetic steps, whereas if there’s a simple, single kinetic pace, you would just see a single exponential crumble, as you indeed do if you, on a particle- by-particle basis, scheme the time between hemifusion and fusion.So to fit the kinetics of the hemifusion episode, we have decided to a relatively simply kinetic planned with two parameters, in which there might be “N” sequential paces , rate-limiting stairs, each with a same charge constant “k, ” or “N” independent parallel stairs. And they turn out under suitable conditions to have basically the same kind of functional behaviour. And as a result, if we fit these histograms that I presented you in the previous slither, we find that the best fit involves an N of 3 and a proportion constant appropriate for the times involved: These experimentations were done at room temperature of about 20 seconds or so, as a kind of mean time to hemifusion under the conditions of this experimentation. Whereas the pore-forming event was a single kinetic stair from the hemifusion country to pore pattern. Now what’s the interpretation of this sort of kinetic analysis? Well, as I said, there were several likelihoods. One might be that there are N sequential stairs, three. Another might be that there are three parallel steps. By looking at the pH dependence, as indicated here, we pointed out that N was essentially independent of the final pH.And it seemed to us unlikely that one could have N distinct sequential gradations that they are able to motley identically with pH, whereas the same is much more likely to be true of N latitude steps. And so we’ve translated N as representing the number of hemagglutinin molecules, the number of hemagglutinin trimers, needed to form a successful fusion pore. That multitude, of course, is likely to be two in some cases and four in others, but on average over the large number of occasions analyzed, the quantity comes out to just about three. In other statements, that the free energy recovered from three hemagglutinin conformational deepens appears to be sufficient to drive the process that I was establishing you in the previous slip. So, which of the various steps in this sort of scheme are we looking at is the rate-limiting step in this sort of analysis? From the pH dependence, and I won’t go into the details, we believe that there’s an initial rapid symmetry between a protonated and unprotonated government, but that as soon as this extended intermediate constitutes, then the process is essentially irreversible. And indeed, as a result of looking at some difference of the hemagglutinin, we’re pretty convinced that it is this step that in the measured I really pictured you we’re looking at.So there happens to be a highly conserved interaction just where the synthesi peptide stows into the trimeric stalking. And mutations now, either in a wholly conserved aspartic acid or a wholly kept glycine residue that stabilising the tucking in, mutations now accelerate synthesi. And so, we make that as evidence that it is this step that we’re looking at. Now, in practice, on the surface area of the virus, there are very tightly compressed hemagglutinin molecules. There are two of them superposed on this electron micrograph. And so it is also plausible that three of these humagglutinins knotted in one region might well be the minimum needed to catalyze this fusion process. Too, because of the close-fisted packing, it’s very unlikely that in the surface of the virus the proteins can move around very much, and so again, the process is probably carried out by a local make of interactions at an feeling part between the viral sheath and the cell surface.So, these sorts of measurements plainly are just the beginning at trying to understand the details of this sort of process, but here, from now just about 50 years ago is an electron micrograph of influenza virus in what the hell is now be called an endosome, to indicate to you, to give you a bit of perspective, and to suggest to you that, from this sort of information, at a theatre when one didn’t even know what the molecules on the surface area of the virus is likely to be, we’re now at a stage … we’re at the level of dissecting the kinetics of the events and, hence, trying to understand sensitive details for neutralization by antibodies, for example. We are truly get at the molecular details of the process that would lead to the release of the nucleoproteins from inside the particle( you can actually verify some of them in cross section now probably) into the cytoplasm through synthesi of the lipid bilayer of the virus with the lipid bilayer of the endosome.I’ve mentioned Dan Floyd and Antoine van Oijen, I should mention too Tijana Ivanovic and John Skehel as traitors in the measured I’ve been exhibition you, illuminating how one can use structure and biophysical appraisals to dissect the fusion mechanism. And I should contribute further approval to Gal McGill, whose living of the HIV fusion mechanism is particularly helpful in trying to understand what we belief these synthesi proteins are doing. Thank you very much ..

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