Virus Structures
Transcript of Part 1: Virus Structures: General Principles
00:00:02.08 Hello, I'm Stephen Harrison of Harvard Medical School, 00:00:05.25 Children's Hospital Boston, and the Howard Hughes 00:00:08.05 Medical Institute. This is the first of three lectures on virus 00:00:14.13 structures. This first lecture will be about general features 00:00:18.28 of the molecular organization of virus particles. The 00:00:22.15 second two will be about specific properties of virus 00:00:26.07 particles relevant to the molecular mechanism of infecting 00:00:30.22 a cell. Viruses are carriers of genetic information from one 00:00:37.04 cell to another; in that sense, they're effectively 00:00:41.16 extracellular organelles. The infectious virus particle, 00:00:46.18 sometimes called a "virion," is a molecular machine that 00:00:50.01 packages viral genomes, escapes from the infected cell, 00:00:54.22 survives transfer from one cell to another, and attaches, 00:00:59.01 penetrates, and initiates replication in the new host cell. 00:01:03.21 It's thus not just a passive package, but rather an active 00:01:12.15 payload deliverer. Now most people know viruses as 00:01:17.17 pathogens because the virus bears the genetic 00:01:21.12 information needed to usurp the cellular biosynthetic 00:01:25.24 machinery and replicate itself. The selective advantage 00:01:31.10 for evolution of the virus may be a selective disadvantage 00:01:34.21 for the host, and as a result, hosts evolve defense 00:01:39.25 mechanisms, the immune system in the case of humans 00:01:45.08 and other higher vertebrates. Now, viruses come in two 00:01:52.17 major flavors: enveloped viruses, in which the infectious 00:01:57.22 virus particle is surrounded by a lipid bilayer membrane 00:02:02.20 derived from host cell membrane; and non-enveloped 00:02:06.27 viruses, rather unimaginatively, that have no lipid bilayer 00:02:11.12 membrane, and the protective coat is just protein. These 00:02:15.01 two structural modes correspond to different modes of exit 00:02:21.09 and entry into cells, different mechanisms of assembly, 00:02:25.13 and different mechanisms of infection, as we'll see in the 00:02:31.18 course of this lecture and the next two. Now, just as 00:02:36.17 quick examples, on the left is an example of a non- 00:02:44.12 enveloped virus particle, a rotovirus particle. This image is 00:02:49.17 based on reconstructions from many electron 00:02:55.00 micrographs, and we'll go into some of the details of that 00:02:59.12 in the third lecture. And on the other side is an example of 00:03:05.25 an enveloped virus particle, also studied by electron 00:03:11.11 microscopy, and in the cross section of the image that 00:03:16.07 you see at the bottom, you can clearly see evidence of 00:03:19.22 the lipid bilayer with α-helical segments of the protein on 00:03:25.25 the outside traversing it. Just to remind you of sizes and 00:03:33.07 distances, both the rotovirus particle and the Sindbis virus 00:03:37.24 particle, as the right-hand one was called, have outer 00:03:42.23 shells that are about 700 angstroms in diameter, or 70 00:03:46.15 nanometers. That's about a millionth the size of a tennis 00:03:50.11 ball. Recall that chemical bonds that are between one 00:03:54.23 and two angstroms in length, and that's why chemists use 00:04:01.06 angstroms rather than nanometers; it's the natural unit of a 00:04:04.26 chemical bond. So when I say 700 angstroms, you can 00:04:08.04 think of that as 500-700 atoms across. Of course it's a 00:04:13.11 volume, and so the molecular mass of these particles is 00:04:17.08 some tens of millions of daltons. So bear in mind during 00:04:23.10 this lecture the following three questions. We'll talk about 00:04:28.15 more than just these three, but the main point of the 00:04:34.00 lecture will be to try to introduce you to the following 00:04:37.26 issues. First, why most non-enveloped viruses and a 00:04:41.29 number of smaller enveloped viruses have highly 00:04:45.05 symmetric structures. Second, what do the building 00:04:49.05 blocks of these particles look like? Turns out that the 00:04:52.14 same kinds of building blocks have been used over and 00:04:54.27 over again in the evolution of different viruses, even 00:04:58.02 viruses with very different replication strategies. And 00:05:01.08 finally, what the outer proteins of some enveloped viruses 00:05:04.27 look like. So let's begin with symmetry. What does 00:05:09.20 symmetry mean? Symmetry, as suggested by the image 00:05:14.00 on the left, means that there's some operation, in the case 00:05:18.12 of physical objects some physical operation like a rotation, 00:05:22.07 that brings the object into self-coincidence. In this case, if 00:05:26.16 you rotated this figure by 120° about the axis represented 00:05:31.29 by that triangle, and you closed your eyes while you did it, 00:05:36.02 you wouldn't realize that you had done the rotation. That's 00:05:39.22 called a threefold axis, and as you can imagine, symmetry 00:05:42.27 of more complicated objects can have other symmetry 00:05:46.14 axes, and so the icosahedron (we'll come back to that in 00:05:50.09 a minute) represented on the right has fivefold axes, 00:05:53.23 threefold axes, and twofold axes of symmetry. Some 00:05:59.06 viruses have helical symmetry. Helical symmetry is 00:06:05.11 represented by a screw axis, and so tobacco mosaic 00:06:09.12 virus, which was studied historically as one of the very 00:06:13.01 first viruses for which detailed biochemistry and detailed 00:06:17.13 structure became available, is a helical array in which the 00:06:22.23 nucleic acid, the RNA, is wound into a groove on the 00:06:27.17 protein subunit and winds up with the protein, which forms 00:06:35.22 this helical array. There are a number of other helical 00:06:39.23 organizations in virus particles: Vesicular stomatitis virus is 00:06:44.16 a much more complicated enveloped virus with an outer 00:06:47.18 glycoprotein, that's what this "G" is, on the right. But as 00:06:52.13 you can imagine, helical symmetry yields elongated 00:06:57.25 particles that get unwieldy. And so far more common is 00:07:04.01 the isometric, that is, roughly spherical characteristics, of 00:07:09.19 virus particles with icosahedral symmetry. The 00:07:13.06 icosahedron, one of the Platonic solids, the fanciest one, 00:07:16.28 so to speak, with 20 triangular faces, is simply a 00:07:20.18 representation of icosahedral symmetry. An object needn't 00:07:25.17 have icosahedral shape in order to have icosahedral 00:07:29.26 symmetry. And likewise, I could destroy the symmetry of 00:07:33.13 this object by painting an asymmetric object on each 00:07:37.17 face, rather than an object with threefold symmetry. The 00:07:44.09 icosahedral symmetry is represented or characterized by, 00:07:53.09 as I said, twofold axes, fivefold axes, and threefold axes. 00:07:58.27 And if you place a single asymmetric subunit into a space 00:08:07.24 governed by icosahedral symmetry and then operate on it 00:08:11.28 with the symmetry axes, you get 59 others, that is, there 00:08:16.21 are 60 locations in all that are related to each other by 00:08:23.27 these various symmetry axes, by these various symmetry 00:08:27.00 operations. And so a particle with icosahedral symmetry 00:08:33.27 will have 60 subunits. What I've flashed in here is a sort 00:08:39.24 of schematic representation of what might be a protein 00:08:43.22 subunit, to suggest that a small particle with 60 protein 00:08:49.28 subunits appropriately interfaced with each other can form 00:08:55.02 an icosahedral structure. Now this is indeed a schematic 00:09:00.21 representation of an actual virus particle. Parvovirus is 00:09:05.14 one of the very smallest and simplest of the viruses, have 00:09:10.04 a single kind of protein subunit that forms a small shell, 00:09:18.25 and 60 of them decorate or assemble into that shell, as 00:09:24.02 suggested here. And so, if we take a slightly closer look 00:09:30.14 at that protein subunit in a traditional ribbon diagram 00:09:35.19 representing the fold of the polypeptide chain, you see 00:09:39.26 that it's based on a quite simple, compact domain 00:09:44.21 represented here in red, with large loops emanating from 00:09:49.23 it. That compact domain has this sort of fold, it's called a 00:09:55.03 "jelly roll β-barrel," or sometimes a "cupin fold," and this 00:09:59.04 particular representation comes from the structure of 00:10:03.10 canine parvovirus, a virus of dogs, as you can imagine. 00:10:08.09 But the parvovirus family includes viruses, such as adeno- 00:10:14.26 associated viruses, now being used as vectors for efforts 00:10:21.04 at gene therapy. So in this case, the simple jelly roll β- 00:10:28.16 barrel structure has been elaborated with loops in order to 00:10:33.09 make a particle of adequate size, as shown here. And 00:10:39.00 that particle can package about a 5 kilobase single- 00:10:45.22 stranded DNA genome. Since the molecular mass of the 00:10:51.14 coat protein is about 50 kilodaltons, there's just enough 00:10:55.23 volume inside to package that genome, of which about a 00:11:00.19 third is actually given over to encoding for the coat 00:11:04.11 protein. So that's a relatively expensive way of spending 00:11:08.29 your genetic information. You've got to dedicate a full 00:11:12.04 one-third just to specifying the cardboard box, if you wish, 00:11:18.18 with which you're going to deliver the payload that you 00:11:22.24 actually wish to deliver. FedEx wouldn't like a system in 00:11:27.26 which a third of the weight were in the box. So what 00:11:38.05 about trying to package larger genomes with larger 00:11:41.24 coding capacity? One example, although still by no 00:11:46.29 means as efficient as the viruses we'll start to talk about, 00:11:55.03 are the so-called picornaviruses. These are small positive- 00:11:59.04 strand RNA viruses, of which poliovirus and the human 00:12:03.10 common cold virus (human rhinoviruses) are good 00:12:06.27 examples. In this case, there are three different protein 00:12:11.26 subunits, each with one of these β jelly roll designs, very, 00:12:17.11 very similar to that red β jelly roll in the canine parvovirus, 00:12:22.21 that assemble as shown into an icosahedral structure. 00:12:27.06 And so one-sixtieth of this structure has three jelly rolls, a 00:12:32.06 red one, a blue one, and a green one, designated in that 00:12:37.10 order, VP3, VP1, and VP2, for the colors as I named 00:12:42.03 them, forming the sort of assembly that you see here. 00:12:48.03 Now, those three subunits, as I said, look strikingly like 00:12:54.22 that same β jelly roll we saw in the parvovirus subunit, but 00:12:59.29 the loops are a little less extensive, because in this case, 00:13:03.05 with three subunits, the size of the particle doesn't need 00:13:09.22 to be additionally augmented by taking up space with 00:13:13.20 those loops, and one can still package adequate 00:13:16.14 amounts of RNA. One other feature of the architecture of 00:13:21.13 this particle that's noteworthy, and we're going to see in 00:13:27.01 various forms as we look at even more complicated 00:13:30.24 viruses particles, is the nature of the interaction among 00:13:38.19 the subunits, which not only involves interfaces between 00:13:46.09 pre-folded, rigid domains of subunits, but an elaborate 00:13:53.07 inner scaffold and a little bit of an outer scaffold made by 00:13:58.17 parts of this protein's subunit that fold up only when then 00:14:04.17 particle assembles. And so on the right, you can see 00:14:06.29 some hint of this in a blow-up of VP1, VP2, and VP3, 00:14:13.02 where you can see that, in addition to the jelly roll 00:14:18.15 domains, there are extended arms, they happen to be N- 00:14:24.06 terminal and extend inward in the particle, that fold 00:14:28.10 together when the particle assembles. Now these viruses 00:14:34.15 manage to package a nine kilobase single-stranded RNA 00:14:38.07 genome, but still use about a third of the genome to 00:14:42.16 encode the coat. The same size of package can be 00:14:50.23 achieved with a single kind of subunit, if that subunit can 00:14:55.13 have multiple conformers. Why does it need to have 00:14:58.22 multiple conformers? I told you that icosahedral symmetry 00:15:05.25 requires that there be 60 and only 60 identical structures 00:15:15.11 that form an icosahedrally symmetric shell, so that if you 00:15:19.21 want to use 180 protein subunits, as in the 00:15:23.14 picornaviruses, either they have to come in three chemically distinct 00:15:29.07 kinds, three colors, if you wish, or they need to have three 00:15:34.18 distinct conformers. In this example, from a simple plant 00:15:39.08 virus called tomato bushy stunt virus, shows that indeed 00:15:44.29 one can make a very similar package with the jelly roll β- 00:15:49.17 barrels packed essentially in the same orientation and the 00:15:54.13 same packing style, so to speak, as in the picornaviruses, 00:16:01.11 but where there's only one kind of subunit, and blue, red, 00:16:05.20 and green correspond to three different conformations of 00:16:09.21 that subunit. Those conformers can be achieved by 00:16:14.11 alternate hinges between rigid domains, as shown here, 00:16:19.18 between the two different major conformations. The red 00:16:25.10 and the blue in the previous slide are actually extremely 00:16:29.10 similar to each other and would be represented by what 00:16:33.08 you see here on the right. And a second conformation not 00:16:38.18 only with a somewhat different hinge but also with an N- 00:16:43.00 terminal arm folded up in an ordered way, whereas it's 00:16:47.04 disordered and hangs into the center of the virus particle 00:16:50.15 on the other conformation. So then again one sees here 00:16:56.29 that there is an elaborate inner scaffold that dictates the 00:17:01.27 assembly formed by parts of the protein subunit that are 00:17:08.17 not rigidly folded, are not ordered, until the assembly 00:17:13.24 comes together. We can have a look at that scaffold, it's 00:17:18.00 instructive, in this blow-up of the particle by just focusing 00:17:23.24 on those 60 of the 180 subunits that have an ordered 00:17:28.11 arm. And if we now focus in on that array of protein 00:17:36.10 subunits, 60 of the 180, and look over here at where 00:17:41.23 three of them interact at a threefold axis of the 00:17:45.27 icosahedral symmetry, you see that there's an inner 00:17:49.10 scaffold formed by the N-terminal arms of the protein 00:17:52.22 subunit that dictates the size and characteristic of the 00:17:59.25 whole assembly. These arm-like extensions, which fold 00:18:05.02 together to form an inner scaffold, also form flexible links 00:18:08.19 to the RNA. This is a good example of the undemanding 00:18:14.07 packaging of a genome, as I like to call it. If the package 00:18:20.04 required either a specific nucleotide sequence for a lot of 00:18:25.14 the RNA, or a defined structure in three dimensions, let's 00:18:30.22 say, for the RNA, then the RNA could not evolve to 00:18:34.19 encode the other functions that matter: an RNA- 00:18:39.21 dependent RNA polymerase, for example. And so 00:18:44.03 packaging of nucleic acids in viruses like these involve 00:18:49.25 both a short packaging sequence or packaging signal 00:18:57.20 that is recognized by a few copies of the protein subunit 00:19:03.14 and that can act as an assembly origin; and then a large 00:19:07.21 of nonspecific, charge-neutralizing interactions to 00:19:11.07 condense the RNA into the center of the particle. And so 00:19:15.19 in the case of tomato bushy stunt virus, at the tip of the 00:19:19.00 arm is a very positively charged polypeptide segment that 00:19:25.14 condenses the RNA and neutralizes the strong negative 00:19:30.02 charge on the phosphates. The specific recognition 00:19:36.15 interactions in the case of bushy stunt virus, we don't 00:19:40.05 have a picture of, but we do have a picture of how that 00:19:44.05 same part of the arm recognizes a specific packaging 00:19:49.24 sequence in the case of a related plant virus called alfalfa 00:19:54.03 mosaic virus. And in that case, a positively charged, 25- 00:19:59.13 residue-or-so, N-terminal segment that is not well ordered 00:20:07.15 on the protein subunit as it folds on its own, co-folds with 00:20:14.05 a short packaging sequence represented here by a 00:20:20.07 standard two-dimensional sequence representation of two 00:20:24.02 stem-loops. And the three-dimensional structure 00:20:28.28 represented here leads to a specific recognition, because 00:20:34.28 the stem-loops form a defined three-dimensional 00:20:38.29 interaction stabilized by their folding together with the N- 00:20:48.04 terminal arm of a small number of subunits. Probably one 00:20:52.22 dimer is responsible for recognizing this pair of stem-loops, 00:20:57.18 and the full packaging sequence might have three such 00:21:00.26 pairs and three dimers, but the protein shell is composed 00:21:07.12 of a much larger number, and the all remaining protein 00:21:09.28 subunits will have nonspecific, positively charged, 00:21:16.12 charge-neutralizing interactions with the RNA. Now, let's 00:21:22.14 go on and talk about still larger and more complicated 00:21:25.11 virus particles. Here's a representation, a surface 00:21:28.20 representation, of a papillomavirus. Papillomaviruses 00:21:34.09 cause warts and, in some cases, cancer in humans and 00:21:41.12 many other animals. The recently introduced vaccine 00:21:46.22 against human papillomavirus 16, 18, and one or two 00:21:50.28 other types, is a vaccine that prevents transmission of the 00:21:56.03 virus, which causes cervical cancer. So this surface 00:22:01.05 representation shows you that these viruses, which 00:22:05.00 package a double-strand DNA genome, are based on an 00:22:13.07 assembly of pentameric building blocks. In this case, the 00:22:20.11 pentameric building blocks are positioned not only at 00:22:26.22 positions of fivefold symmetry in this icosahedral shell, but 00:22:31.29 also at a general nonsymmetrical position so that this 00:22:37.09 pentamer is actually surrounded by six other pentamers. A 00:22:42.02 fivefold peg in a sixfold hole, so to speak. This sort of 00:22:48.24 assembly can nonetheless be stabilized by the same sorts 00:22:53.09 of principles that we've seen in the simpler viruses, 00:22:57.04 namely, the tying together of rigid or relatively rigid building 00:23:02.20 blocks by flexible, and hence potentially multidirectional, 00:23:11.00 arms. So here the pentameric assembly of the protein L1 00:23:17.22 that forms this structure is represented here, and as you 00:23:22.27 see, there are loops coming out of it with dotted lines 00:23:26.22 here, that form the interactions between the pentamers 00:23:33.08 shown here. And of course, since this is a fivefold peg in 00:23:36.19 a sixfold hole, its arms have to be directed in different 00:23:41.18 ways, but the pentamer itself is a rigid, fivefold-symmetric 00:23:47.01 object, just like its chemically identical mate here on a 00:23:53.20 fivefold position. Now, this subunit, the L1 subunit, is also 00:24:01.27 based on the same sort of β jelly roll building block that 00:24:06.15 we saw in the positive strand RNA viruses that we were 00:24:11.05 just talking about, and it's elaborated by various loops that 00:24:17.19 vary from virus type to virus type, just one of the reasons 00:24:24.08 that these viruses come in a great variety of serotypes, of 00:24:31.25 immunologically distinct types, because these loops, 00:24:36.19 which are on the outside of the virus particle (that is, this 00:24:39.24 is the part of the pentamer that faces outward, and this is 00:24:46.11 the part that would face inward, this would be the inside 00:24:49.13 of the virus, this the outside of the virus)... these loops are 00:24:53.25 free to vary evolutionarily because they're not so critical 00:24:59.07 for the formation of the stable assembly or for forming the 00:25:05.04 rigid pentamer, and hence can respond, if you wish, to 00:25:10.05 the pressures of their coevolution with the human immune 00:25:16.17 response, or the immune response of the particular animal 00:25:19.28 that they infect. Now, a similar principle, if you wish, 00:25:29.14 namely, reuse of the same kind of building block but in 00:25:35.01 environments that don't have a simple symmetry, is 00:25:43.02 exemplified by the adenoviruses. These are even much 00:25:46.27 larger structures, and I will try to make a few points by 00:25:53.06 talking about the adenovirus structure. The particle has a 00:25:58.28 strikingly icosahedral shape with fibers coming out of the 00:26:04.13 fivefold positions that are responsible for cell attachment. 00:26:08.20 The main part of the coat is represented by a protein 00:26:12.29 called "hexon" because it forms these sorts of 00:26:16.14 hexagonally packed arrays, but in fact the hexon is not a 00:26:19.20 hexamer, it's a trimer. It's a trimer, however, with two of 00:26:24.05 these β jelly roll domains, rather similar in their overall 00:26:28.00 shape, next to each other, so it has a kind of hexagonal 00:26:31.14 outline. As a result, the face of the icosahedron does 00:26:39.13 have threefold symmetry, and the whole structure has 00:26:42.20 threefold symmetry, but the hexon itself actually is only a 00:26:49.15 threefold, and not a sixfold, symmetric entity. Now one 00:26:54.25 quite interesting aspect of the structure here is that there 00:27:01.26 is a bacteriophage called PRD1 (and indeed several 00:27:05.13 other bacteriophages now known) that has essentially 00:27:09.19 exactly the same design. Adenoviruses are viruses of 00:27:13.11 humans and vertebrates and actually a large number of 00:27:17.06 other animal species, so with this structure, one can make 00:27:25.08 the point that even viruses of bacteria have strong 00:27:32.23 resemblances in their design to those of humans and 00:27:41.20 plants, for that matter. Indeed you saw similarity between 00:27:45.19 the plant viruses, like tomato bushy stunt virus, and the 00:27:51.08 picornaviruses, such as polio and the human common 00:27:54.02 cold virus. This doesn't mean, in my own view, that these 00:27:59.10 viruses are so ancient, if you wish, in their design, in their 00:28:05.12 structure, that they antedated the divergence of bacteria 00:28:10.17 and animals, or animals and plants. Rather, we know that 00:28:15.11 viruses can jump species. They can jump from insects... 00:28:19.20 indeed, there are viruses that infect both insects and 00:28:22.28 people, and there viruses that infect both insects and 00:28:26.29 plants. And so the transfer of genetic information that I 00:28:31.17 alluded to at the very beginning of the talk, the notion that 00:28:35.06 a virus particle is package that gets genetic material from 00:28:41.09 one kind of cell to another, may well be true not just for 00:28:45.13 the cells within you or between you and another individual 00:28:50.23 of the same species, but across species. We know that 00:28:55.05 flu jumps from swine to people, as we all learned from the 00:28:59.09 2009 pandemic, or from birds to people. But also, 00:29:05.09 ultimately, through eons of time, from one kingdom to 00:29:10.24 another. At any rate, it does means that the structures 00:29:15.20 we're talking about show a striking similarity and a striking 00:29:19.11 unity, whatever the evolutionary details. In the case of the 00:29:24.04 adenoviruses, the subunit on the fivefold axis is a different 00:29:31.27 protein subunit from the hexon. It's got one β jelly roll 00:29:37.10 domain instead of two, so that again there's a kind of 00:29:42.21 duplication and elaboration as this structure develops into 00:29:48.29 a much larger shall to package, in this case, a 35 00:29:54.01 kilobase-pair, double-strand DNA genome, much, much 00:29:58.04 larger genome. And indeed there are viruses based on 00:30:02.09 very similar kinds of protein subunits, the same double jelly 00:30:07.08 roll structure with a separate, related but genetically and 00:30:13.02 chemically distinct, single jelly roll pentamer on the fivefold 00:30:17.20 axes. There are even much larger viruses based on this 00:30:23.25 kind of subunit. Now, an interesting point in relating the 00:30:31.02 adenovirus structure to the bacteriophage that I 00:30:36.21 mentioned is based on or has a similar kind of major outer 00:30:44.00 shell subunit, are the mechanisms by which the virus 00:30:53.16 forms a defined and specific structure. As you can 00:30:56.27 imagine, in this sort of structure, how in the course of 00:31:00.26 assembly is the relationship between one fivefold position 00:31:06.03 and another fivefold position determined? How is the size 00:31:09.22 of this structure determined, rather than allowing, let us 00:31:14.19 say, multiple hexons to start forming much bigger and 00:31:18.11 bigger triangles? In the case of the phage, the answer is 00:31:26.15 particularly simple. One stripped off the outer shell of 00:31:31.15 hexon-like subunits and penton-like subunits, and 00:31:34.27 discovered that, from the x-ray crystal structure of this 00:31:38.22 particle, that there is an extended protein called a "tape 00:31:44.11 measure" protein by the investigators who discovered 00:31:47.14 this, that in effect stretches from a fivefold position here to 00:31:54.17 a twofold position here and then meets another one, 00:31:57.28 twofold symmetric to the next fivefold, and that 00:32:01.15 organization, think of the scaffolds that we talked about 00:32:06.28 before, governs the fixed size of the particle. In this case, 00:32:12.27 the scaffold protein is not an arm of the same protein 00:32:17.15 subunit, it's a separate protein, but the same principle 00:32:21.25 applies, and likewise, in the adenovirus particle, there are 00:32:26.12 several different so-called "glue" or "cement" proteins 00:32:30.19 that form, in effect, a scaffold that knits together the 00:32:34.18 structure in a way that leaves no ambiguity for the size 00:32:44.07 and characteristic of the final particle. In all of these 00:32:50.24 structures, the papillomaviruses, the adenoviruses, the 00:32:55.15 picornaviruses, the plant viruses such as tomato bushy 00:33:02.21 stunt, we see a simple construction principle at work that 00:33:09.16 is a little bit like an assembly line, like a factory assembly 00:33:13.23 line. There is in all cases a fixed assembly unit, happens 00:33:19.03 to be a dimer in the case of the coat protein of TBSV. 00:33:24.05 You saw that it was a pentamer in the case of the L1 00:33:26.21 protein of the papillomaviruses. The same of the 00:33:30.14 polyomaviruses like SV40. And you saw that the 00:33:35.04 adenovirus hexon, the trimeric adenovirus hexon, is 00:33:38.24 likewise a mass-produced assembly unit. But in order to 00:33:44.18 determine how that mass-produced assembly unit fits into 00:33:49.16 a defined structure of larger size, how the positioning of 00:33:57.18 that subunit doesn't simply lead to errors in the building of 00:34:03.09 a larger or smaller particle, there's a framework or scaffold 00:34:07.07 just as in the construction of a building, let's say, that 00:34:10.10 ensures accurate placement of these mass-produced 00:34:15.15 assembly units. And we've also seen that, interestingly 00:34:19.07 enough, there's a recurring architectural motif that has 00:34:23.17 appeared in the evolution of these structures (and it's a 00:34:26.09 complicated one, so it probably evolved only once) over 00:34:30.21 and over again. Now you might well ask, is this the only 00:34:34.22 architectural motif? Why are all viruses based on a so- 00:34:41.12 similar building block, and the answer is, that isn't the 00:34:44.00 case. There's at least one other, and that sometimes is 00:34:47.09 called the "HK97" fold, after the bacteriophage HK97 in 00:34:55.05 which it was discovered. You can see that this protein 00:34:57.13 subunit looks quite different, it's got some α-helices, it's a 00:35:00.20 somewhat irregular-looking structure, and it's found in the 00:35:07.12 bacteriophage P22 and a large number of other double- 00:35:11.01 strand DNA bacteriophage, where it forms a shell with a 00:35:22.10 number of these subunits forming both hexamers and 00:35:25.15 pentamers, so that there are 60 hexamers and 12 00:35:30.14 pentamers (there are always 12 pentamers in any 00:35:33.12 icosahedral structure), as suggested here. These viruses 00:35:39.03 assemble with an inner scaffold, but the scaffold in this 00:35:43.16 case is discarded by proteolytic digestion in some cases. 00:35:48.24 In this case, it's actually reused; it exits from the particle 00:35:53.08 and gets reused in the case of P22. And the particle then 00:36:00.24 changes some details of its organization as the scaffold 00:36:04.27 exits, as part of the process by which the double-strand 00:36:09.18 DNA is injected, actually pumped, if you wish, into the 00:36:14.20 particle at the next stage in assembly. So these are cases 00:36:19.02 in which the shell preassembles around a scaffold. The 00:36:23.20 scaffold is ejected, either chewed up or literally ejected 00:36:31.17 and reused, and a series of events involving motor 00:36:36.10 proteins are responsible for inserting DNA into these 00:36:42.22 structures. Now you could ask whether this is true only of 00:36:48.16 bacteriophage, answer: "no." You might anticipate that 00:36:52.09 the answer would be no from what I told you about 00:36:55.04 adenovirus and PRD1 for example. Here are two 00:37:01.07 bacteriophage protein subunits that have this sort of 00:37:05.04 structure, but the herpesviruses, of which the herpes 00:37:09.05 simplex 1, the cold sore virus, is one example, are based 00:37:15.24 on a much more elaborately looped, elaborately 00:37:21.13 decorated, version of the same fundamental fold. The 00:37:26.04 structure that we have at the moment is from electron 00:37:28.25 microscopy and not yet at the same resolution that the x- 00:37:33.04 ray structures of these subunits have yielded, but you can 00:37:37.29 probably see in this relatively low resolution representation 00:37:43.03 of the herpesvirus that this part, for example, there's a 00:37:47.20 long α-helix, corresponds to the much simpler, 00:37:53.03 undecorated fold you see here. And then these are loopy 00:37:56.04 structures that stick out and make the protein subunit 00:37:59.10 much larger and have to do with other interactions that 00:38:03.11 the protein subunit of the herpes particle makes. The 00:38:07.12 herpesvirus particle is more complicated, it's both larger 00:38:09.27 and more complicated than the phage particles, and so 00:38:13.25 there are other interactions of those surface loops that are 00:38:18.18 important. Herpesviruses like the phage have very tightly 00:38:25.20 coiled DNA that is inside, that's pumped into them in this 00:38:31.23 reconstruction from electron cryomicroscopy, you can 00:38:34.23 actually see the coiling of the DNA. The DNA is actually 00:38:39.20 coiled this way, that is, circumferentially about the axis of 00:38:45.00 the particle, it's injected through one vertex, and as you 00:38:48.09 see, there's a specialized internal structure here, to which 00:38:53.19 then the tail of the phage that ultimately injects it back 00:38:58.04 into a new host cell, is attached. The cross section here 00:39:04.21 looks as if you have circumferential layers of density in the 00:39:12.12 other direction because, as you can see from this 00:39:15.05 diagram, DNA coiled about a vertical axis, if the order is 00:39:25.23 such that, from particle to particle, there isn't exactly a 00:39:29.16 piece of DNA here, but this one might be here or here, 00:39:34.10 then on average, you will get radial shells of density as 00:39:40.07 you see here, fitting tightly into the interior of the particle, I 00:39:46.03 like to say, with a gardener's analogy (it might not be 00:39:50.10 relevant for all people listening), like winding a hose into a 00:39:54.06 hose pot, or rope into a bucket. Now finally, let's talk a 00:40:01.26 little bit about enveloped viruses. Enveloped viruses 00:40:08.25 acquire their envelope, in general, their membrane (this is 00:40:12.04 not true of all enveloped viruses, but true of almost all of 00:40:19.08 them), by budding out of the cell, either out of the cell 00:40:22.22 surface, or into an intracellular compartment such as the 00:40:29.03 endoplasmic reticulum or the Golgi apparatus, and then 00:40:33.16 being transported out. And in that budding process, wrap 00:40:38.19 themselves, if you wish, in a membrane that's derived from 00:40:42.23 host cell lipids, although host cell proteins are in general 00:40:47.21 excluded. Some of the smaller enveloped viruses have 00:40:53.21 icosahedral symmetry, and their structure and assembly is 00:40:58.15 determined by regular interactions within an icosahedral 00:41:03.08 shell, just as the ones you've seen in the non-enveloped 00:41:06.26 viruses. But larger and less regular enveloped viruses are 00:41:12.08 also seen, such as HIV or influenza, in which the protein 00:41:21.12 interactions are less perfect, but that doesn't matter for 00:41:26.11 protecting the nucleic acid nearly so much, because the 00:41:30.03 lipid bilayer in effect is an impermeable barrier against 00:41:35.07 agents that might get in and degrade or damage or 00:41:41.00 cleave the nucleic acid. So the budding process that I 00:41:48.06 mentioned can either involve, as in the case of the so- 00:41:52.02 called alphaviruses, or which Sindbis virus is one of the 00:41:56.18 prototypes and well studied... and a recent human 00:42:02.01 outbreak of an alphavirus is the chikungunya virus, which 00:42:07.03 had a major outbreak in the French island of Réunion, 00:42:12.22 and led to considerable interest and publicity about the 00:42:16.21 properties of that virus. The alphaviruses have a core that 00:42:22.25 preassembles in the cytoplasm and then two species of 00:42:28.05 glycoprotein that are synthesized on the rough ER, 00:42:33.29 exported to the cell surface, and then the particle buds 00:42:39.03 out through a process by which the inward-directed C- 00:42:47.01 terminal tips of the glycoprotein, which stick through the 00:42:52.09 membrane in a single α-helical segment (you might 00:42:55.04 remember very early on, I showed you a cross section 00:42:58.05 that showed that), interact one-to-one with the 00:43:05.04 icosahedrally symmetric core that's assembled rather like 00:43:09.21 a non-enveloped virus in the cytoplasm, and buds out. In 00:43:16.07 other cases, such as influenza, there's no preassembled 00:43:21.03 inner particle, but rather, the assembly occurs at the 00:43:24.20 membrane, as you see here, where the inner structures 00:43:28.18 and the glycoproteins that incorporate in the membrane 00:43:33.20 come together as part of the elaborate budding event. 00:43:38.17 Separate cellular machinery, in some cases, is then 00:43:42.05 needed to the finish the pinching off, whereas these 00:43:44.27 viruses don't seem to need a separate pinching off 00:43:47.23 mechanism. In the case of HIV, these micrographs show 00:43:54.15 particularly dramatic examples of HIV budding. It's 00:43:58.27 directed in this case by the interaction of the N-terminal 00:44:04.21 domain of the inner protein, the so-called "Gag" gene 00:44:09.10 product, and that protein has a myristoyl group at its N 00:44:17.06 terminus and a very positively charged surface, and 00:44:22.10 interacts with the membrane to drive budding, as shown 00:44:25.20 here. In these micrographs, you can see that the HIV 00:44:33.01 particle is rather sparsely decorated with an envelope 00:44:38.25 glycoprotein that has the function of attaching the virus 00:44:44.17 particle to a new host cell and mediating viral entry. In the 00:44:51.14 case of the smaller icosahedrally symmetric enveloped 00:44:57.11 viruses, like dengue virus for example, the outer coat is 00:45:03.29 much more tightly packed, it forms a very regular array, in 00:45:08.14 case with 180 subunits of the protein whose structure is 00:45:14.21 shown up here, forming a perfect icosahedral array, and it 00:45:18.26 is an assembly of that array that drives particle budding. In 00:45:27.23 all cases of enveloped viruses, the entry process (and 00:45:33.21 that will be the topic of the next part of this set of lectures) 00:45:39.28 involves fusion of the viral membrane with a membrane of 00:45:44.17 the host cell. So just as the assembly process, the 00:45:48.24 maturation process, the exit process, involved budding 00:45:53.19 out and pinching off, so entry involves the reverse 00:45:58.15 process: attachment and fusion of the two membranes. 00:46:03.17 We'll talk about fusion in great detail in the next part of 00:46:07.02 this series, but just to give you a hint of what's to come, 00:46:10.20 an important of all of these viral envelope proteins is that, 00:46:16.17 under suitable circumstances, they can be triggered to 00:46:20.24 undergo a major conformational rearrangement. It's that 00:46:25.26 rearrangement that drives the fusion event, so that in the 00:46:31.14 case of the dengue virus particle, there is a 00:46:33.24 rearrangement from the dimeric structure shown here, a rather plate-like 00:46:42.14 organization of two somewhat elongated protein subunits, 00:46:48.27 into a trimer in which hydrophobic residues at the tip of 00:46:54.05 one of the domains, this yellow domain, so-called "domain 00:46:57.05 II," cluster together at one end of the trimer and interact 00:47:02.18 with the target cell membrane in order to begin the 00:47:06.03 process by which the two membranes are brought 00:47:09.03 together. In the case of dengue virus, this conformational 00:47:13.10 change is triggered by proton-binding, a signal that the 00:47:17.26 virus has arrived in the low pH compartment of an 00:47:21.19 endosome. In other cases, other signals are read out, so 00:47:27.00 to speak, by the fusion mechanism. We can look at this in 00:47:36.00 one more slide, where the interaction with the target cell 00:47:40.13 membrane is shown, and there is a zipping-up process of 00:47:44.08 the C-terminal part of the subunit that actually is part of 00:47:49.28 the pinching together of the two membranes, and leading 00:47:54.10 to an elaborate bit of molecular machinery. Now not all 00:48:04.06 enveloped glycoproteins form such a regular array. In the 00:48:08.11 case of the influenza virus particle, the proteins on the 00:48:13.03 surface of the virus particle sticking out from the 00:48:17.15 membrane are rather spike-like. There are two of them, as 00:48:22.14 you probably know, or two species, the hemagglutinin 00:48:26.06 and neuraminidase, the "H" and "N" of H1N1 or H5N1, 00:48:32.18 that you read about when pandemics threaten. The 00:48:38.12 hemagglutinin is the protein that undergoes a low pH- 00:48:44.16 triggered conformational rearrangement to drive fusion. 00:48:50.05 We'll be hearing quite a lot about that in the next part. 00:48:54.17 The hemagglutinin shown here is a spike-like structure as 00:48:58.13 I mentioned, its molecular design doesn't look anything 00:49:02.10 like that of the envelope protein of dengue virus, it's a 00:49:08.14 stalk-like structure, and long α-helices project the 00:49:19.04 receptor-binding site at the top about 120 or 130 00:49:24.23 angstroms away from the membrane. We'll use that 00:49:30.13 structure to discuss fusion mechanisms in much more 00:49:33.22 detail in Part 2 of this series. See you then. 00:49:40.17
