hello I'm Steven Harrison of Harvard Medical School Children's Hospital Boston and the Howard Hughes Medical Institute this is the first of three lectures on virus structures this first lecture will be about General features of the molecular organization of virus particles the second two will be about specific properties of virus particles relevant to the molecular mechanism of infe in a Cell viruses are carriers of genetic information from one cell to another in that sense they're effectively extracellular organel the Infectious virus particle sometimes called a virion is a molecular machine that packages viral genomes escapes from the infected cell survives transfer from one cell to another and attaches penetrates and initiates replication in the new host cell it's thus not just a package a passive package but rather uh an active payload deliverer now most people know viruses as pathogens because the virus Bears the genetic information needed to user the cellular biosynthetic machinery and replicate itself The Selective advance Vantage for evolution of the virus may be a selective disadvantage for the host and as a result hosts evolve defense mechanisms the immune system in the case of um humans and other higher uh vertebrates now viruses come in two major flavors envelope viruses in which the Infectious virus particle is surrounded by a lipid bilayer membrane derived from a host cell membrane and non-enveloped viruses rather unimaginatively uh that have no lipid bil layer membrane and the protective code is just protein these two structural modes correspond to different modes of exit and entry into cells different mechanisms of assembly and different mechanisms of infection as we'll see uh in the um in the course of this lecture and the next two now uh just as quick examples on the right is an example of a uh non- envelope virus particle a rotavirus particle this uh image is based on uh reconstructions from many electron micrographs and we'll go into that in the some of the details of that in the third lecture and on the other side uh is an example of a of of an envelop virus particle also studied by electron microscopy and in the cross-section of the image that you see at the bottom you can clearly see evidence of the lipid bilayer with Alpha helical segments of the protein on the outside traversing it just to remind you of sizes and distances both the rotavirus particle and the synbus virus particle as the um right hand one what's called um have outer shells that are about 700 angstroms in diameter or 70 nanometers that's about a millionth the size of a tennis ball recall that chemical bonds are between one and two angstroms in length and uh that's why chemists use angstroms rather than nanometers it's the natural unit of a chemical bond so when I say 700 angstroms you can think of that as 500 to 700 atoms across of course it's a volume and so the molecular mass of these particles is some tens of millions of uh of Daltons so bear in mind during this lecture um the following three questions will talk about more than just these three but the main point of the lecture will be to try to introduce you to uh the following issues first why most non- envelope viruses and a number of smaller envelope viruses have highly symmetric structures second what do the building blocks of these particles look like turns out that the same kinds of building blocks have been used over and over again in the evolution of different viruses even viruses with very different replication stru strategies and finally what do the outer proteins of some envelope viruses look like so let's begin with symmetry what does symmetry mean symmetry as suggested by the image on the left means that there's some operation in the case of physical objects some physical operation like a rotation that brings the object into self coincidence in this case if you rotated this figure by 120° is about the axis represented by that triangle and you closed your eyes while you did it you wouldn't realize that you had done the rotation that's called a three-fold axis and as you can imagine symmetry of more complicated objects can have other symmetry axes and so the icosahedron we'll come back to that in a minute represented on the right has fivefold axes three-fold axes and two-fold axes of symmetry some viruses have helical symmetry helical symmetry is is uh represented by a screw axis and so tobacco mosaic virus which was studied historically is one of the very first viruses for which detailed biochemistry and detailed structure became available is a helical array in which the nucleic acid the RNA is wound into a groove on the protein subunit and uh and and winds up with the protein which forms this helical array there are number of other helical organizations in virus particles vascular stomatitis virus is much more complicated envelop virus with an outer glycoprotein that's what this G is uh on the right but as you can imagine helical symmetry uh yields elongated particles that get unwieldy and so far more common is the isometric that is roughly spherical characteristics of virus particles with icosahedral symmetry the icosahedron one of the platonic solids the fanciest one so to speak with 20 triangular faces is simply a representation of icosahedral symmetry an object needn't have icosahedral shape in order to have icosahedral Symmetry and likewise I could destroy the symmetry of this object by painting an asymmetric object on each face rather than an object with three-fold symmetry the icosahedral Symmetry uh is uh represented or characterized by as I said two-fold axes fivefold axes and three-fold axes and if you place a single asymmetric unit asymmetric subunit into a space governed by icosahedral Symmetry and then operate on it with the Symmetry axes you get 59 others that is there are 60 locations in all that are related to each other by these various symmetry axes by these various symmetry operations and so a particle with with icosahedral symmetry will have 60 subunits what I've flashed in here is a sort of uh schematic representation of what might be a protein subunit to suggest that a small particle with 60 protein subunits appropriately interfaced with each other can um form a nedal structure now this is indeed a um schematic representation of an actual virus particle parva virus is one of the very smallest and simplest of the viruses um have a single protein kind of protein subunit uh that uh forms uh a small shell and 60 of them decorate that or or assemble into that shell as suggested here and so uh if we take a slightly close closer look at that protein subunit in a traditional ribbon diagram representing the fold of the polypeptide chain you see that it's based on a quite simple compact domain represented here in red with large Loops emanating from it that compact domain has this sort of fold it's called a jelly roll beta Barrel or sometimes a cupin fold and this particular representation comes from the structure of Kine parvo virus a um a virus of dogs as you can imagine but uh the parva virus family includes viruses now being used such as adino Associated viruses now being used as vectors for um efforts at gene therapy so uh in this case the simple jelly roll beta Barrel structure has been um elaborated with loops in order to make a particle of adequate size as shown here and that particle can um package about a five kilobase single stranded DNA genome since the molecular mass of the code protein is about 50 kilodaltons uh there's just enough volume inside to package that Genome of of which about a third is actually given over to encoding for the coat protein so that's a relatively expensive way of spending your genetic information you've got to dedicate a full onethird just to specifying the cardboard box if you wish with which you're going to deliver the uh the payload that you actually wish to uh to deliver FedEx wouldn't uh like a system in which uh fully uh a a third of the the weight were uh in in the Box um so what about trying to package larger genomes with larger coding capacity one example although still by no means as efficient and as uh as as the uh viruses will start to talk about uh are the so-called PNA viruses these are small positive strand AR RNA viruses of which polio virus and the human common cold virus human Rhino viruses are good examples in this case there are three different protein subunits each with one of these beta jelly roll designs very very similar to that red beta jelly roll in the Kine porvo virus that assemble as shown into icosahedral structure and so 160th of this structure has has three jelly rolls a red one a blue one and a green one uh designated in that uh order vp3 vp1 and vp2 for the colors as I named them uh forming the sort of assembly that you see here now uh those three subunits as I said look strikingly like that same beta jelly roll we saw in the parav virus subunit but the loops are a little less extensive because in this case with three subunits um the uh the size of the particle doesn't need to be additionally augmented by taking up space with those loops and one can still package adequate amounts of RNA one other feature of the architecture of this particle that's noteworthy and we're going to see in various forms as we look at even more complicated virus particles is the nature of the interaction among the subunits which not only involves interfaces between prefolded rigid domains of subunits but an elaborate inner scaffold and a little bit of an outer scaffold made by part of this protein subunit that fold up only when the particle assembles and so on the right you can see some hint of this in a blowup of vp1 vp2 and vp3 where you can see that in addition to the uh the the jelly roll domains there are extended arms they happen to be uh n terminal and extend inward in the particle that fold together when particle assembles now these viruses manag to package a 9 kilobase single stranded RNA genome but still use about a third of the genome to encode the coat the same size of package can be achieved with a single kind of subunit if that subunit can have multiple conformers why does it need to have multiple conformers forers I told you that icosa hedral symmetry requires that there be ex 60 and only 60 identical structures uh that form an icosahedral symmetric shell so that if you want to use 180 protein subunits as in the picor viruses either they have to come in three chemically distinct kinds three colors if you wish or they need to have three distinct conformers this example from a simple plant virus called tomato bushy stunt virus shows that indeed one can make a very similar package with the Jelly Roll beta barrels packed essentially in the same orientation and the same uh packing style so to speak as in the picorna viruses but where there's only one kind of subunit and blue red and green correspond to three different conformations of that subunit those conformers can be achieved by alternate hinges between rigid domains as shown here between the um two different U major conformations the uh red and the blue in the previous slide are actually extremely similar to each other and would be represented by what you see here on the right and um a second conformation not only with a somewhat different hinge but also with an end terminal arm folded up in an ordered way whereas it's disordered and hangs into the center of the virus particle on uh the other conformation so that again one sees here that there is an elaborate inner scaffold that dictates the assembly formed by parts of the protein subunit that are not rigidly folded are not ordered until the assembly comes together we can have a look at that scaffold it's instructive in this um blow up of the particle by just focusing on those 60 of the 180 subunits that have an ordered arm and if we now just uh focus in on that array of protein subunits 60 of the 120 and look over here at where three of them interact at a three-fold axis of the icosahedral Symmetry you see that there's an inner scaffold formed by the end terminal arms of the protein subunit that uh dictates the size and characteristic of the whole assembly these arm-like extensions which fold together to form an inner scaffold also form flexible links to the RNA this is a good example of the undemanding packaging of a genome as I like to call it if the package required either a specific nucleotide sequence for a lot of the RNA or a defined structure in three dimensions let's say for the RNA then the RNA could not evolve to encode the other functions that matter an RNA po dependent RNA polymerase for example and so packaging of nucleic acids in viruses like these involve both a short packaging sequence or packaging signal that is recognized by a few copies of the protein subunit and that can act as an assembly origin and then a large number of non-specific charge neutralizing interactions to condense the RNA into the center of the particle and so in the case of tomato bushy stunt virus at the tip of the arm is a very positively charged polypeptide segment that condenses the RNA and neutralizes the uh strong negative charge on the phosphates the specific interactions in the case the specific recognition interactions in the cas case of bushy stunt virus we don't have a picture of but we do have a picture of how that same part of the arm recognizes a specific packaging SE sequence in the case of a related plant virus called alphalpha mosaic virus and in that case a positively charged 2 residue or so n terminal segment that is not well ordered on the protein subunit as it folds on its own co- folds with a short packaging sequence represented here by uh a standard two-dimensional sequence representation of two stem loops and the three-dimensional structure represent presented here leads to a specific recognition because the stem Loops form a defined three-dimensional interaction stabilized by their folding together with the inter terminal arm of a small number of subunits probably one dier is responsible for recognizing this pair of stem loops and the full packaging sequence might have three such Pairs and three dimers but the protein the the uh protein shell is composed of a much larger number and all the remaining protein subunits will have non-specific positively charged uh charge or charge neutralizing interactions with the RNA now let's go on and talk about still larger and more complicated virus particles here's a representation a surface represent presentation of a papiloma virus papiloma viruses cause warts and in some cases cancer in humans and uh many other animals uh the recently introduced vaccine against human papiloma virus 16 18 and one or two other types is a vaccine um that prevents transmission of the virus which causes cervical cancer so this surface representation shows you that these viruses which package a double strand DNA genome are based on an assembly of pentameric building blocks in this case the pentameric building blocks are positioned not only at positions of five-fold symmetry in this icosahedral shell but also at a general nonsymmetrical position so that this pentamer is actually surrounded by six other pentamers a five-fold peg in a six-fold hole so to speak this sort of assembly can nonetheless be stabilized by the same sorts of principles that we've seen in the simpler viruses namely the tying together of rigid or relatively rigid building blocks by flexible and hence um uh mult potentially multi-directional arms so here the pentameric um assembly of the protein L1 that forms this structure is represented here and as you see there are Loops coming out of it with dotted lines here that form the interactions between the pentamers shown here and of course since this is a five-fold peg in a sixfold hole its arms have to be directed in different ways but the pentamer itself is a rigid five-fold symmetric object just like its identical chemically identical mate here on a five-fold position now this um subunit the L1 subunit is also based on the same sort of beta jelly roll building block that we saw in the positive strand RNA viruses that we were just talking about and it's elaborated by various Loops that vary from virus type to virus type is one of the reasons that these viruses come in a great variety of of serotypes of immunologically distinct types because these Loops which are on the outside of the virus particle that is this is the part of the uh the pentamer that faces outward and this is the part that would face inward this would be the inside of the virus this the outside of the virus these Loops are free to vary evolutionarily because they're not so critical for the formation of the stable assembly or for forming the rigid pentamer and hence can respond if you wish to the pressures of um their co-evolution with the human immune response or the immune response of the particular animal that they infect now a similar um principle if you wish namely reuse of the same kind of building block but in in environments that don't have um a simple symmetry is exemplified by the adov viruses these are even much larger um structures and I will try to make a few points by talking about the adav virus structure the particle has a strikingly icosahedral shape with fibers coming out of the five-fold positions that are responsible for cell attachment the main part of the coat is represented by a protein called hexon because it forms these sorts of hexagonally packed arrays but in fact the hexon is not a hexamer it's a trimer it's a Trier however with two of these beta jelly roll domains rather similar in their overall shape next to each other so it has a kind of hexagonal outline as a result the face of the icosahedron does have three-fold symmetry and the whole structure has three-fold symmetry but the hexon itself actually uh is a three-fold is only a three-fold and not a six-fold symmetric entity now one quite interesting aspect of the uh the structure here is that there is a bacteria Fage called prd1 indeed several other bacteria fages now known that has essentially exactly the same design adov viruses are viruses of humans and vertebrates and actually a large number of other animal species so that that with this structure one can make the point that even uh viruses of bacteria have strong resemblances in their design to um those of of humans and and um and and plants for that matter indeed you saw a similarity between the plant viruses like tomato bushy stunt virus and the um the picorna viruses such as polio and the human common cold virus this doesn't mean in my own View that these viruses are so ancient if you wish in their design in their in their structure that they anti the Divergence of bacteria and animals or animals and plants rather we know that viruses can jump species they can jump from insects indeed they viruses that infect both insects and people and they're viruses that infect both insects and plants and so so the transfer of genetic information that I alluded to at the very beginning of the talk the notion that a virus particle is a package that gets genetic material from one kind of cell to another May well be true not just for the cells within you or between you and another individual of the same species but across species we know that flu jumps from swine to people as we all learned from the 2009 pandemic or from birds to people but also ultimately through eons of time from one Kingdom to another at any rate it does mean that the structures we're talking about show a striking similarity and a striking Unity whatever The evolutionary details in the case of the adov viruses the subunit on the fivefold axis is um a different protein subunit from the hexon it's got one beta jelly roll domain instead of two so that again there's a kind of duplication and elaboration as this structure um develops into a much larger shell to package in this case a 35 kilobase pair double strand DNA genome much much larger Gena and indeed there are viruses based on very similar kinds of protein subunits the same double jelly roll structure with a separate related but genetically and chemically distinct single jelly roll pentamer on the five-fold axes there are um much l even much larger viruses based on this kind of subunit now an interesting um point in relating the adenovirus structure to the bacteria Fage that I mentioned is based on a similar kind of or um has a similar kind of major outer shell subunit is the mechanism or are the mechanisms by which the virus uh forms a defined and specific structure as you can imagine in this sort of structure how in the course of assembly is the relationship between one five-fold position and another fivefold position determined how is the size of this structure determined rather than than uh allowing let us say multiple hexons to start forming much bigger and bigger triangles in the case of the Fage the um the answer is particularly simple when stripped off the outer shell of hexon likee subunits and Penton likee subunits and discovered that from the X-ray crystal structure of this particle that there is an extended protein called a tape measure protein by the investigators who discovered this that in effect uh stretches from a five-fold position here to a two-fold position here and then meets another one twofold symmet to the next fivefold and that organization think of the scaffolds that we were talking about before governs the fixed size of the particle in this case the scaffold protein is not an arm of the same protein subunit it's a separate protein but the same principle applies and likewise in the uh adenovirus particle there are several different so-called glut or cement proteins that form in effect a scaffold that knits together the structure in a way that leaves the um uh leaves no ambiguity for the the um the size and uh and characteristic of the final particle in all of these structures the Pepa viruses the adov viruses the picorna viruses um the the um plant viruses such as tomato bushy stunt we see a simple construction principle at work that um is a little bit like an assembly line like a factory assembly line there is in all cases a fixed assembly unit happens to be a dier in the case of the code protein of tbsv you saw that it was a pentamer in the case of the L1 protein of the papala viruses the same is true of the polyoma viruses like sv40 and you saw that the adenovirus hexon the trimeric adenovirus hexon is likewise a mass-produced assembly unit but in order to determine how that mass-produced assembly unit fits into a defined structure of of of larger size how the the positioning of that subunit it uh doesn't simply um lead to errors and the building of a larger or smaller particle there's a framewor or scaffold just as in the construction of a building let's say that ensures accur accurate uh placement of these mass produced assembly units we've also seen that interestingly enough there's a recurring architectural Motif that has appeared in the evolution of these structures and it's a complicated one so probably evolved only once um uh over and over again now you might well ask is this the only architectural Motif why are all viruses um based on a so similar a building block and the answer is that isn't the case there's at least one other uh and that sometimes is called The hk97 Fold after the bacteria Fage hk97 in which it was discovered you can see that this protein subunit looked quite different it's got some Alpha helices it's somewhat irregular looking structure and it's found u in the bacteria Fage P22 and a large number of other doubl strand DNA bacteria Fage where um it forms a a shell with um a u uh a number of these subunits forming both hexamer and pentamers so that there are 60 hexamer and uh 12 pentamers there are always 12 pentamers in any icosahedral structure as suggested here these viruses assemble with an inner scaffold but the scaffold in this case is discarded by proteolytic uh digestion in some cases in this case it's actually reused it exits from the particle and gets reused in the case of P22 and um the particle then changes some details of its organization as the scaffold is exits as part of a process by which the double strand DNA is injected actually pumped if you wish into the particle at the next stage in assembly so these are cases in which the shell pre assembles around the scaffold the scaffold is ejected either chewed up or or or literally ejected and reused and a series of events involving motor proteins uh are responsible for inserting DNA into these structures now you could ask whether this is true only of bacteria Fage answer no you might anticipate that the answer would be no from what I told you about adenovirus and um prd1 one for example uh here are two bacteria phage protein subunits uh that have this sort of structure but the herpes viruses of which the herpes simplex one the cold sore uh virus is is one example uh are based on a much more elaborately looped elaborately decorated version of the same fundamental fold the structure that we have at the moment is is from electron microscopy and not yet at the same resolution that the X-ray structures of these subunits uh have yielded but you can probably see in this relatively low resolution representation of the herpes virus that this part for example there's along Alpha Helix uh corresponds to the much simpler undecorated fold you see here and then these are loopy structures that stick out in make the protein subunit much larger and have to do with other interactions that the U that the protein subunit of the herpes particle makes the herbes virus particle is more complicated both larger and more complicated than the Fage particles and so there other interactions of that surface um of those surface Loops that are important heres viruses like the Fage have very tightly coiled DNA that is on inside that's pumped into them in this Reconstruction from electron cryomicroscopy you can actually see the coiling of the DNA the DNA is actually coiled this way that is circumferentially about the axis of the particle it's injected through one vertex and as you see there's a specialized internal structure here uh to which then the tail of the Fage that ultimately injects it back into a new host cell is attached the cross-section here looks as if you have circumferential um uh layers of density in the other direction because as you can see from this diagram uh DNA coiled about a vertical axis if the resolution uh is or if the order is such that from particle to particle there isn't exactly a piece of DNA here but this one might be here or here then on average you will get radial shells of density as you see here fitting tightly into the interior of the particle I like to say with a Gardener's analogy might not be relevant for all people listening like winding a hose into a hose pot or a rope into a bucket now finally let's talk a little bit about enveloped viruses envelope viruses acquire their envelope in general their membrane this is not true of of all envelope viruses but true of almost all of them by butting out of the cell either out of the cell surface or into an intracellular um comp compartment such as the endoplasmic reticulum or the GGI uh apparatus and then being transported out uh and in that budding process wrap themselves if you wish in a membrane that's derived from host cell lipids although host cell proteins are in generally excl in general excluded some of the smaller envelope viruses have icosahedral symmetry and their structure and assembly is determined by regular interactions within an icosahedral shell just as the ones you've seen in the non envelope viruses but large and larger and less regular en envelope viruses are also um seen such as HIV or influenza in which the protein interactions are less perfect but that doesn't matter for protecting the nucleic acid nearly so much because the lipid bilayer in effect is an impermeable barrier against agents that might get in and degrade or damage um or cleave the um the nucleic acid so um the budding process that I mentioned can either involve as in the case of the so-called Alpha viruses of which um synbus virus is one of the prototypes and well studied and a recent outbreak human outbreak of an alpha virus uh is the chickun virus which has um had a a major outbreak in in the French island of reuno and led to considerable interest and publicity about about the properties of that virus the alpha viruses uh have a core that pre assembles in the cytoplasm and then two speci PES of glycoprotein that are synthesized on the rough ER exported to the cell surface and then the particle buds out through a process by which the um inward uh directed C terminal tips of the glycoprotein which stick through the membrane in a single Alpha helical segment you might remember very early on I showed you cross-section that showed that interact one to one with the um the iOS Rel symmetric core that's assembled rather like a um like a non- envelope virus in the cytoplasm and and buds out in other cases such as influenza the there's no pre-assembled inner particle but rather the assembly occurs at the membrane as you see here where the inner structures and the um and the glycoproteins that incorporate in the membrane come together as part of the elaborate budding event separate cellular Machinery in some cases is then needed to finish the pinching off whereas these viruses don't seem to need a separate pinching off mechanism in the case of HIV these micrographs show particularly dramatic examples of hi V buding it's directed in this case um by the interaction of the inter terminal domain of the inner protein the so-called gag Gene product and uh the that protein um has a marisole group at its end Terminus and a very um positively charged surface and interacts with the membrane to drive budding as shown here in these micrographs you can see that the HIV particle is rather sparsely uh decorated with an envelope glycoprotein that has the function of attaching the virus particle to a new host cell and mediating viral entry in the case of the smaller icosahedrally symmetric envelope viruses like Deni virus for example the outer coat is much more tightly packed it forms a very regular array in this case with 180 subunits of the protein whose structure is shown up here forming a perfect icosahedral array and it is assembly of that array that drives particle budding in all cases of envelope viruses the entry process and that will be the topic of the next part of this set of lectures uh involves Fusion of the viral membrane with a membrane of the host cell so just as the assembly process the maturation process the exit process involves butting out and pinching off so entry involves the reverse process attachment and fusion of the two membranes we'll talk about Fusion in great detail in the next uh part of this series but just to give you a hint of what's to come an important feature of all of these uh viral envelope proteins is that under suitable circumstances they can be triggered to undergo a major conformational rearrangement it's that rearrangement that that drives the fusion event so that in the case of the Deni virus particle there is a rearrangement from the dimeric structure shown here a rather platelike organization of two somewhat elongated protein subunits into a trimer in which hydrophobic residues at the tip of one of the domains this yellow domain so called domain 2 cluster together at one end of the trimer and interact with the target cell membrane in order to begin the process by which the two membranes are brought together the case of Deni virus this conformational change is triggered by proton binding a signal that the virus has arrived in the low PH compartment of an endosome in other cases other signals are um read out so to speak speak by the fusion mechanism we can look at this um in one more slide where the interaction with the target cell membrane is shown and there is a zipping up process of the C terminal part of the subunit that actually is part of the pinching together of the two membranes and leading to a an elaborate bit of molecular Machinery now not all envelope glycoproteins form such a regular array in the case of the influenza virus particle the proteins uh on the surface of the virus particles sticking out from the membrane are rather like there are two of them as you probably know are two species the hemog glutenin and nurin a the H and N of H1N1 or h5n1 that you read about when pandemics threaten the uh hemog glutenin is the protein that undergo a low PH triggered conformational rearrangement to drive fusion and we'll be hearing quite a lot about that in the next part the hemog glutenin shown here is a like structure as I mentioned its molecular design doesn't look anything like that of the envelope protein of Deni virus it's a stock-like structure uh and Long Alpha Hiles project the receptor binding site at the top about 120 or 130 angstroms away from the membrane we'll use that structure to discuss Fusion mechanisms in much more detail in part two of this series see you then