 So I'd like to thank the organizers for inviting me to come and share our work with you. So I am not a physicist, but I am a biologist slash biochemist and I do believe that the physical properties that guide a lot of our biology are important for us to understand a lot of the biological mechanisms that we're looking at in alpha viruses. So I welcome any sort of feedback or comments that you might have as I show you the data that we have now on our alpha virus work. So what are alpha viruses? Many of you might not be familiar with where they are. What we have here is just a list of all different animal viruses and as with all viruses you can categorize things based on shape, based on components, based on genomic material and what you need to remember for what I'm going to tell you today is that alpha viruses are icosahedral viruses, they're enveloped and they contain a positive strand, single stranded RNA genome. They are arboviruses, which means they're transmitted by arthropods, most likely mosquitoes. And the reason this is important for us when we're in the context of assembly is when we study assembly and disassembly we have to remember that the virus has two different hosts. It has a mammalian host and it also has a mosquito host. So as we compare and contrast different mechanisms we want to know are those conserved in the two different hosts or are they different? Some examples of alpha viruses would be Chick and Gunyer, Ross River, which is the model system that our lab uses, Venezuelan equine encephalitis and Zika is not an alpha virus. Just everyone thinks mosquito, Zika, we're all the same but we're not. And in particular for today's talk what I'd like you to keep in mind and why do we care about alpha viruses is it's actually one of the simple assembly systems for looking at an envelope virus and I'll explain this in just a minute. And we also have an in vitro assembly system for studying the nuclear capsid core. So this is really nice because not only can we do cellular and animal studies, I'm not going to share any of those with you today, but we can also do some in vitro studies to kind of dissect out the mechanisms of the different capsid and euclidic acids and lipid interactions that are taking place. So first of all let's start out with what does an alpha virus particle look like because if we want to assemble a particle we need to know what the final goal is or what that final particle looks like. And this is just a schematic and I'll show you a cryo reconstruction in just a minute. But what we have over here on your left are purified Ross River virus particles that are frozen in vitreous ice ready for cryo-em reconstructions. And this is just a schematic. You're going to have a nuclear capsid core in the center of the particle. This gray is your lipid bilayer. And then these yellow are your glycoprotein spikes. The spikes are actually embedded in the lipid bilayer and connected to the core. And if you take a cross section of the virus, you have the RNA inside, you have your capsid and green, your lipid and gray and your spikes again in yellow. And really the main thing you need to know is that there's nuclear capsid core and there are spikes. And then if we look at the cryo reconstruction this looks exactly like the schematic. These spikes that you see here on the surface contain two different glycoproteins. These spikes are absolutely required to cause an infection. They bind to the host cell receptor and mediate fusion. And that's something we're not going to talk about. And then on the inside you can again see your spikes on the outside. These two gray circles are your outer head group and your inner leaflet of your phospholipid membrane. You have your capsid protein. You have this gray medium density, which is a combination of capsid protein and RNA from the genome, and then you have the inside of the particle. What's interesting about our virus in terms of its assembly is that we have both icosahedral symmetry on the glycoprotein layer as well as on the interior capsid. And so you can see the symmetry up here on the spikes, but since we're focusing in on the capsid, this is how a section of the cryoem density map looks. In the four different colors you can see four different monomers of capsid protein that have been fit into the asymmetric unit. So both the glycoproteins and the spike have T equals four symmetry. More importantly is the symmetry between the core and the spikes are actually aligned. And so if we look at a sampling of different envelope viruses that are here, coronaviruses and retroviruses have depicted what that nucleocapsid core would be in orange and those are non-icosahedral. So I've just shown them as an oval or as a circle. And if you look at the glycoproteins around them, they also don't have icosahedral symmetry. Things such as herpes virus do have an icosahedral core, but their glycoproteins so far have not been shown to have icosahedral symmetry. Flaviviruses are actually the reverse where the core has no icosahedral symmetry, but the glycoproteins on the surface of the particle are symmetrically arranged. Hepadnaviruses such as hepatitis B, they do have an icosahedral core, they do have an icosahedral glycoprotein shell, but those two shells are not aligned with each other. And what that means is that the five-fold symmetry axis of the glycoproteins is not aligned with the five-fold symmetry axis of the core. In contrast, alphaviruses seem to have both their symmetry axes aligned. The reason for this, structurally, as far as we can tell, is because the glycoproteins have a transmembrane region, and then they also have a cytoplasmic tail that's on the interior of that lipid membrane, and that 34 amino acid portion of one of the glycoproteins has shown to make contact with the capsid protein. So you're actually anchoring the two together. I bring this up because one, some of our recent data shows that this model might not actually always be correct, and I'll show you that data, but also it's important to think when you're assembling a particle, you now no longer just assemble the core by itself, but that core that you're assembling actually has to interact and has one more component to interact with to make an infectious particle, and that is the glycoprotein spikes. So if your core is okay, but it doesn't interact or align with these glycoprotein spikes, then you have a non-... possibly have a non-infectious particle, and so that's important to keep in mind. The same thing is when we look at disassembly is how then is the release or the conformational changes that take place in your glycoprotein, how does that affect the disassembly of the core that the spikes are bound to? Okay, so now if we focus in on the nucleocapsid core, it's made up of 240 copies of the capsid protein, and over here, the capsid protein has a C-terminal domain and an N-terminal domain. Our N-terminal domain is very disordered. We haven't had structures by NMR or X-ray crystallography, and even in cryo-EM, we haven't been able to resolve the N-terminal tail coupled to RNA. In that N-terminus, we have in Ross River, 34 basic residues, and in general, the alpha viruses will have between 29 and 32 basic residues in only this 120 amino acid sequence. What you can do is you can very clearly and very nicely fit in the C-terminal domain of the capsid protein into the cryo-EM density of the nucleocapsid core. This N-terminal domain then is what I'm showing you here in this green region, and what we have fit in there is we know and we've modeled in what the N-terminus would be, and in that density, there's still extra room, and so we're speculating and hypothesizing that in that N-terminal, you will also be binding to RNA, which is not any brand new idea for this audience, but that RNA capsid that you are forming might actually have some sort of order in the variant particle because you have a pretty high amount of density there, but the N-terminal itself has not been resolved, and none of the RNA has been resolved either in the cryostructures. Okay, so let's talk a little bit about how do we assemble these alpha virus particles, and more particularly, how do we use an in vitro system for assembly? So Bill Gelbert introduced to you the genome of alpha viruses yesterday, and really what I want to focus on is the second open reading frame. The most important thing, and I think what makes the alpha virus is a very nice mechanistic system for studying assembly of an envelope virus, is you have two parallel pathways that you can recapitulate independently, and you can add those pathways in trans if you need to. So the first thing is your capsid protein is translated, it cleaves itself from the rest of the proteins, and that's in pathway A here, and as that capsid protein is translated and you have replicated viral RNA, you go on and you form a pool of these cytoplasmic cores, and that's what I'm showing you here. In a totally different pathway, you have the spike complex, so all those proteins that are required for the outside of the variant, those are targeted to the ER and the Golgi, they go through the whole cellular secretory system, and at the end what happens is you have those spikes that are showing up on the plasma membrane. The very last step is budding, and that's when you have these cytoplasmic cores that were the first step I talked about that interact with the glycoproteins, and you have a release particle. And if you, Mike Hagan's talk yesterday, this is what he was talking about, the simulation of, and the results of how budding is with and without a nucleocapsid core. So what we could do, because we have two parallel pathways, is we went ahead, and this was work that was actually done long ago in Richard Kuhn's lab by Tim Telling-Husen, is he first showed that you can express capsid protein in a recombinant system. This was first done in E. coli, but since then it's been done in insect cells and mammalian cells. You can add cargo, and I'll talk about what kind of cargo is necessary. You can use pretty much any sort of buffer. It needs to be in a pH range between six and eight, and you can form core-like particles. And what do these particles look like? We have various assays to look at them, but the two assay methods that are important for today's talk is we can look at them by TEM, and if you look at that negative stain or cryo, they're about 40 nanometer particles and 40 nanometers in diameter of particles. They're spherical. They're pretty homogeneous in what they look like. And then we can also look at these by DLS, or dynamic light scattering. And what we know with dynamic light scattering is if you add capsid protein alone, you have the blue line and you really don't see any self-assemble particles, but as soon as you add cargo, you'll see a peak between 40 and 50 nanometers. And then if you actually take this sample and look at it by TEM or any of our other assays, you see that you have core-like particles. So something that's interesting about AlphaVirus core-like particles is even though it's thought it's known or it's been predicted in some of the AlphaViruses to have a packaging signal, you don't need that packaging signal to form core-like particles or CLPs. We can make CLPs with single-stranded RNA various lengths. We can use single-stranded DNA oligos as long as they're longer than 14 nucleotides. We can take gold nanoparticles that have been coated with PEG and DNA. We can even take negatively charged molecules such as heparin. And when you look at these, and we do a whole bunch of different properties, whether we look at melting or salt sensitivity and all of these things, they're very similar to the cores that you will isolate from a cellular infection. So there doesn't seem to be a specificity for what that cargo is, except for if it is nucleic acid, it needs to be single-stranded. The other thing is if we look at these CLPs, they're arranged like the nucleocapsic core in the virion. And so this is a reconstruction actually from like 2012, but we can see that the CLPs have T equals four symmetry, and that's what we saw in the virus particle itself. Work that I don't have time to go into is this is great. We structurally have something that looks like a core, but what we care about, does it actually function like a core? And the answer to that is yes. So work from Richard Kuhn, Bill Gelbert and our lab have all shown that you can take CLPs and you can put them into cells that express glycoproteins and those particles that come out have now incorporated your in vitro CLP. And more importantly, those VLPs you've made can go on to infect a new cell. So you're not only making a virus particle, but you're making a virus particle that is now infectious or is able to release its genomic material in a new cell. So structurally and functionally, the system we have for CLPs seems to be mimicking very well what is found inside a cell during a natural infection. Okay, so our big question and what I wanna talk about today is what is the role of cargo during alpha virus assembly? It doesn't have to be specific, but yet if you don't have it, you don't form CLPs. And if it doesn't have to be specific, but we know that the core has to also interact with glycoproteins, what exactly is happening? What is it doing? So we used, and what we're gonna talk about today is I'm gonna do two things. I'm gonna address this where cargo is the variable. So we're looking at differently single-stranded DNA oligos and then also where I've modified some of the basic residues on the capsid protein. So before we start, and as we looked at the cargo, there were some points about cores, nucleocapsid cores, that both experimentally and theoretically had been shown. And we wanted to actually look at this with our alpha virus cores, which are an envelope core. And a lot of this was done with viruses that are not enveloped. So we know that in single-stranded RNA viruses, electrostatics are important for core assembly. It's also been shown that weak subunit interactions are important. The idea is that if the capsid misassembles, it can readily disassemble and reassemble or fix itself. And when I mean subunits, that can either be protein-protein interactions or protein-cargo interactions. We also know that many capsids will have hysteresis to disassembly. So as you assemble it, that's great, but then as you try and disassemble it, it actually takes a lot more effort to pull that capsid apart. Whether that means that once upon assembly, there are conformational changes that have taken place or secondary interactions that have taken place. And it's generally thought that capsids are spherical closed shells. What we've done in our system is we've taken Ross River virus capsid protein that's been purified from E. coli, and we've added a 27-mer oligo. And we've done a simple titration where we've kept our capsid protein at 1.5 micromolar and we've titrated in 27-mer oligo and we've measured the output of CLPs by DLS. And what you see is that, yes, you get an increase in CLP formation and then it stops. And then if we do this in the presence of different amount of ionic strength or different amount of salt, as you increase the salt, you see that the maximal amount of CLP that you're forming is actually going down. Interestingly, the ratio at which you get maximal CLP formation, if our capsid is at 1.5 micromolar, our cargo is also at 1.5 micromolar. So from here, there's a couple take-home messages. One is that electro interactions seem to be key because the assembly is disrupted in the presence of higher ionic strength. There seems to be a one-to-one mole ratio at all ionic strains in terms of capsid and cargo that are being formed. And we can look at this in a very simplistic way of Q minus to Q plus as being one-to-one because we're adding 27 negative charges from our cargo and we know that in our interminous of Ross River, we have 31 positive charges. So to one significant figure, they're all the same. I'm gonna, we've done a lot of experiments like this and one way that I'm gonna show that data is over here. And what we do is we go ahead and say if the red is the maximal amount of CLP that we have, that as we vary the salt concentration, what percentage of that maximal is actually forming? And we'll see graphs that look like that. And then the other parameter that we look at is what we call CLP 50. And what CLP 50 is what is the salt concentration? Cause we're primarily gonna be looking at ionic strength, but what is the salt concentration where you get 50% of your CLPs that are being formed? So these are the two things I'd like you to keep in mind. Okay, so then we wanted to test for hysteresis. And ideally, if you assemble a particle in the presence of salt, you should have this black curve. But then once those particles are formed, it should be more difficult to break them up. And so you would expect your disassembly curve to look like this. And with disassembly, you're mixing your cargo and your capsid in a tube. You have a certain, and it's a very low salt concentration. You have CLPs and then you would titrate in salt and that should initiate the disassembly of the CLPs. So this is what we expected to see, but this is what we saw. So, and you laugh, you will laugh more throughout the time. So really with our 27 more, there's no hysteresis. And then we thought, okay, maybe we just don't have the right amount of cargo to capsid ratio. So we took away some of the cargo, we added some more cargo. And what I'm showing you up here is the different ratio. Our capsid protein was held constant for all of these. It didn't really matter. Assembly and disassembly showed the same sensitivity diionic strength, and your CLP 50 for all three of them also was the same. Yeah. What is the reason why you picked the 27 more? Yeah, so initially we picked the 27 more because we had this very simplistic view that we just want to neutralize whatever chargers are there. And in about two slides, I'll show you what happens with longer cargo as well. So from this, yes, there's no hysteresis taking place upon disassembly, but this could also suggest that we do not have intact closed shells in our core. And that could explain this. So we went ahead and these are 2D images that have been classified. And when you look carefully, you can actually see that, yeah, part of the core is complete, but then there are some parts that are open. Zhou Wang, who's in our EM, our electron microscopy center and also works with Adam Slotnik, we tried to look at these and figure out if there was some sort of pattern. Did you always have an opening in a certain part? We had this idea that maybe there was a portal formation or something going on, and we couldn't find it. It seemed to be pretty random of where you had areas of discontinuity. But this also made us think, you know what, maybe our in vitro system is completely artifactual. Maybe everything that we've set up until now, what we think is a true in vitro system for CLPs and for alpha viruses is not really true. Cause I just told you that we can take those cores and make VLPs and structurally, they look the same. So what we did, and I don't have time to go into all the data, but we went ahead and took Ross River virus in tagged variants and we did our reconstructions in different ways, either masking out the core or masking out the glycoprotein spikes. And actually what we found there is that 15% to 30% of those particles actually showed variants that had incomplete cores. In the upshot of why we always would seem to get the core in the spikes having icosahedral symmetry, seemed to be because we imposed icosahedral symmetry on the entire particle and the dominance of that glycoprotein shell seemed to mask some of the disordered features of the core. This also explains a lot of little details and frustrations that we had when we would have our variant particle. There were certain parts of peptide chains that we weren't able to trace and we couldn't understand why, even at four-onkstroms resolution. And part of that now could be explained by our core actually being disordered. So whereas before I showed you that both the core and the glycoproteins have icosahedral symmetry that's intact, let's modify that a little bit and say that the core might have partial icosahedral symmetry. Okay, so right now our model so far for 27-mer is very simple. This is our capsid protein, C-terminal domain and terminal domain that's basic. It binds to an acidic cargo and really the function of the 27-mer is to neutralize and to go on and form CLPs. And so then Bill, coming to your question, what happens if you change the length of the cargo? And we didn't go too much longer but we went ahead and we used a 48-mer that was predicted to be a hairpin. 48-mer that was predicted to be linear and then a 90-mer. These are M-full predictions which can either be right or wrong depending on your luck. And so our collaborators at Champagne Urbana went ahead and did some simulations to see at different salt concentrations what the conformation of our oligos would be. So we did the same sort of assembly reaction, cargo plus capsid, looking at CLPs. So there's a couple things I want you to look at here. First of all, this is our 27-mer. This is the same data that I've shown you before but if you look at that in reference to the 48-mer, hairpin linear or 90-mer, you get less CLPs being formed with the shorter oligo. The second thing is your CLP 50 for your 27-mer containing CLPs is much lower than your CLP 50 if you have longer oligos. So structurally there seems to be a difference even if you double the length of your, one and a half times the length of your oligo. And there doesn't seem to be a big difference between 48 and 90. And I understand that in a virus particle we actually have 11,700 bases of RNA and this is a very small view of that but we're trying to focus in on what is happening at that end terminal region and not so much at the entire genome itself. Okay, then we look to see with the longer cargo now because you have differences in the assembly and the kind of CLPs that are forming. Do you have anything with hysteresis? I think that the answer is no, there might be something with your 48 hairpin. And so again, we wanted to look at these structurally. So we picked the 27-mer as a comparison and we looked at both the 48-mer linear and the 48-mer hairpin to kind of do cryoEM and to look at what these particles look like. So the first thing I want you to look at here you can't really see the particles but if you look at this inset here you can see a few things. First with the 27-mer the particles are slightly smaller than either of the 48-mer particles. The second thing you'll notice is you, these are rotational and translationally average particles. You have three concentric layers of density in your 48-mer's but you only have two in your 27-mer. It is very exciting to speculate. This is pure speculation, but it's what I'm gonna share with you that maybe the C-terminus, C-terminal domain in both the 27-mer and the 48-mer fit into that cryoEM density but as you have longer cargo you're able to form additional contacts in that N-terminal domain with your longer cargo compared to your shorter cargo. We have no evidence for that. We just think it's kind of cool if it was true. We went ahead and looked at the particles and you can see that regardless of the cargo length you still don't have complete cores. Knowing that, we still wanted to know if you impose icosahedral symmetry, what kind of particle do you have and you have T equals four particles. So take homes and I think I've discussed these along the way. The CLPs of 27-mer are slightly smaller. You have fewer shells of density in the 27-mer compared to the 48-mer but you still don't have a continuous core that's being formed. Okay, and one last thing that I wanted to look at was or we wanted to look at and I'll share with you is that the 27-mer CLPs compared to the 48 CLPs they actually have a difference in sensitivity to thermal temperature. So the CLPs made with 27-mer not only are they more sensitive to ionic strength but they also seem to disassemble or fall apart sooner in the presence of heat. Yes, slightly stronger. Yeah, so our model here is the following for the 48 and 90-mer that not only is the cargo acting to neutralize but it's acting to scaffold. And you could see that maybe upon binding of the nuclear capsid in the cargo together you might have a slight conformational change. You make particles that again are not completely closed but they're larger than what you see with the 27-mer. And so just trying to look at all of this together. Yeah, we found that in our envelope core electrostatics are still important but and I didn't get into this but Chad actually in the initial work with 27-mer found that our Delta G values were at minus six kcal per mole. So those are rather strong interactions. We don't seem to have hysteresis and that could be really due to capsids not being closed spherical shells. Okay, now that's kind of the work we have with cargo. What if we take a different approach and say let's look at what's happening with the capsid protein? So we went ahead and made four mutants or I'm sorry, made two mutants. One is called 4D where we've taken this patch of four lysines and mutated those to aspartic acid. So not only are you removing four basic charges but you're now introducing four negative charges. So it's almost like a Delta of minus eight. And then we made a 10D where we've also mutated these two patches of three additional lysine residues next trip. And I wanted to ask two questions from this. We wanted to say what is the best ratio of using only the 48 hairpin cargo? What is the best ratio of 48 mer hairpin to cargo that's needed to make CLPs with our 4D and our 10D compared to our wild type? And then once you make these CLPs how much of that cargo is actually packaged in them? Because now you're changing the amount of basic residues in there and work that was done by Cheng-Kel's lab and Bogdan Dragnea on bromoseic virus show that if you increase or decrease the amount of basic residues in the end terminus you also increase or decrease the amount of nucleic acid that's packaged. Again doing our assembly reactions monitoring by DLS and other methods as well but this down here is showing you the ratio of hairpin to capsid. We're keeping capsid constant in these experiments. And what you find is that for wild type the best sort of ratio seems to be about 0.5 so that would be one cargo molecule interacting with two capsid molecules. You see with the 4D it's also pretty similar and with the 10D you don't really see very many core like particles that are being formed at all. And then we went ahead and I'll walk you through this table because it's a little confusing at first and this table right now is incomplete but just I think it gives you a general idea of what's happening. If your input is having these ratios of 48 murder capsid so anywhere from 0.4 to one so that means along this black line we're looking at different ratios here. What you get in the output and these are different ways of looking at them is how many 48 murder capsid it's about 0.4 which really is then about two and a half capsid proteins per cargo molecule. And if you look and just count plus and minuses you're getting about 62 to 66% neutralization from your cargo to those capsid protein monomers. If you do the same thing with 4D and here we're looking actually at this beginning part of the slope you can see that at the end you're getting about 2.7, 2.6 capsid proteins binding to one 48 murder and the amount of neutralization is a little bit more. At this point we're like is there really a difference? Does it matter how much of a difference do we consider significant when it comes to these packaging experiments? But before we went any farther we actually had a pretty big surprise and our surprise was this and this came because we were trying to purify our 4D and our 10D and we're running into a lot of trouble and what we found out actually was that our 4D protein without any cargo on its own can now form core like particles. Our 10D particle so where we've removed 10 of the basic residues can also form some sort of spherical shaped thing. I wouldn't really say their core like particles. Definitely the 4D were much more homogeneous and they're smaller than what you see if you have wild type capsid with 48 mer hairpin that's going on. When we compared the 4D to kind of characterize it because in our very simplistic mind we said oh maybe this is a purely electrostatic interaction because we've taken away basic residues and we're adding acidic residues but it doesn't seem to be because as you're changing the salt concentration as depicted here you still able those 4D no cargo particles still persist. So there's something else going on besides electrostatics there. And again when we do the temperature this is what I've shown you before for 27 mer and 48 mer and when you put in the 4D cargo less particles you actually get this sort of biphasic structure. So we're not really sure what those particles look like yet and that's something that we're looking into but to us it was rather surprising that they could self assemble. And so the sort of model we have going there if you look at 4D and 10D with no charge is yeah in the middle of your tail you have acidic residues and now you might be forming some sort of confirmation maybe it is initially mediated by these electrostatic mutations these changes in the charged residues in the interminous but the particles you're forming are definitely smaller than what we see with the 27 mer and the 48 mer and we're speculating that they're not complete cores yet but we don't know that. But based on our salts data or ionic strength data they actually might be packed more tightly together and you might be getting more capsid-capsid interactions in that sea terminal domain and that could be why it's more difficult to disassemble them under ionic strength or under thermal conditions. But at the end of the day we wanna know does the virus really care if we've mutated these four residues or these 10 residues in the interminous because that's great we can notice all of these things that are happening in vitro and we can see small differences but we also know there's caveats to our system and that we're using DNA oligos not single-stranded RNA we're not in a presence of a cell where there might be a cellular membrane where replication takes place we don't have high concentrations of protein and RNA we don't have cellular factors that might be important for some of our assembly work. So I'm just gonna show you two slides maybe even one, two on cell work. The first thing we went ahead and made the mutations in the whole virus genome and the first thing we looked at was viral titer viral growth in plaque size. So viral growth is how does the wild type in the 40 mutant, how do they grow in cells? How much virus is released over time? Those were the same. The number of infectious particles that are coming out were very similar but what was different was the plaque size. So in 10D we always get much smaller plaques and what this suggests is that we're having a deficiency in spread and perhaps our deficiency in spread is actually due to new particles disassembling and maybe they're not disassembling as well as the wild type in a cellular context. Or even based on some of our in vitro work we know that the 40 is more stable so maybe that's playing a role in disassembly inside the cell. However when we go ahead and purify the variants they look kind of the same. So now this sort of beg the question okay maybe there's something wrong with our core but maybe the envelope that contains those glycoproteins is a second measure to make sure that okay everything is being packaged okay and that's kind of the regulatory role of some of the envelopes or the spikes. The other thing I wanna share with you is we then said okay we've made a mutation in the capsid protein. So what does our nucleic capsid core look like? And there's two ways that we can do this. So the first thing we did is we took our purified virus and we put it on a sucrose gradient so we applied it here you spin it for a long time and then it'll sediment and the sedimentation is primarily due to the genome that's inside of the core and the nucleic capsid core itself. And if you take fractions where fraction one is at the top and fraction 12 is at the bottom for wild type you can see these are virus particles cause we're looking at capsid and spikes primarily at starting at fraction eight. With our 4D and our 10D well for this for our 10D we always see particles that are coming out at one fraction earlier. If we then look at cytoplasmic cores cause I told you that inside the infected cell you form cytoplasmic cores independently of your spikes. So you can take cells that are infected you can break them open and you can take those cytoplasmic cores which contains genomic material and put them on a gradient. And what we find for wild type is you have a peak at around fraction six or seven where you have the most cytoplasmic cores but in your 10D you don't have that you have things that are that are at earlier fractions which suggests you don't have complete cytoplasmic cores that are even forming in 10D. So one model we have is that perhaps in wild type you have a lot of virions where you have closed cores and closed glycoproteins and you have a few of them about 15 to 30% where you have these incomplete cores that are being formed. Whereas in the 10D that balance has switched and you actually have more of these incomplete cores and that's why they're sedimenting at a different place. Yeah. Oh, okay, perfect. And then another reason why some of your virions might be sedimenting at a density that's not at a less, that the virions aren't as dense is maybe our 10D is actually packaging random RNA in the cell. And so that's something we want to look at as well. So let's come back to why would an envelope virus actually want an incomplete core? Maybe this is actually how it should be or maybe there's some cases where you want a complete core and some cases you don't. So we know that for a virus infection, timing and cellular location are absolutely critical. If your virus falls apart in the wrong place, you're not gonna be able to propagate your genome and keep going with the life cycle. And it has to be also have the correct cellular factors with you. And so in all of these where we seem to have cores that, let's start with this and the ones that have cores that are icosahedral, these two actually have to traffic to the nucleus to release their genomic material. Whereas things like coronaviruses, slavy virus and alpha viruses don't. Retroviruses, I guess technically they're replicating in the cytoplasm, but they're in a core. So it's slightly different. So for all of these, slavy, alpha and corona, there's no intracellular trafficking that's required before your genome is released. And here structurally, maybe you wanna release your genome independent of when your capsid protein or how your capsid core can actually disassemble, whether it disassembles into individual parts or not. We also know that in order to get that initial translation step, the host ribosome is very important. So if there's sort of an opening or a way that the genome can either be extruded or the ribosome can make contact, that might be a great way for the ribosome to start pulling out that message RNA and to start its initial translation. And the other thing is this is a lot faster than having to disassemble your capsid. And what that means is you're able to start replicating your genome and you're able to evade your immune system. So one of the key points, one of the key double-stranded RNA which is a replication intermediate is something that's recognized by the cells. And so if you're able to really start your infection earlier, it just delays that recognition by your innate system. And so 10D we think is disrupting this balance in all of these different ways and we've already talked about that. And with that I'd like to finish. This is really our team capsid. I told you about the three different steps of the virus life cycle. That's pretty much what my lab studies, the core, the spikes and them coming together. These are the people past and present that have been involved in team capsid. And the work I showed you today, a lot of it was done by Vomsi, a former postdoc in the lab and Alan a current graduate student with a lot of collaborative work with the Zlatnik nap and these former members of Klaus Shultz in the lab. So I can take any questions if you have them.