Should I share my screen now? You can share your screen. We wait for some more minutes that people can join and once you once you already let me know I’ll share my screen. Okay. So audience can pose their questions in the comment box below. I’ll be moderating it at the end of the presentation. Think you can share your screen Okay, let me know when to start talking because I really can’t see anyone so I like to show you can see the screen right again sorry. Yeah. Think I’ll start. Hello, everyone. Welcome to the second chapter of buyer battalion webinar series. Today we are extremely delighted to host Dr. Arthur ceren. Research Associate Professor Loyola University Chicago. But before we begin with the presentation, I would like to talk a few words about via battalion. We are a relatively nascent non for profit group started by a group of like minded science students at the early phase of their career in science. We try to organize webinars on a perpetual basis, and work closely with academicians, industrial researchers and science communicators. We’re also inviting original creations from interested people to freely host on our bio battalion science blogs. This initiator we believe, would eventually help them become better science communicators. Also, if you haven’t already subscribed to YouTube channel, please do so to receive instant notifications on our upcoming events. Also, as I told our audience can pose the questions in the comment box, and I’ll be moderating it at the end of the end of the presentation. Now without further delay, let me introduce Dr. Arthur’s parents you all doctor either starting as a trained cell biologist and biologist currently working at the Department of microbiology and immunology, at Loyola University Chicago, USA. As a research associate professor, Dr. Durham obtained a Bachelor of Technology degree in biotechnology and biomedical engineering in July 2008 from the University of Kerala, India, and a master’s degree in genetic manipulation and molecular cell biology in January 2010. From the University of Sussex the United Kingdom, Dr. Darren Andrews and the infectious biology PhD program at Hanover medical school Germany, where his thesis was an understanding how microtubules and its associated motors aided HCV replication and in the formation of HCV induced replication factories. Upon completing his PhD, Dr. Darren moved to Loyola University Chicago to start his postdoctoral training under the guidance of Professor Edward Campbell. at Loyola Dr. Darren’s primary focus was to better characterize the early stages of HIV one replication with focus on viral trafficking and up or import. In 2019, Dr. Darren was promoted to his current position. His research is primarily funded through the National Institute of Health grants. Apart from his research duties, Dr. Darren is also involved in mentoring students in their research projects, teaching two courses for Masters and PhD students in the depart Now over to you, Dr. Anderson. Thank you, Pooja, thank you for the kind introduction. So So today I’m actually going to talk to you about one of the steps which is involved in during the HIV one life cycle which is a nuclear import of the virus. So as Pooja mentioned, I’m right, currently, I’m a research associate professor with Viola University and we are in this we are in this building, which is called the Center for translational research and education. So it is it’s a, it’s a big multidisciplinary Institute where, you know, microbiology is just one part of it. There’s, it’s It comprises of a number of Institute’s and basically, like I said, I’m in the microbiology department. So, one of my research focuses, like Pooja mentioned is to better understand how HIV one infects a cell. So, as most of you are aware of HIV, one is the virus that causes AIDS. So, and what happens to like once the virus infects is it results in a gradual or a drastic decrease in your CD for positive T cells or, and what happens is like once the person acquires some virus, or once the person acquires aids, you are mostly immunocompromised. So and as you can see from this most recent update from the who are around 38,000,030 8 million people are living with the virus right now. And so that brings the importance of why, you know, we need to better understand this virus and to make more suitable drug targets. So now coming on to more biological, more cell biology into the wireless, so HIV one, it’s a positive strand, single stranded positive sensor virus. So you have actually the RNA virus, which is basically the genetic material for the for the virus, which is mostly enclosed within this conical structure, which is made up of a protein called p 24. So this conical structure basically houses the viral RNA, we have two copies of the viral RNA, along with a number of other proteins, which the virus requires, and some of them are the reverse transcriptase. So one of the hallmarks of HIV one is like it, which makes it different from other viruses. Like for example, like, you know, everyone right now should be familiar with SARS. COVID, too, right? So it’s an RNA virus. So HIV one is also an RNA virus. But what makes it different is it’s able to convert its RNA virus into a DNA, which can then integrate into our host chromosome. And how the virus does is the virus has an enzyme called reverse transcriptase, which converts the RNA to DNA. And then it has the integris protein, which integrates the reverse transcribed DNA into the host chromosome. So all this components along with the viral RNA, it’s enclosed within this conical capsule structure. So the conical capsule has a number of significant so it basically it’s a it’s like a shell, which protects everything within the structure from our host, our from our host body. So like, like most of you are aware of like our host has a number of defense mechanism, which which protects us from many different pathogens, right. And so this structure, basically, it’s kind of like a shell, which protects everything within the structure from any host mechanism. Now, surrounding this the viral capsid, you have the viral matrix for protein, and outside the matrix protein, you have a lipid bilayer, which the virus gets from the host cell and onto the slip advisory around the world glycoproteins, which the US uses. Excuse me. So are you changing the slide, sir? No, I’m not changing the slides. Alright. Thank you. So you can see you can see you can see my pointer, right? Yeah. Yeah. Okay, I’m not changing the slides. So yeah, onto this wire, onto the slip by layer, you have the viral glycoproteins, which the virus uses to gain entry into the cell. So now, just to give a little bit more depth into the viral life cycle before we actually move into the, into the talk, so like I said, the ones the virus finds its target cell. And in case of HIV, when it’s mostly the CD for positive T cells or the dendritic cells, the virus binds to its receptor, which is the CD four, along with the core scepters, the CCR five or the cscf. And once the glycoproteins bind to this receptors, there’s a structural change happening at the glycoprotein level. And this structural change actually results in the wider, wider membrane actually fusing with the plasma membrane, and once it fuses, it releases this conical capsule, which has all the water, land and all accessory proteins into the cytoplasm. And once the water capsule is in the cytoplasm, it traffics from the site of entry. All the way Down to the nucleus. And during this process, like I said, the viral RNA gets reverse transcribed into DNA. And once it’s inside the nucleus, the DNA it’s integrated into the, into our host chromosome. So what the virus uses is the our own host translation machinery to make more copies, right, and once it makes more copies, it’s then exits out from the nucleus, and then goes over to the plasma membrane, where the virus assembles, and then the newly assembled viral viral particles are then released, which then goes ahead to infect more cells. So this is a this is a very general overview of how the virus life cycle works. But so my aim today is actually to show you like, all the steps of the viral life cycle, I mean, I mean, what we have contributed to, you know, better understand the steps of the virus life cycle. So there’s a number of host factors, as well as restriction factors, you know, which plays a critical role during the virus infection. So when I say host factors, those are factors which the virus needs to infect a cell, right? I mean, the virus carries some of them, like it has its own proteins, but it also needs some host for proteins, like proteins with the novel cell to help in its replication. But in the, if you look into the, you know, the into the other side, there are restriction factors, our body’s trying to always, you know, defend this infection. So there are restriction factors which tries to tries to prove, which tries to prevent the virus infection. And one of the reasons the virus recruits a number of factors is to evade this restriction factors. So for example, what I can show you here is like, you know, the, in this cartoon, you can see, like, a, it’s a normal virus infection, and why does it get sin, it recruits a number of host factors, it goes all the way to the nucleus gets into the nucleus, it has its infection happening, right. But let’s say you have a defective virus, a virus, which is not able to run across factors, it gets actually sensed by our first mechanism, and you know, it This can prevent infection. And similarly, you know, like I said, there are restriction factors does, which binds to the viral capsid or the, you know, the conical structure, and it can actually degrade the viral capsid. And thus it prevents infection. So, for one example, the virus recruits the host factor called cyclophilin. A, and the recent LED, there has been a number of studies going on in the last several years. But recently what they have found is the reason the virus recruits cyclophilin a, is our, our, our body has a protein comes from fiber. So true five alpha can actually bind to the virus capsid and degrade the capsule does it province. Yeah, sure. Right. Actually, this slide is still in the I mean, it’s actually the first lifestyle now, which has not been changed it. Oh, really? Yeah. You’re not. So it’s still in the first way right now. Yeah. Sorry, I’m so sorry. But let me actually stop sharing. So you don’t see the slides now, right? No. Okay. Let me share the screen. Right. Whereas I’m not so familiar with stream yard, how it works. Okay, let’s see. Let’s see if this works. I okay, now I can really find my slides now. Okay, so opening up the presentation. Okay. So do you Did you see the slides? No. Not yet. So, yeah, we can see. Okay, you so I’m making it full screen right now. All right. Now you see it as full screen. You do or you don’t? Not yet. Oh, so there must be a lag. I mean, okay. Can you try changing the slides, right. So you see the slides moving right? Yeah, can you see? But do you see the slides on the side or used See the way it’s not in the full screen mode? We can see the slides on the side. Okay, so I okay, that means Should I go in this way then? Yeah. But the slides are moving. Okay. Okay. You know, I’ll go ahead and I’ll go in this way, that’s fine. You know, the way it has to be. Okay, I’m sorry. So, so if you were really stuck on the first slide. Okay, let me let me get back to this point, I think that kind of clarifies most of the situation here, okay. So, what I was saying is, like, you know, once the virus infects a cell, it recruits a number of false factors to you know, to successfully complete the, you know, the process of infection. So, so, one sample I was talking about is the factor called the host factor for the cyclophilin a, so, the reason the virus recruits cyclophilin A is our our body has a protein called trim five alpha, so, trim five alpha can actually bind to the virus capsid and then degrade the capsule. So it can act it prevents infection. So, what the word does is it recruits cyclophilin A, which prevents 10 five alpha from binding to the capsid. So, the take home message here is like, you know, there is a there is a number of hosts factor recruitment, which the wireless does to successfully, you know, complete the process of infection and to evade our host sensing mechanism. And this becomes more clear, actually in the next slide. So, you know, this is just three publications, but you know, there’s like, there’s like a ton of publications right now. So, any any kind of abnormality in the world events like you know, the the wires, the traffic and all the stages of the wire cycle is perfectly timed, it has to be done in the right sequence. So any abnormality you have in this world Leavens can actually stimulate our host sensing mechanism. So for example, you know, this paper which is from Greg tawas lab at UCL London, what they have shown is like, you know, if you deplete certain host factors, which the virus requires, it will actually result in the virus getting sent via our host body, and then the virus actually being degraded by the host body. So, this first factor recruitment is like very critical for the virus for a successful infection. And, and other lab, what we have shown is the trafficking events. So, like I said, once the virus binds our or once the virus enters, it needs to traffic from the site of entry to the nucleus. And if we prevent this trafficking, like you, or if we actually strand the virus in the cytoplasm, this will actually result in the virus being sensed by our host, and then the virus being degraded by our host. So again, the the take home message here is like all the steps, which I talked about in the early stages of the virus infection is perfectly timed, or it has to be done in the right orientation otherwise, that can actually result in our host body being recognizing the virus and triggering a response. So this makes it more interesting why we need to understand every step of the virus lifecycle, because if you understand every step of the words, lifecycle, it’s more easier to find ways how we can actually moderate those steps. So our host body can actually sense the virus and then you know, prevent infection. So like I said, in my in so my talk, what I’m interested in is how the virus accomplish the process of nuclear import. So like I said, once the virus and just the cell, traffic’s all the way down to the nucleus. So once at the nucleus, the virus has to enter the nucleus, right? And what it does is it travels through the nuclear pore complex, so I’m not sure how many of you I think, I think most of you must have heard of the nuclear pore complex. So, the nuclear pore complexes are giant macro molecule austrac chains which are made up of more than 30 different proteins. So these proteins are arranged like there are multiple copies of the same protein within a nuclear pore complex. And this nuclear pore complexes are like a gatekeepers, they are like the gateways into the nucleus. So, proteins are certain molecules, which are below a certain size limit, can freely diffuse through this nuclear pore complexes, but proteins about a certain size they once they have a nuclear transport receptors, they can be actively transported to the nuclear power complex. So what I’m interested in, and it has been shown that the virus capsid uses the nuclear pore complexes to gain entry into the nucleus. So what I’m interested in is like, what are the factors like how does the virus accomplish this process of nuclear import? Like what are the factors that helps the virus to complete this process? And if we can actually find those factors, you know, there, there is a chance that we can affect the process and then does inhibit the virus infection. So when I started my work with Dr. Campbell in 2013, I mean, the first kind of project we were really working on was the draft. Looking at events like once the wire senders, how does the traffic along microtubules. So I mean shown here, it’s a VPN VPN is one of the accessory protein of the virus labeled with the GFP. And you can see that the VPN label was particles on microtubules. So what I was interested in is like, what are the factors that helps the worst to traffic along microtubules. And we were made basically interested in this to microbial motors, kinesin and dynein, I’m pretty sure you must have heard about this one in your cell biology classes. And the first kind of paper I had from from Dr. Campbell’s lab was we looked at how viral uncoating which I will talk in more detail in my later part of the talk. We showed how uncoating is actually facilitated by these two motor proteins. So when we were actually studying this, when we were actually doing this process, what we kind of noticed for one of the kinesin protein was was quite interesting. So when we knocked on kinase and one key five B, which is a kinesin, one motor domain using sa RNA, so si RNA, you can actually, you know, insert a knockout, it’s a knockdown of your protein, and a new 358, which is a which is a protein which is present in the nuclear pore complex. So what we saw was, if you look at the infectivity, you’re looking at the wild type infection, this is scram, we’ll just say, when you knocked on key phi B, or new 350, do you see a drastic reduction in infection. And when we look at the wireless reverse transcription, so we can measure reverse transcription, or the nuclear import using specific primers. So when we looked at reverse transcription, there was no change in reverse transcription. So the virus is able to convert its RNA into DNA. But But when we looked at the nuclear import, this decrease in infection correlated to a defective nuclear input. So somehow, the virus is not able to get into the nucleus, when you deplete out, kinase in one or the kinesin, one motor domain or nuclear 58. And when we looked at the virus by microscopy, so what we did here was we took killer cells. And normal cells, which most people use in the, in the in the virus field, and we infected these cells with HIV one. And after three hours post infection, we, we fixed the cells and we stained the cells for the virus capsid protein p 24, using an antibody. And what you can see here is in the control, you see the that the virus is like all the way in, in the cytoplasm, but when we know tonkinese in one, or nu 358, you can actually see the viruses stuck around the nucleus, it’s as if like, it’s not able to get into the nucleus. And we could kind of find this one, like, you know, we could quantify this red signal. And what you can see here is that if you concentrate just on the three hour time point, either for the key fi key five nocturno, for the new 350 can see, compared to the control, there’s like an increased wires around the parent in the in the parent nuclear location. So what it shows is like, somehow, when you when you knock down this kinase and one or nuclear 50, it results in the virus from like increases it, it results in the virus being stranded at the nucleus and not being able to get into the nucleus. And we also saw a different, we also saw a different phenotype with with the new 358. So if you look at uninfected cells, like I said new 358 it’s a it’s a, it’s a member of the nuclear pore complex. So if you look at if you stay in a normal cell, it will be you know, right around the nucleus. But what happens after a virus infection, so this is infecting with a wild type HIV one virus, either in its two different cell lines, it’s either monocyte derived macrophages or HeLa cells. But you can see here is like there is a drastic reorganization of this protein into the cytoplasm, when you comparing it with our uninfected cells. And you lose that phenotype, you lose the phenotype, which I show which which you have seen before when you knocked on kinase and one. So when you knock on kinase, and again, you see all the wires stuck around the nucleus, and you prevent this new 358 from being relocalized into the cytoplasm. And this is also like, you know, we quantified this phenotype, you can you can see here, the amount of new 358 in the cytoplasm. Like if you look at a three hour time point, compared to the control when you knocked on kinesin. One, there’s a drastic decrease in the amount of new 358 in the state of Assam. So at that time, we actually put forward a model where both kinesin one a new 358 how it mediates the nuclear input of the virus. So what we thought at that point what was happening is the virus once it enters, it comes all the way down to the nucleus. Right now the nuclear power has a size limitation, right, and the wires is too large for the nuke to pass through the nuclear pore complex. So what the wires We think is happening is once it’s at the nuclear pore complex, it actually gets inserted into the nuclear pore complex. And then kinase and one, so kinesin one, it’s a motor protein which moves towards the plasma membrane. So it by and so on to the virus capsid. And then it puts the virus caps in, in the other direction means towards the plasma membrane. And by pulling the wires caps, it was the plasma membrane, it’s actually breaking out the virus caps, it’s actually like, reshaping the virus capsid to a size, which can pass actually through the nuclear pore complex, right? So I hope I think I’m assuming you’ve got that thing. So and this is the I mean, we were not the first people to actually put forward such a such a mechanism. I mean, this has been proposed by a different group in 2011, for different words for adenovirus. So what they see a similar phenotype, so add no virus, it’s a huge virus, right? it and once it comes to the nuclear pore complex, it can’t really pass through the nuclear pore complex. So what happens is a similar mechanism where kinesin comes and binds, and it puts the virus capsid in the opposite direction, thereby, you know, breaking out the virus capsid. And what happens is, whatever is inside the genetic material inside the virus capsule is then transported through the nuclear pore complex. So, we were like, Okay, this is this is, this is pretty interesting, we see a similar phenotype with HIV, we see a similar phenotype, if not worse, that means something is happening. So somehow, these viruses are able to utilize a mechanism which is which our host body has, like, as probably most of you are aware of, like, you know, viruses doesn’t invent anything new, they actually hijack our host mechanism, right? They utilize whatever is present in our host body to for its own benefits. So we thought, okay, maybe maybe we came up with an hypothesis, saying, okay, maybe a child even and maybe add a virus, it’s hijacking a host repair mechanism, whereby if there’s a huge thing, which is sitting right at the nuclear power complex, then it’s recruiting kinase and want to actually remove this rock, and the virus is hijacking this mechanism to gain entry into the nucleus. And we wanted a way to somehow, you know, study this mechanism. So but you know, for today’s talk, I want to really go into this hypothesis, this is a completely self biological question, which, you know, we are pursuing But to do that, actually, we were looking for, you know, systems, which, you know, by which you can study this process. So, during that time, we came around this paper from a different lab at University of Michigan, in the US. So what they did is, they used a nuclear protein called nuke 62. So, new objects to do, what they did was they fused the new HP 62 onto a dimerization domain, along with a few copies of GFP, which is a fluorescent protein. And what they did was you can express this construct into any cell types. So, once you express this construct nibh 62, goes to the nuclear pore complex, because it it belongs, it’s a nuclear protein, right, so it goes and sits at the nuclear pore complex. Now, what you can do is, since it has a diamond session domain, you can add a drug which I’m calling here as a homodimers intro, right, once you add the drug in dimerizes, all those all these domains, the dmrb domains, so, once a diamond has all these domains, so, the new six to two has to be quite flexible in the central channel for you know, things to pass inside and outside. But if you dimerize or the new six door right at the central channel, it prevents active transport. So, they use this construct to basically block active nuclear pore transport. And so we were like, this is this is pretty cool like we can use this construct to study our hypothesis we can clock nuclear pore complexes and ask if kinesin is actually required to bear this block bore and this would really support our you know, virus mechanism that you know, the viruses utilizing the mechanism which the cell has enough to gain entry into the nucleus. Now, the figure here basically shows you know, the system works. Like, you know, estrogen receptor actually is basically it’s in the, it’s in the cytoplasm. And if you add the estradiol, which is which is the hormone, the estrogen can like, you know, translocate into the nucleus. But once you block the nuclear pore complex using this technique, you no longer prevent estrogen translocation and when you add the hormone, so this this is a proof of principle to show that, you know, the system wants to block nuclear power transport now, so I was like, we were like, super excited. We were like, okay, we’re going to study the cell biology process and we were like, you know, me and Dr. Campbell. Were You know, talking over this one, and we were like, Okay, what happens? Like, what happens if you use this blockade? What happens to virus infection, right? This virus infection goes down. So what we did was we made a number of cell lines. So this is HeLa cells. php wants to learn a number of different disciplines, we expressed all these cell lines with our construct. And what what we did was we infected all these cell lines with the virus with HIV one virus, and we blocked the nuclear pore complex, we added the drug to block the nuclear pore complex. And what we saw was when we added the drug, there was a drastic decrease, you can see here, if you compare this blue and, and the red bars here, you can see there’s a drastic decrease in infection. Regardless of what cell type you’re doing, when you when you block the nuclear pore complexes, you block infection. And this blocking infection is actually at the level of nuclear input. Because there is no change in reverse transcription. If you look at the later 30 copies, there is no change. But if you look at the two ltr circles, which is a measure for the nuclear import, you see that when you add the drug, there’s a significant decrease in the amount of two ltr copies, which correlates with the defective infection. So this artificial NPC blockade can inhibit virus infection at the level of nuclear input. So we were like, okay, I mean, we kind of like we, we put a halt on to our cell biological question, and we were like, Okay, this is something we should pursue, we should, we should use this technique to, you know, now monitor nuclear import. And that’s, that’s what we that’s what we did. So what we did was, I’m pretty happy to explain this essay again, at the end, if you didn’t understand it’s a little bit complicated. I say, it’s not complicated when you’re doing it, but it’s a little difficult to understand. So what we did was we took a cell line, like any cell lines, which are expressing our nucleus to do demonstration construct, and we infected the cell and with a reporter virus. So when I say a reporter virus, it’s a virus where you know it, this virus is fused to injury. So at the end of the essay, if you see an injury positive cells, that means it’s infected with the virus. And what we did was once you, in fact, it says, we added the drug at different times first infection. So you can basically consider this as a drawbridge, right? So if you look, if you think this is an mpcs, when you add the drug at different time points, you’re basically raising the gate, you’re not allowing anything to get inside the nucleus. So we wanted to ask when the virus enters the nucleus, right, so let’s say we add the drug at six hours post infection, and we don’t see any change in infection, right? That means the viruses already inside the nucleus. So by by adding the drug or by basically, or by basically blocking the nuclear power at different times post infection, you can actually see a kind of ticks of how or when the virus actually enters the nucleus. So like I said, we added the drug at different times first infection, depending on whatever time we had drug was person for the first 24 hours, and after the 24 hours, the drug was changed the vocab for another 24 hours. And then we measured the amount of mp3 positive cells, which is a measure for infection by by fax. And this is how a normal result would look like. So this is in HeLa cells. So you can see all the time points where we had the drug on the x axis, the y axis represents the number of positive cells or the infection. So the time point zero represents the time where you add the drug immediately after you add the virus. So this is a longer time the drug is on the cell. That’s why you see a greater inhibition. So this is an order of control, right? So as you keep adding the drug at different times postinfection, you can already see that when you add the profit 10 hours or when you basically block the nuclear for at 10 hours post infection, there is no significant changes in infection, that means the virus is already inside the nucleus by tenovus. Right? So this is this is basically what we’re thinking like this is basically the idea we had was like, by using this essay, we can measure the nuclear input kinetics of when the virus gets entered into the nucleus. And we can you know, this the graph on the on the right, basically, it’s a it’s a normalized graph from here, where we actually set the time point zero as the baseline infection and the non drug as the 100% control. And then you can normalize every other time points, and you can actually get a nice nuclear import kinetics curve of the virus in that specific cell line. And as you can see, in HeLa cells, it the half time for the virus to get into into the nucleus is close to seven hours, by by by around nine hours, 10 hours, the virus is already inside the nucleus. And we did the same for a number of different cell lines. For a number of actually relevant cell lines I would say like macrophages are a more relevant target cells which the virus infects T cells definitely. And what we observed was was quite interesting in the cell. And so comparing it to heal ourselves, HeLa cells are it’s a good model cell line, you know, to study different processes, but they are not actually the real target cells for infection, right. But if you look at this relevant cells, the off time for the nuclear input is like way faster. It’s like Iran firewalls, the virus is already inside the nucleus, compared to HeLa cells, like, like for macrophages, around 3.5 is the half time for T cells around the same like, by around six hours in mostly all the cell lines, the virus is already inside the nucleus. So this kind of like raised immediately a number of questions for us, like this cell and actually have been shown to have a very delayed reverse transcription, like you know, reverse transcription where you know, the viral RNA is converted into DNA, it takes a long time in the cell lines to complete. And any classical life cycle died. Rom if you you know, Google, let’s say Google, HIV one lifecycle diagram, they all tell you that the virus was transcription is completed in the cytoplasm before it enters into the nucleus. So we were like, no, this is not true, because by by since there was the viruses already inside the nucleus, and reverse transcription cannot be completed by six hours, it’s been shown that it takes almost 24 hours for reverse transcription, you know, for the process to happen. So somehow, all the scar turns, which you have been seeing for the last decade, or 12 years is where like, Oh, that’s wrong. I mean, it’s not really Right. Right. So we were like, okay, we should we should be really now looking, we should be asking if reverse transcription is really completed prior to nuclear import. And also, the next question we were asking is like this, the capsid actually play a role inside the nucleus. So to kind of address the first question, what we did was, we did kind of a very similar essay, which I talked about, we took cell lines, which are expressing our new HP 62 dimerization construct, we infected with the virus and report a virus. And on the first condition, we added the drug like the same as before, we added the homodimers in drug to block the nuclear force and different times first infection. But on a second condition, what we did was we added a drug called v rapid so we’re wrapping is HIV one, it’s a very specific HIV one, reverse transcriptase inhibitor. So and so what we and we added this burlap in a different times post infection, again, respective of what time you add, the drug was kept for the first 24 hours. And then after under 24 hours, you measure infection. So what we hope to see was, if reverse transcription is really completed in the cytoplasm, before nuclear import, so by the time the virus is already inside the nucleus, when we add the wrapping the drug, which blocks reverse transcriptase, it should have no effect on infection, because it’s already inside the nucleus and, and this will really support the last 12 years of the virus life cycle. But that’s exactly not what we saw. So if we look into this graph here, the blue and the red one, that’s basically so there are two different cell lines. One is mdms. And other one our CD for positive T cells, which are infected with the virus. And then the blue and the red are basically you’re adding the homodimers in drug at different times for infection. So they’re basically measuring the nuclear input candidates. And as you can see, for both the cell lines, by around six hours, the virus is no longer sensitive to this block, it’s all that tells you like the virus is already inside the nucleus. But if we look onto the green and the orange graph, where you add the verapaz, in a different times first infection, you can see that even at six hours, it’s still getting inhibited. So virus infection is still affected when you add the drug, even then I was even at 12 levels. So this tells you that even though the viruses inside the nucleus, the drug is still affecting the virus infection, or in a different term that the virus was transcription is still an ongoing process. It’s not completed in the nuclear, it’s not completed in the cytoplasm, it’s still unknown, it is still an ongoing process that happens inside the nucleus of the cell. So this was like this was literally a game changer because this was like completely you know, redrawing or re drawing all the life cycle diagrams, you know, please see in all the textbooks, and we confirmed this also using a different essay, more microscopic essay. So what we did here was, we took we took, we took thp one cells which are differentiated into macrophages, we infected with the virus and we fixed the cells at different times post infection. So fixing means you’re basically rendering the cells metabolically inactive. We do an illness treatment. I think it’s not Really important at this point. So what we did was we used fish props. So these are props which are able to detect specific sequences. And these props are labeled, right. So what we did was we use fresh fish props to detect either the negative strand, or the positive one a DNA strand. So once the viral RNA is converted into a DNA, you know, DNA has a negative and a positive strand. The neck strand is the first strand that’s getting synthesized. And then it’s the positive strand. So we used fish probes, which either this detects the negative, or the positive strand. And we also cause change the same cells with a capsid protein and a lemon using antibodies. So again, this is kind of confirming what I just told you, as you can see here is by until like six to nine hours, what you can see here is you only detect the negative strand, you don’t detect the positives. But by around 12 hours, you start to detect the positive strand, which is a red strand, all the way down to the bottom here. And even that’s inside the nucleus. So the new color boundary is marked by the slammin stain. And you can see that only by around 12 hours, you start to detect the second strand of the water DNA. And by around six hours as you can see in cell and nuclear input should be completed. So this again, like you know, this is quantified in this graph here that you start to detect the the second strand by around nine to 12 hours. So this shows that the nuclear import proceeds the completion of reverse transcription in in all the relevant cells, which the virus infects. Now the second question we wanted to ask was, does the virus capsid fully disassemble before nuclear and break before nuclear entry? So once what is virus capsid assembly now, if you remember the life cycle I talked about, once the virus enters the cell, it releases the, the capsid shell into the cytoplasm, right, and the capsule, Shell has all the viral RNA and all the extra proteins. Now, the water, RNA gets converted into DNA, but it was transcription. Now at some stage of the wires lifecycle, right during the early stage, the capsid has to disassemble, it has to break out for the reverse transcribed DNA to get outside and integrate with our host chromosome. And so that process of you know, breaking out the word scabs and that’s gonna capture this assembly. Now, the field of capsule disassembly it’s it’s it’s quite complicated. I mean, it has been going around for the last 20 years, like where does this disassembly happens, people were talking, like initially, they were talking about the capsule disassembly happening right after entry, which I think most people don’t regard at this point. Because it doesn’t make sense if the capsid get disassembled immediately, because then you’re you’re making your RNA to be sensed by our host mechanism. There’s a second model where they say is the capstone course, once it travels down to the new place, like it’s a gradual uncoating, it slowly start to shed its shell, once it travels down to the nucleus, and once it’s at the nucleus, you completely lose the absurd, and whatever that’s inside the capsule gets into the nucleus. And then there was a third model, which shows that the capsule is mostly intact, which this is the this was the most preferred model until recently, that the capsule capsule remains mostly intact until it reaches the nucleus, because it makes sense that you have to protect everything which is inside the capsule. And once it’s at the nucleus, the or once it’s at the nuclear pore complex, the capsule disassembles and then everything which is inside the capsule gets into the nucleus. And so like Like I said, there’s a number of models actually for the sceptre disassembly. And we were like, okay, maybe maybe our essay could actually tell us something about the capsule disassembly process. So, and this is again to show you that people have shown that there’s a lot of gaps inside the nucleus like people, they can they can detect a small amount of capsule inside the nucleus, but they were they were having no idea of what this capsule actually plays a role inside the nucleus. So what we did was we used we used a compound called bfcm before so bf stands for it’s a capsid binding compound. I mean, it’s right now in a number of clinical trials to actually block infection. So what we have Sandy for binds is the so a little bit into the into the structural biology of the virus capsid, the virus capsid it’s actually made up of a number of hex hammers and Panama’s right. So the all the hexagons and panoramas are arranged to form this conical structure. So PMC four is actually known to bind to this hexameric structure. So the hexameric structure is already present in our in our assembled cap set, like when you have a conical cap set. So what we did was kind of a similar approach. As the art experiment, so we took cell line which is expressing our musics to do our dimerization construct, we infected with the virus. And after infection, we either we added either the homodimers syndrome, in order to block the nuclear pore complex or in a second set, we added this drug at various times plus infection. So, what we were thinking is like, you know, if the virus is completely disassembled before nuclear import, then when you add the drug at those time points, when the virus is already inside the nucleus, it should not affect infection, because this drug would only bind to an assemble form of capsule. So, again, depending upon what time you add, the drug is kept for the first 24 hours then after another 24 hours you measure infection. And what we see here is again, more interesting. Again, the blue and the red are two different salons where we measured in training for kinetics and again, you can see by six hours, most of the virus it’s not the you know, it’s no longer sensitive to the block. So the virus is already inside the nucleus. But at this time points even when you add the drug, the PF 74 which binds to an assemble form of capsule it still inhibits infection. So that tells you that there is an assembled like almost a fully assembled form of capsid which is passing through the nuclear pore complex and all the way into the nucleus so all the models which have shown where you know there’s a gradual loss of capsule in the cytoplasm is to be frank not true. It there’s, there’s it’s almost like a whole capsule is still present when the viruses all the way like when the when the virus has finished its process of nuclear import. So just to summarize, I think I might be really on time that using this essay, we were able to actually monitor the nuclear mechanics in a number of different cell lands. And our whole point when we actually you know, develop the system was just to monitor nuclear import candidates, we wanted to see when does the virus finished nuclear important member of survive, but you know, as like, like you said, you know, when you keep doing experiments, it opens up more and more, you know, relevant questions. And we were able to show actually, that, like we were kind of the first lab to show that you know, the reverse transcription is a process which is not completed in the cytoplasm and it’s still an ongoing process once the virus has finished nuclear import. And also, there’s a presence of an assembled form of capsule inside the nucleus, which is relevant for a productive infection. And slowly I would say, the viral life cycle is changing based on all our studies and also from a different also from a number of studies from other labs, that you know, this virus capsule, like if you look at the lifecycle from a from a review last year, you won’t see an assemble captured in the cytoplasm. But now things are changing, they are kind of drawing an assemble capsule in the cytoplasm basically from our study and from other study, that there is an assemble capsid even inside the nucleus, and reverse transcription is still happening once the viruses has completed the nuclear import process. So with that, I would I would, I would basically stop I would be happy to take any questions. I mean, this is all the people in the lab, all our collaborators, which you know, we are collaborating for constructs for help, I mean for experiments, my funding sources and definitely rely on for the position and I’ll be happy to take any questions. Are you guys here? Sorry, I can I can’t hear you Yeah, hello. Yeah, yeah, sure. No. Shall we move to the show? Okay. So the first question I’ve been asked by devices. Personally, why are vaccines not effective against HIV as it is against other RNA viruses? I mean, it’s it’s it’s it’s that that’s because of the exactly kind of one of the thing he mentioned in its in his question. It’s an RNA virus. So there’s more chance I mean, so I can I can actually give you an example of from our current virus, you know, the virus, which we have right now, the source code we do. From from the time the virus came in, in Jan, 2020. Until, until until yesterday, there’s already four different variants, right? There’s the alpha, beta, gamma delta, right? And that’s because it’s an RNA virus, it rapidly mutates in the body. Right? And all the mutations in tsarskoe? We do, it’s linked to the spike protein. And that’s the same thing with HIV one, that there, there there, there has been a number of like, so if you look at the vaccine, the vaccine history for HIV one, it has been progressing for the last 20 years. I mean, there’s a number of companies which are, you know, making vaccines, but the problem is this vaccines work for six months. And then like, it’s not even like, I mean, it’s not even in market when they do the trial, the vaccine works for six months, and then suddenly the virus just mutates the virus adapts to the vaccine. And that’s, that’s because it’s it’s the most intrinsic nature of an RNA virus to mutate itself. So that that’s that’s answer why vaccines are not effective. So the best strategy, I mean, it’s easy to make vaccin but it’s still a long trial, you know how to make it. But the best approach right now people are trying is to actually inhibit infection or inhibit the viral load by targeting the different pathways the virus uses to infect yourself. Okay. So the next question is, as my wife Sharky, we should not wear the jacket itself that should positive for infection tested for subsequent infections. Where the drug treated cells, which are which drug, are you talking the drug, the hope the homodimers in drug I used? I kind of did not get that question. If you’re actually talking about the homodimer rising drug, so this once you trade it’s actually not so sorry, if I if you didn’t actually get it, it’s so that drug is basically when I say the word homodimers. In drug, it’s a drug which basically blocks active transport through the nuclear pore complexes. So it actually prevents infection. So we did not so with all the viruses which we have been using in our study, there are single round infection. So these viruses are only able to infect the cell phones because it lacks unnecessary viral protein called Neff. So it can subsequently infect a cell. But we haven’t we haven’t tested you know, to see what are viruses which which, what about cells which survived infection, can they be infected? I can, if that answers his question. Okay, so is there any more questions? So I get upset. Okay. Yeah. Thank you. Thank you, Dr. Better. No problem. Thank you so much. Thank you. Thank you for hosting me. I appreciate it. Thanks so much.
