In HPV31-positive keratinocytes (CIN612), TOP1α and TOP3β are upregulated and required for viral transcription and replication, acting via distinct effects on R-loop accumulation and topoisomerase-mediated DNA breaks.
0:00Welcome to Base by Base, the papercast that brings genomics to you wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. Good to be here. All right, so today we're diving into a problem that, uh, it sounds more like it belongs in an engineering class, but it's actually a central crisis for one of the most common viruses on the planet.
0:19Right. I want you to picture a physical cable. You know, like one of those old school coiled telephone chords. Okay, got it. Now, imagine you hold both ends and you just start twisting. You're not stretching it, just twisting, and you get twisting and twisting.
0:34Eventually it doesn't just get tight. It starts to nod up on itself. It kinks. And if you keep cranking up that tension, eventually. You snap, it fails. That's a classic example of torsional stress. Super coiling.
0:47Exactly. And here's where the biology comes in, because that isn't just a metaphor. That exact physical stress is happening inside your cells every single second. Your DNA is a double helix. Two strands twifted together.
1:00And every time a cell needs to, you know, read a gene or copy its DNA, it has to pull those strands apart. Which, by definition, overwinds the DNA ahead of the machinery. You can't avoid it. The more you unzip, the tighter the knot gets down the line.
1:13Yeah. For a human cell, this is business as usual. We've got enzymes to handle it. But today, we're looking at this from the virus's point of view. Specifically, human pepaloma virus. HPV. Okay. So if you're HPV, you're inside a host cell and you need to replicate like crazy.
1:30You are generating a massive amount of this torsional stress on your own little circular genome. And if that viral DNA snaps or gets so tangled up that the copying machine just falls off, well, that's game over for the virus.
1:42So the question for our deep dive today is, how does HPV solve this huge physics problem? I mean, it has a tiny genome. It doesn't rum its own molecular scissors. It has to steal the host. Right. But as we're about to find out, it doesn't just borrow them.
1:55It hijacks them, turns them up to frankly dangerous levels, and then uses them to break things on purpose. Which sounds completely backwards, but it turns out to be this brilliant survival strategy. To understand it.
2:10We're breaking down a paper called Differential roles of Type Bite to Poisom races in regulating HPV pathogenesis. And we should definitely give a shout out to the team here. This is fantastic work from Arushi Vats, Conor W. Templeton, and Lehman's.
2:25All coming out of the Department of Microbiology Immunology at Northwestern University. Yeah, they're a great group, really reshaping how we think about the molecular machinery behind these viruses. So before we get into the mechanics, Let's just set the stage.
2:37HPV isn't some rare virus. Oh, not at all. It's responsible for about 5% of all human cancers. We're talking cervical, oralpharyngeal, anal cancers. It's a huge clinical problem. And the really tricky part is it's life cycle, right?
2:52It's not a quick in and out kind of virus. No, it's a squatter. It infects what are called basal caratinocytes. Think of them as the stem cells at the bottom layer of your skin. Now, normally as these cells move up to the surface, they differentiate, and crucially, they stop dividing.
3:08basically, yeah. But HPV needs that replication machinery to be active, so it can make copies of itself. So it forces these retiring cells to stay on the job. It shoves them back into the cell cycle. And that's where the stress comes from.
3:22A cell that should be dormant is forced to run a marathon. That's where you get all that DNA super coiling. Exactly. And that's why the cell and the virus need these tools we mentioned, the tapoisomerouses.
3:35The untwisting enzymes. Right. And we're focusing on a specific class today. Type I to poisomoruses. So what makes them type I? It all comes down to how they cut the DNA? A type I to poise Amorys makes a nick in just one of the 2 DNA strands.
3:51It holds onto the ends, lets the other strand rotate through the brake to relieve tension, and then it glues it back together. So it's a controlled brake. Clip, spin, seal. Perfect analogy. It's much safer than a type two, which cuts both strands snapping the whole chromosome.
4:04Now, we've known for a while that some viruses, like Kirpes, really depend on one of these called TOP1 Alpha. But for HPV, it was kind of a question mark. A huge one. I mean, our cells have 3 main type I enzymes, TOP one alpha, TOP 3 alpha, and TOP 3 beta.
4:21We just didn't know which ones HPV was using, or if it just grabbed whatever was around. And that's the mystery this Northwestern team wanted to solve. It is. And I really want to highlight their methodology here because the cell model you use is everything in this kind of research.
4:34Right. They didn't just use standard hela cells. No, and that's so important. Heila cells have the HPV genome actually integrated, you know, stuck into the human DNA. But that's not how a real infection works.
4:46In an active infection, the virus exists as an episode. A little free floating loop of DNA. Correct. So they use a cell line called CIN 612, which comes from a real pre-cancerous lesion. And in these cells, the HPV genome is maintained as those free floating loops.
5:02So the physics of the problem, the twisting of a loop, is actually authentic to what's happening in a patient. Precisely. The model is right. So they have the right cells. How do they figure out which enzyme is the important one?
5:12This is where the cool tools come in. Yeah, 1st up is something called lentiviral HHRNA. You can think of it like a genetic sniper rifle. unless you specifically knock down, or silence, the gene for each poise hom race, one by one.
5:24So you take away a tool and see if the virus factory grinds to a halt. Exactly. Then they use Chen AP assays. This basically tells you where the enzyme is. Is it sitting on human DNA or is it parked on the viral DNA?
5:37Very cool. And they also used a couple of others to look for damage, right? They did. Comet assays, which are just as visual as they sound. You put the cell's DNA in a gel, and if it's broken, the fragments stream out like a comet's tail.
5:50A longer tail means more damage. So a very direct way to see if things are breaking. And DRIP assets, which we'll get back to, because they detect a very specific kind of genomic mess called an R loop.
6:01Okay, so let's get to the findings. The very 1st thing they noticed was just the sheer quantity of these enzymes. Yeah, it was. It wasn't a small difference. In the HPV positive cells, and they check this in actual cervical cancer biopsies, too.
6:13The levels of all 3 enzymes, one alpha, 3 alpha, and 3 beta were through the roof. How high are we talking? Up to 6 times higher than in normal healthy cells. Six times. So the virus gets in and just cranks the production of these things way up.
6:26It absolutely does. The viral onco proteins, E6 and E7 are directly responsible. They're telling the cell, flood the place with these scissors. So your 1st thought is, okay, the virus must need all 3 of them.
6:37Why else make so much? That would be the logical assumption. But when they use that genetic sniper risel to knock down TOP 3 alpha, nothing happened. Nothing. The virus just kept going. Didn't miss a beat.
6:50Replication was fine. And it turns out TOP 3 alpha mostly lives out in the cytoplasm and mitochondria. It's not even really in the nucleus where the action is. So it's like the virus just orders a combo meal, and TOP 3 alpha is the side dish it doesn't even eat.
7:05That's a great way to put it. It's just a byproduct of this blunt command to make more depoisom races. But the other two, TOP1 Alpha and TOP3 beta. That's a different story. A completely different story.
7:16They are absolutely essential. When the researchers knock down either of those, The whole viral lifecycle just collapsed. Replication stops, transcription stops. Everything. And the GPP data showed exactly why.
7:27They were both bound directly to the HPV genome, and not just anywhere. They were at the URR, right? The upstream regulatory region. Which is the command center, is where the origin of replication is. It's where the major promoters are.
7:41These enzymes were sitting right at the controls. So we know the virus is addicted to TOP1 alpha and TOP3 beta, but the really cool part of this paper is that they aren't interchangeable. They do different jobs.
7:52This is where it gets really elegant. They started looking at what else went wrong when they removed one of them. For instance, when they took out TOP1 alpha, they saw this huge drop in a cytokine called IL6.
8:04Interlucan 6, that's an immune signal, right? It is, a pro-inflammatory one. And you'd think a virus would want to quiet the immune system down, but it seems like HPV needs to fine tune it. TOP1 alpha helps regulate that environment, and it also helps manage P53 levels.
8:20So it's not just untwisting DNA. It's also a diplomat, managing the cell's internal politics. In a way, yes. But TOP 3 beta. Its job is, you could argue, even more critical. It's the janitor. Okay, this brings us back to that genomic mess you mentioned before, the R Loops.
8:39The R-Loops, yes. So very simply, DNA and zips, a strand of RNA is made, and then that RNA is supposed to float away. The DNA zips back up, clean. But sometimes it's not clean. Not always. Sometimes that new RNA strand is a bit sticky and it actually hybridizes back with the DNA template strain it was just copied from.
8:57So you get this 3 stranded structure. One strand of RNA wedged into the DNA double helix. sounds like a major problem. It's a disaster waiting to happen. It a physical roadblock. When the replication machinery comes racing along.
9:08It slams into that R loop and the whole thing and just collapses. You get DNA breaks. It's incredibly toxic. And since HPV is forcing the cell to work overtime, It must be making a ton of these R loops.
9:18A ton. And this paper shows that TOP 3 beta is the specific solution. When they knocked down TOP 3 beta, our loops went through the roof everywhere. So without it, the virus literally chokes on its own transcriptional garbage.
9:31Exactly. And what's fascinating is that TOP3 beta doesn't work alone. It acts as a recruiter. It brings in other proteins, like DHX 9, which are hella cases that specialize in physically unwinding these knots.
9:43So TOP 3 beta is the foreman that spots the problem and calls in the specialized crew to fix it. You've got it. So now you see the division of labor. POP1 alpha handles the primary twisting and immune signaling.
9:55POP3 beta is the RLU cleanup crew. But there's a paradox here we have to talk about. We said these enzymes prevent DNA from snapping, but the paper shows that having such high levels of them is actually causing more DNA breaks.
10:08This is the most counterintuitive and maybe the most brilliant part of the whole story. Remember, the enzymes job is to cut, let's spin and reseal. Right. But if you have way too many of them working in a high-speed environment, sometimes they get stuck, they get trapped in the cut phase, covalently linked to the broken DNA.
10:25This is called cleaved complexes or TOP1CC. They are a form of DNA damage. So by cranking its own helpers up to 6 x normal levels. The virus is creating more damage. That seems self-destructive. It would be, except for one thing.
10:39When a cell detects DNA damage, it activates its alarm system. The DNA damage response or DDR, specifically pathway is called ATM and ATR. The emergency repair crew. The emergency repair cruise. You'd think a virus wouldn't want those crews showing up.
10:54That it does. It does. HVV hijacks those repair factors. It needs them. It recruits them to its own replication centers and uses the cells repair machinery to help replicate its own viral DNA. Oh, wow.
11:06So it's creating a controlled crisis. It intentionally breaks things a little bit using these access to poisomeruses just to summon the repair crew that it then enslaves to build more virus. That's the model.
11:18It's weaponized DNA damage. Break things just enough to get the help you need, but not so much that you kill your host cell, at least not right away. That is just, it's diabolical. And it totally reframes these enzymes.
11:29They're not just maintenance tools. They're strategic weapons in this viral takeover. Which brings us to the clinical take-home message. Yeah. Because we have drugs that inhibit depoisomorases. Right. Some chemotherapies, but they're like sledgehammers.
11:42They hit all dividing cells. Incredibly toxic. But this study reveals a specific vulnerability. These HPD driven cancers have a unique, profound addiction to TOP3 beta and it's our loop cleaning service.
11:55Something a normal, healthy cell probably isn't as desperate for. Exactly. So the idea is, what if you could develop a drug that doesn't just block TOP 3 beta itself, but maybe blocks its ability to recruit that cleanup crew, DHX 9?
12:10You could, in theory, let the virus drown in its own our loops. While leaving healthy cells, which are moving at a slower pace and have other backup systems relatively unharmed. You'd be targeting the virus's specific addiction.
12:22Moving from kill all fast growing things to exploit this virus's specific bad habit. And that's really the future of targeted cancer therapy. It's just amazing how drilling down into the basic physics of a DNA molecule can point us towards such a clever therapeutic strategy for a major cancer.
12:39It's why basic science is so fundamental. You have to understand the machine before you can figure out how to break it. A perfect place to leave it. What an incredible piece of molecular detective work.
12:48It really is. A pleasure to walk through it. This episode was based on an open access article under the CCBY 4.0 license. You can find a direct link to the paper and the license in our episode description.
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