Human Papillomavirus: from basic mechanisms to a new antiviral paradigm

January 31, 2019

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Yale Cancer Center Grand Rounds | January 29, 2019

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00:00In the mail and Department of genetics just wanna get my timer starting.
00:08So I'm going to talk to you about some recent work. We've done on human papilloma virus infection and I want to turn the lights down so if you want it fall asleep that's OK try not to snore.
00:28OK.
00:31So papilloma vocal first off I should say have a disclosure. I'm inventor on a patent application related to this work.
00:40So papilloma viruses are important human pathogens of these are small and simple viruses with a single DNA molecule genome. The only encode about 10 proteins and like most DNA viruses. They replicate in the nucleus. The very important medically 'cause they cause cancer. In fact about 5% of all human cancer deaths are.
01:01Cancers caused by these viruses in the most prominent of which is cervical cancer.
01:07However, if you heard last week, Doug Lewis. There are wonderful vaccines that prevent infection by many of these viruses and we're hopeful that within the next few decades, will be able to eliminate the major cancers caused by these viruses. But we have a long way to go, but many people are not vaccinated still.
01:25As I said the simple viruses. Here's a picture tomorrow. Particle does not have a membrane envelope is in an envelope virus there. Only 2 proteins in the capsid both coded by the virus. The L one major protein and then the L 2 protein which is so called minor capsid protein's I'll convince you I hope it's actually a very import.
01:46As I said a DNA genome.
01:49The bars particles actually a very efficient DNA delivery machine. It does many different things that protects the genome from the elements when it's outside cells, but it binds to cells gets taken up by cells deliveries of genome to the nucleus aside of replication disassembles to release the genome so it can replicate then innovate.
02:10I'm going to tell you about one particular aspect. We've been focusing on and that is the delivery of the viral genome to the nucleus when we began this work almost nothing was known about this process. In fact, our knowledge to be summarized by this cartoon HD into the endosome the miracle occurs and it gets to the nucleus.
02:31Here we felt we should be a little bit more explicit in step 2, so we decided to undertake a genetic analysis of HIV entry so Elvis Lapaz. KUSA grass seed in my laboratory conducted in SIRNA whole genome screen to identify cellular proteins required for infection and based on Alex is results.
02:51Studies done by ways hang in the laboratory of very unexpected picture emerged and that is the virus takes a very unusual. In fact, unprecedented wrote to the nucleus after sinus. I toast it travels through transport vesicles to the Trans Golgi Network. They need the Golgi apparatus. They did to the ER before it finally gets into the nucleus.
03:12So the viruses is inside membrane bound organelles his entire route to the nucleus. This is probably a good immune evasion strategy avoids cytoplasmic commute sensors, but really is a quite unusual path. This pathway is called a retrograde transport pathway of refer to it as that we got interested in this first step.
03:33Paris goes from the endosome were many viruses are to the retrograde pathway with nothing. No other viruses known to utilizing this way, and in particular on approaching call retromer, which is a cellular proteins that normally carries other subtle proteins into the retrograde pathway and what's the role of red shimmer.
03:54Affection.
03:55So we study this we did some image NG Studies. Initially and ask what's the consequences not be bound rectum are for infection.
04:06We use them as a Co PLA for proximity ligation assay in this a resident signal is generated if the virus is in a particular organelle. So here we're looking at virus in the end assault as you can see at 8 hours after infection. Awhile type cells virus gets into the endosome acentos. I toast, but by 8 by 16 hours is left in that store.
04:27Did the endosome and gone to the trans golgi net?
04:31However, if you look in knockdown cells where he knocked down the rectum are you see a very different picture of the virus gets in just fine, so richer knockdown does not affect internalization. However, it never leaves the endosome. In fact, it accumulates here. So when you knock down the rectum are the virus is impaired horrendous on exit.
04:50So that's schematically here we knocked on the rectum are the virus accumulation the endosome. It never makes it to these more distal compartments.
05:01So that suggests that perhaps the the viruses using the rectum are to enter cells in some way we wondered is the virus. A actual cargo of the rectum are so we asked does record bind to the virus. If so I'm going to pay my laboratory looked at the sequence of this L 2 minor capsid protein and found a short sequence here.
05:22To look a little bit like a rectum are binding site. So we decided to test set, she constructed peptides that covered 3 different sections of the L 2 protein label. The peptides with Barrett in as in did a pull down experiments so she added. Each one of these peptides. This whole extracts pull down with Jeff evident and then blooded forever and as you can.
05:43Featural peptide that contains this computer binding site. In fact, Lions rectum are very well by these other peptides failed to bind rectum are suggesting that in fact, the virus this segment. In fact does bind to the sailor trafficking molecule. She then are constructed. Mutations in this binding site or in this site and asked that black.
06:04And it does so that site is required for binding and with Chris Byrd, a colleague in cell biology. We show the finest direct using recombinant proteins. She then inserted this mutation into virus and ask does it block infection and in fact, it does block this binding site is required for entry and in fact if you take this site the virus Accum.
06:25Just like what happens if you knockdown rectum are so if you knockdown retromer or mutate the binding site get the same phenotype, namely the virus is trapped in the endosome an infection can progress so this says that in fact, the rectum are deliveries HPV if the retrograde pathway.
06:45To the nucleus that then raises the question? How does this happen? How does the richer access of HPV.
06:54This represents the particle we see mostly L, one which forms the shell. The part of the virus and maybe this little nubbin avail 2 sticks out so combine the retromer, but there's a problem and the problem is if the membrane in the way so the viruses in the end is in the lumen of the endosome whereas Arete Rumor is in the cytoplasm.
07:14Molecules can't see one another so that then let us to propose an unusual model that basically this L 2 protein which is normally part of this non envelope virus is actually able to stick through the the initial membrane protrude into the cytoplasm where combine rectum are and this is a very unusual thing protein.
07:35This so we thought it was important to test whether or not the L 2 protein actually protrudes into the cytoplasm.
07:43To do that pink anyways hang in my laboratory set up a split GFP S 8 and the principle is the following or you could take GFP, which is a fluorescent protein, but cut it into 2 parts a short part of only 16 amino acids and then the rest of the protein. Both of these segments are they don't do not fluoresce on their own, however, if they're expr.
08:04Apartment they can self associate reformed reconstitute GFP and reconstitute fluorescence, so this could be used as a measure for whether these 2 halves of GFP or in the same cellular compartment, so the idea. Then is to fuse this little bit of GFP to the L 2 approaching the end of the L 2 protein that we think for truth.
08:25And express GFP one to 10 in the cytoplasm, then ask whether or not. We can generate fluorescence. So Pangui made virus particles that contain GFE a fuse to L 2 infected cells. These cells have an engineer to express GFP went to 10, only in the cytoplasm so if L 2.
08:46Endosome there's no fluorescence, but if this end of L 2 sticks through the end of swimming to the cytoplasm. It can reconstitute GFP and generate fluorescence. So we did. The experiment and in fact, it worked. This shows if we infect cell if you use a reporter cells with GP one to 10 in them if we in fact with?
09:06Doesn't have GFP 11 there's no fluorescence? But if you fuse L 2 to the end of GFP 11 then you get this nice cytoplasmic fluorescence exactly as you would predict if the L 2 protein is protruding into the cytoplasm. This is true for HPV types 16, which is the most common type in human cancers and what I've talked about so far.
09:27Valve 16, however, it's also true for HP 5, which the quite distinct HIV type that insects skin.
09:36So then how does this protrusion occur again as it is a very unusual reaction? Well Penguin look at the sequence of the end of L 2 and here's this rectum are binding site that I mentioned before this al foil sequence represents directly to this, but she notices another sequence downstream here. It's very basic R Ray.
09:56Presents are getting K Rivers and slicing to basic amino acids and this sequence or a close variant of it is absolutely conserved. In all 400 sequence papilloma viruses, so this emerged. Hundreds of millions of years ago and has been maintained in papilloma viruses. So we wonder. Perhaps that this sequence of might be important for infect.
10:18In fact, it looks to us to our eyes it looked like a secretly already knew something about with recall cell penetrating peptides. So so penetrating peptides are short protein sequences that can deliver proteins into cells. They were first discovered over 30 years ago, the most common type. In fact, the first identified, was an HIV.
10:38Similar sequence to the papilloma viruses and these are called cationic. In fact, even probably are Janine can suffice to deliver proteins into cells.
10:49Uh sell picturing peptides have been widely explored's drug delivery tools to deliver peptides into cells, though, as far as I know they're not actually in clinical use. Yeah, however, despite 30 years of study really don't know what they do in biology. The natural role of CPS is basically very poorly understood and we thought, maybe we stop.
11:09A real activity of these sequences to deliver the papilloma virus into the retrograde pathway, so first. We asked whether or not the sequence acquired for infection. So he just simply replacing with 6. Allah names and this totally abolishes infection. Not surprising as I said, This is a very conservative sequence. Similarly, 3 Arjun.
11:30Active in 3, Arginines is not long enough to serve as a good CPP so then we then decided to actually test directly is the sequence does it have cell penetrating activity using the standard assets? Which is diffuse it GFP purify Fusion proteins in bacteria than out of the cells.
11:51The wildtype sequence with the wild type basic region and then a couple of mutants. And as you can see when you add them to Hackett keratinocytes. The Wild type protein is very efficiently taken up by cells, causing the cells to florists and we're looking here at intracellular fluorescence on the other hand, the 6 a mutant is totally defective for uptake.
12:12Mutant is almost totally defective is quite in period for uptake in this parallel is exactly the phenotype of viruses that carry these mutations in at the wild type virus. I was the infect cells well. It's a mutant is totally effective for infection in the 3 R Mutant is quite impaired for infection. His Susan with the idea that this segment might be important.
12:33Infection by acting as itself penetrating peptides, so, based on this we hypothesize the L to self care training peptide drives. The C Terminus of L 2 through the end of cell membrane into the cytoplasm where it's in a position to bind read trimmer.
12:48We can test that will display GPS so here this shows a split GF. PSA the wild type protein generates fluorescence as shown here. The Westerwelle type virus generates lessons is shown here. But if you take this cell penetrating peptide sequence. You almost entirely block protrusion. So you actually do need the self penetrating peptide sequence in or.
13:09Rain into the cytoplasm and if you build these mutations into virus at the self penetrating mutant fails to bind rectum are in infected cells as we predicted this in the wrong place doesn't get into the cytoplasm and Furthermore dispute accumulates in the end assault.
13:27So based on all this week and propose a somewhat coherent model for how this step of infection takes place when the viruses innisai toast. It initially goes into the lumen of the endosome then under the action of the cell penetrating peptide. The protein protrude through the membrane into the cytoplasm where.
13:47Amber and other trafficking factors that are essential for infection. Furthermore, once this happens. We think there may be a trend membrane domain at the end N terminus of the L 2 protein that Sam Campos described that perhaps anchors L 2 into the internal membrane so this is really is. Remarkable reaction if you think about it.
14:08Tombola virus that's composed a sizable proteins under some trigger perhaps low PH. The CPP becomes activated allows us to 2 sticks of the membrane bina. Central factors that be converted from a cyber protein into a transmembrane protein so that character in that first cartoon was right. This is a miracle that happens during insect.
14:30So to summarize this part of the talk HPV traffic through the retrograde pathway to the nucleus of RL sell pantry in peptide transfers. A segment of L 2 from the end of some into the cytoplasm once it's in the cytoplasm. The Seidel Plasmic. Rectum are combined to the L 2 protein and transfer it into the retrograde pathway, therefore issues and uh.
14:51Mccargo that utilizes a unique mechanism to access the retrograde pathway use works been published in it in a series of papers.
15:00It also tells us something about what is the role of self penetrating peptides in biology as I said, since 1988. They've been thought to transfer proteins into cells. That's not what this one does rather it transfers. A protein or protein segment from one intracellular compartment into another and possibly conversa cycle protein into a transmit.
15:21So maybe the reason, people have been able to figure out what these segments do that are looking for the wrong activity. Maybe they don't serve to take proteins into cells, but rather to move proteins around inside cells and perhaps are misnamed and rather than being considered self penetrating peptides really should be called membrane penetrating peptides.
15:41So, in the last few minutes. I'm going to buy some unpublished work. How can we use this basic mechanistic understanding to imagine a new way of preventing virus infection and tell you about the progress we made and again this is work by Penguin.
15:55So here's our model L 2 sticks into the cytoplasm binds rectum. Ernest essential for infection. If that's true. We imagine that if we could flood cells with short peptides that buying Rhett rumor that might they might all by in the rectum and titrate retromer away from incoming virus so the incoming vice.
16:15First would not be able to accept so the first goal. There was to design a peptide that might do this so we designed a short design a short peptide that contains record binding site. That's essential for this idea to work now. The problem with peptide. Therapeutics and they don't get it sells very well.
16:36Played into cells where the viruses figure that out for us, namely has itself penetrating peptide. So we simply can take a short bit of L 2 that has the greater binding site. This cell penetrating peptide. We would predict this would drive the pep 8, into the cytoplasm where this goodbye in Gresham are in black infection as controls we?
16:56Built to mutant peptides one with a mutant in the Richmond binding site and another with a mutation in the penetration sequence. Well, this peptide very efficiently blocks infection here. We're looking at infection by wild type. HPV 16 virus at different concentrations of peptide you're very nice dose dependent inhibition of infection with.
17:1750 year round for micromolar so the peptide does block infection. It doesn't block SC 4 DLC for is totally unrelated bars that does not use restroom are so that's as predicted, and if these doses of our peptide doesn't display any obvious toxicity.
17:36The next couple of experience I'll show you experience using but this level of peptide around 14 micromolar, which is quite effective as you see here.
17:46First off we ask does it also block other HPV types? What I showed you before was type 16 but it also blocks type 18 like 60 in a high risk general type and also type 5, which I mentioned before is a skin type so this peptide effectively blocks several cancer associated HPV types and then look at the mutations here are 2 mutation.
18:07Trading peptide they both eliminate activity 'cause It can't get into cells and this is a mutation in the rec room binding site. This also eliminates activity 'cause It can't find retro.
18:18So then we wanted to perform the experiments to confirm this proposed model of action mechanism of action so first. We wanted to say does this peptide really prevent finding of retroverted virus and it does here. This is a PLA experiment. So the wild type virus 8 hours after infection you can see nice finding of.
18:39To the L one purple actually to be able to protein, but you measure L one here, however, if you had the peptide that inhibits infection. You see you eliminate binding of the virus to to the rector, so as predicted just peptide displaces retamar from virus.
18:56We've also done expresses a wild type peptide. We've also done experiments with mutant peptide with automatic binding site. And this does not displace director as predicted well. This is blocking the way we think it does. We have a prediction where the virus gets stuck in that is issued accumulate in the endosome just like happens when you take the record binding site and again that's exactly.
19:1618 hours after infection by this time, the wild type virus has less the endosome. However, if you had the peptide. You prevent endosome exit in the virus that accumulates in the endosome.
19:29So just as I told you before. This sort of is sort of common Kathleen when you black infection. At this step. We can cause and assemble accumulation and prevent the virus and getting into these distal compartments by knocking down rectum are by mutating the rectum binding site by mutating that cell penetrating peptide so the binding site never guessed into the cytoplasm or now.
19:49By treating cells with a peptide has a self penetration sequence to get the record by new sites in the cytoplasm so this is sort of. As I said it's a common pathway inhibition.
20:03So to summarize this why I showed you as at AC terminal L 2 peptide specifically inhibits infection by several HPV types, but not unrelated viruses. In addition, requires an attack binding site and install penetration sequence.
20:18The L 2 peptide inhibits the interaction of incoming HPV to the restroom are in this causes HPV to accumulate in the end. It's all so we think this may at some point be really a new way of preventing virus infections new paradigm for antiviral activity. There are very effective antiviral.
20:39No most of them work the vast majority by inhibiting viral enzymes so for example, in case of HIV. We have inhibitors up block reverse transcriptase protease and integrates they're very effective in combination therapy. There's some inhibitors that block binding viruses to cells. There are some that Buck disassembly viruses such as amantadine with influenza virus.
20:59That this may be a new way to black infection, namely to inhibit intracellular trafficking the virus. So it doesn't get too excited replication by blocking the binding to an essential trafficking factor.
21:12So this is a Roach motel approach the virus checks in the endosome that doesn't check out so just close by acknowledging that people did all the work Alex took the leap into HIV entropy never study entry before Alex SIRNA screen using eminent biology graduate student that is terrific job and work with ways.
21:33To illuminate the retrograde pathway, Andrea Post Doc in the lab showed that HPV HBL 2 bikes replica rectum are nest important for Anderson, exit an infection, then really a lot of praise to ping ways. Ang post doc in the lab. Currently, who discovered this cell penetrating peptide develop display GPS say.
21:54I told you I had envisioned a terrific job. I haven't had time to talk about John's work. But he is now looking at more mechanistic detail and identified proteins that regulate rectum are function and block infection.
22:06Gabriel work limit with paying way do some sequence analysis and we did a terrific collaboration with Chris Burden. Some of our retro more work and this is supported by grants in the NCI in the National Institute of allergy infectious disease. So I'll stop here and I'm very happy answer questions. Thank you.