Wisconsin Public Television University Place  Virus Takeover of Host Cell Functions  Paul Ahlquist  4/17/13 UP859  cc >> I'm happy to have the opportunity to talk to you, and thank you all for coming out on a somewhat inclement evening. So, I'm going to talk today about several different aspects of viruses, and we work on a variety of different viruses, as will become clear as we proceed. I'd like to touch on three main directions tonight as we go forward. I want to tell you a little bit about why we study viruses, what our motivations are in this regard. And I want to give you, basically, two different answers to that. One that has to do with the expanding disease burdens that are associated with viruses, both in terms of acute diseases and also chronic diseases, including cancer. I also want to tell you about, on the more positive side of things, what sort of discovery opportunities viruses provide in terms of fundamental research and understanding. I want to move from that foundation and tell you about one of the most important fundamental facets of studying viruses and that's that viruses are amazingly poor in genetic terms. So poor that it's difficult to understand how they accomplish what they accomplish. And I'll tell you how viruses really can only survive by masterfully exploiting host cell functions at nearly all steps of their replication. 

And I want to move on from that then to finish with the concept that these many host genes that viruses exploit and depend on for their replication provide opportunities for controlling viruses in ways that can be further improved beyond the controls we have now. Okay, why we study viruses, in brief. One of the most fundamental motivations we have is the sheer toll associated with viruses. Infectious diseases worldwide cause about 1500 deaths per hour. Now, half of those are children under five. About half of that total is viral. So, before we're done with tonight's evening, another approximately 750 people will have died from viral diseases. So we can put a clock on our efforts, and that gives us a powerful motivation to understand better how these agents work and how we can control them. Now, we normally think, most people normally think in terms of acute diseases in terms of this death toll, but there are also chronic disease issues, and the best established of these are in relation to human cancers. And, as I'll expand a bit more in a moment, viruses are now recognized to cause at least 15% to 20% of human cancers. 

So there's a tremendous burden there which we're also interested in better understanding and alleviating. And in fact, despite our best efforts, the balance of this situation is not moving in a positive direction, and the emergence of new viral diseases is accelerating. Now, let me expand on that just a little bit and bring that home to the situation in the US. If we think in terms of infectious disease mortality, of which, again, viruses are a major component, and think about the 20th century, in the first half of the 20th century, infectious disease mortality declined about tenfold. So, this was a time of great hopes. It was a time at which many people predicted that we would soon seen the end of infectious disease. It was a time when some prominent universities got rid of their microbiology departments because this was obviously soon going to be a solve problem. And, unfortunately, that has not been the way things have matured. From 1950 through about 1980, the mortality stayed steady, and from then until the present it's been increasing. As you can see from the graph here, that's true whether you include HIV, which is the main red curve, or whether you take HIV away. 

We still have a slowly increasing death toll in the United States from infectious diseases. As I mentioned before, in general we're seeing the emergence of new viral diseases, and the World Health Organization and others confidently predict that this is going to continue and probably accelerate. The classic example of this is HIV, and HIV, like the other diseases on this list, is a zoonotic infection, which means that it's moved from animal hosts into humans. And in the current environment as we have increasing expansion of the human population across the globe, we've seen in recent years and decades many other zoonotic infections that have spread into the human population, and some prominent examples would include the SARS coronavirus. You probably have heard in the news recently that there's a new coronavirus that's been emerging in the Mideast and has moved into some other parts of the world including Britain. West Nile virus in the US. Influenza, as Tom was mentioning a bit earlier, moving into human population in several different strains from birds. Ebola and other viruses. Again, all of these zoonoses from primates and birds and bats and other sources. And all of the trends that we see at present indicate that these disease incursions from the animal sphere into the human population are really only going to increase for all of the reasons that are listed here. 

The expansion of the human population and its collection increasingly in densely packed urban centers, providing a perfect environment for a new disease to move in and then spread through the population. The encroachment of the human population on other environments, creating new interactions between humans and animals. The increasing globalization of trade and travel, ensuring that when new diseases emerge, they're going to quickly spread across the globe. And other factors such as climate change, which is expanding the territory of many important insect vectors and other reservoirs for virus diseases. Now, I mentioned cancer a moment ago. I want to just expand a little bit on the issue of viruses and cancers. As I mentioned, it's now recognized that at least 15% to 20% of human cancers are caused by viruses. And these are relatively recent recognitions in most cases. And for that and other reasons, many people suspect that viruses are involved in additional human cancers and this will become clear as we go forward. Now, when this connection was first recognized it was expected that the virus was contributing to the early stages of tumor development but probably not later stages. 

And as research has advanced, it's become clear that in fact viral genes are continued to be required for the survival of tumors even after they've matured and acquired many other genetic changes. So, the implication of these results is that if we could inhibit preferentially the viral infection in the first place, or, if necessary, the right viral functions after infection with these human tumor viruses, we could prevent or potentially cure many different cancers, again providing powerful motivations for understanding how viruses work, interact with their hosts, and can better be controlled. This slide shows the really well established human tumor viruses at the present time. I want to just speak for a moment on a couple of these. I'll start with hepatitis B and C. These viruses cause the great majority of liver cancers. Liver cancer is the fifth or sixth most common cancer worldwide, but the prognosis is rather bad. And so it's the third leading cause of cancer death. Now, more than half of those liver cancers are caused by hepatitis B. The majority of the remaining liver cancers are caused by hepatitis C. 

These are viruses that have somewhat different molecular biology but some clinical similarities. And, again then, together represent a tremendous burden of death and suffering. The other virus I want to mention in this context is human papillomavirus. So, human papillomavirus, as I think is well known, causes essentially all cervical cancers. Cervical cancer is relatively well controlled in the developed countries through population-wide screening. It is not well controlled in the developing world and there remains a major killer. By many estimates, the second leading cause of cancer death in women. But papillomavirus has also caused other cancers, and one of the best established now are head and neck cancers. So, this is an important cancer in men as well as women. And currently, approximately 25% of head and neck cancers, and actually a growing percentage of head and neck cancers, are caused by papillomavirus. 

Other cancers are also caused by this virus. Esophageal cancers and others. If you add all of this up together, this is more than 5% of all human cancers caused by this single virus. So if you think about these viruses, hepatitis B and papillomavirus, the only thing you can compare them to as single identified causes of cancers is smoking. So, controlling these viruses, for example through the recently introduced papillomavirus vaccines, is a tremendous need and opportunity for controlling many kinds of cancer. Okay, so, there are all of these potent reasons in terms of public health and disease burdens to study these viruses. There are also some more positive views of why we study viruses, and that's, of course, to advance the understanding and the treatment and the prevention of these viral diseases, and, beyond that, to enhance the understanding of normal cell and organismal biology. Viruses have proven extremely good at revealing aspects of general biology. And viruses are also very good at a number of things that we're interested in in controlling for medicine and biotechnology and nanotechnology. So, viruses are the premiere natural agents for moving genes from one cell to another, and they're the premiere agents for expressing genes at high levels. 

They're also extremely good at building structures on the nanometer scale. So they're already being utilized in a number of procedures in that regard. The potency of viruses in promoting, again, general biology understanding and such tools is sufficiently powerful that many of my colleagues ask not why study viruses but why would you study anything else. So, if you think about, again, in terms of biology, much of what we know about cell biology came initially from studies of viruses, many aspects of gene expression, general understanding of the secretory pathway in cells, a key factor in higher organismal gene expression, the presence of what we call introns and RNA splicing, and aspects of powerful new genetic technologies called RNA interference. All of these things came largely out of studies of viruses. In terms of cancer, both oncogenes and tumor suppressor genes, arguably they're key pillars of our understanding of cancer now, came from studies of viruses were initially identified in the studies. The current success in controlling HIV, or at least forestalling death from HIV, through multi-drug combinatorial therapy has provided a paradigm that many people think will serve to illustrate how we can move into a new phase of cancer control similarly using well designed combinatorial therapies to turn cancer from a death sentence, in many cases, to a manageable disease where you can maintain and extend a reasonable quality of life with continuing treatment. Not a cure but at least a useful intermediate stage on the way to a cure. And other areas, such as immune studies and so forth, have also greatly benefited from work with viruses.  So, we've talked a little bit now about why we study viruses, and let me go on to focus on a couple of aspects of viruses and their interactions with cells in conjunction with the topic tonight. 

So I want to emphasize now, again this concept that despite all of the problems that viruses cause us, despite all of our difficulty in controlling them, if you look in genetic terms, viruses are really amazingly simple and amazingly poor in resources. They're very poor in genes, and they can really only survive by exploiting host genes. So, here, a cartoon illustration of a typical human cell and a virion, the infectious particle in which a virus spreads its infectivity from cell to cell. This is not to scale. The virus is illustrated as much too large. So if you look at some of the depictions here, the scale of these objects in terms of the cell is in micrometers, for the virus particle, nanometers. So a thousandfold difference in the scale of these two objects. If genetic terms, in terms of the amount of genetic material that the virus has, then in terms of typical viruses, are on the order of a hundred thousand to a millionfold smaller in terms of their genomes than the cell. So, for example, humans have about 3,000 megabases of DNA; whereas, HIV has .009, I've rounded up to 10 here, megabases of DNA. This human cell has about 25,000 genes; HIV has nine or 10 genes. 

So this is an amazing difference, and I want to try to bring this home in a couple of ways. So, one is I want to give you an idea of the scale of this kind of a genome. So this is, again, we're using megabases here but we can shift this over to kilobases. So this is, the HIV genome is 9.2 kilobases. 9,000 bases. 9,000 steps on a DNA double helix ladder. So, I wanted to get the HIV sequence up but didn't have time to do that, so I settled for polio virus. Polio virus and HIV, like many viruses that you're familiar with, have genomes in this range of about eight to 10 kilobases. So this is the polio virus sequence, that's here, is 74th, 41 bases. Again, HIV is about maybe 20% bigger at about 9,200. 

The point I'm making is that the nucleotide sequences of these viral genomes can easily be written on a page. This is enough information to remake the virus. So, with common technologies now involving DNA synthesis and cloning, almost any reasonable molecular biology laboratory could take this page or the equivalent page for HIV and remake the virus. So, what does this mean? Well, let me try to pose this in a different way. We're on a university campus, so let me give you an assignment. Okay? So the assignment is I want you to go home and write a complete detailed molecular instruction manual for HIV. It has to tell everything that HIV needs to do to infect and replicate and survive. So that's at least, minimally, everything that's written here. It has to tell how the virus is going to recognize the right cells to infect, successfully attach to them, enter, initiate its replication, replicate its genes, express and regulate those genes, make new progeny infectious particles, release them, escape from host defenses, spread across the human population, overcome all of our attempts so far at vaccines and our individual antiviral drugs, all of these things you have to write down. That sounds like a big assignment. 

For comparison, you can think about all of the literature that's been written about HIV, all the scientific literature, probably the best studied virus now. So, we could fill much of this room with books on HIV. So, why am I stressing all of this? I'm stressing all of this, as some of you doubtless realize, because we have this instruction manual. It's the genome. As I told you, that's enough information to remake the virus. Everything the virus knows how to do is in that genome. And, going back to our stand-in here, polio virus, but imagine this just slightly expanded for HIV. Again, it's all in one page. A further point is that the DNA language, the steps on the double helix ladder of course, only involve four letters: A, C, G, and T. So this one page, but it's written in a four-letter alphabet. 

So that's less information than written in a 26-letter alphabet of English, for example. So, how can this possibly be enough information to do everything that HIV does? How can one page of instructions be enough to prevent us from coping with the HIV epidemic? Well, I have to say first off, I don't understand it completely. It's one of the challenges that keeps us working, trying to understand how this can be. But some of the answers are that viruses are extremely good at using the genetic resources they have to the maximum. So they have unusually high density coding, they code information in the genomes that are much higher density than cell genes do. Also important in this is that HIV in most other viruses mutate at a ferocious rate. So, in consequence, they have the ability when necessary to evolve, and they also have in their populations, even within a single patient, tremendous diversity, and diversity is the source of robustness and response for a virus just as it is for a human society. So, when the virus encounters challenges like antibodies and antiviral drugs, it calls on that diversity to produce a resistant variance and can escape from those drugs. But a really important part of this answer that I want to emphasize in the remainder of the talk is that the virus is calling up subroutines from the host genome. So the complexity of the problem is not just the viral genome, it's the host genome plus the virus. 

And an analogy that I think is useful here is that of classical guerrilla warfare. So, -- style guerrilla warfare. A small, poor insurgency against a much larger power. And, in that situation, for the guerrilla the best source of supply typically is the enemy. That's where you get most of your food, your weapons, your supplies. That's what the virus does. The virus uses all of the resources of the cell to accomplish its own goals and missions. And that's what I want to move on to discuss in the rest of the talk. This take over of host cell functions, and to keep this within our time limits, I'm going to give you just a few illustrations from aspects of how viruses replicate their genomes. And, as a foundation for that, I'm first going to set up a little bit of introduction to molecular virology and talk a little bit about how viruses work with their genes. 

So, everybody knows that cells store their genes in DNA and replicate that DNA. So the DNA serves as an archive, the essential archive of information that every organism has, and that DNA is used, and this was one of the early triumphs of molecular biology was to establish this information flow pathway here that the DNA is expressed by producing a related molecule, RNA, which serves are working instructions or blueprints to produce proteins. In many cases, the final product that carries out enzymatic reactions or makes structures that cells use, etc. So, again, DNA and RNA are related molecules. This production of RNA, this reading of the DNA information into RNA is called transcription, as most of you know, signifying relatively modest change from one kind of script into another script. This reading of the RNA information into a rather different molecule, protein, is called translation, signifying a much bigger change in structure. So, again, this is often referred to as the central dogma of molecular biology for cells, and it turns out for one category of viruses, DNA viruses, the same approach holds. That the archive of information is in DNA which is replicated through other DNA forms and expressed in this matter. An example of such DNA viruses would be the herpes viruses. Moderately large DNA viruses that infect humans and many other animals. 

But many other viruses, in fact most viruses, don't follow this pattern. In fact, most viruses don't even have DNA, they only have RNA. So they're using RNA as both the archive of their information and the working instructions to make protein, and they replicate that RNA through other RNA forms with no natural DNA intermediates. So viruses break free of this paradigm into a new model, and, again, most viruses follow this pattern. There's another group of viruses, the retroviruses, which are somewhat intermediate. They use DNA as well as RNA, but they replicate in a very different fashion than the cells or the classical DNA viruses. That is, they replicate the DNA by producing a complementary RNA, and then they copy that RNA back into DNA. Now, as many of you know, this act of copying RNA into DNA was discovered by Howard Temin who worked in McArdle laboratory building just next door to us here, and Howard Temin received the Nobel Prize for that work, what's often referred to as the discovery of reverse transcription of information from RNA into DNA. But today I'm going to primarily discuss these RNA viruses and their genome replication as an example of host interactions and the exploitation of host functions. So I want to talk about how, in a general sense, this RNA replication works and where it occurs. 

So, go back to our picture of the cell, cells carry their DNA and replicate it in the nucleus, depicted here, a membrane-bounded compartment in the cell that separates that genetic material and it's replication from other parts of the cell. Now, the viruses, these RNA viruses, I want to tell you today, we and others have discovered make new virus-specific, infection-specific membrane-bounded compartments, what we increasingly think of as mini organelles, in which they carry out their genome replication, even though this is an RNA replication not DNA replication. And they use these virus-induced compartments to organize the factors and the templates and the steps of the replication process and to protect that replication process and the replication intermediates from a variety of host defenses, including the interferon machineries and some other defenses that are activated by RNA molecules. So, today I want to tell you a little bit about the structure and the assembly and the function of those replication compartments and about the crucial roles of a number of host proteins in the generation and the function of those compartments, and as particular examples I'll use some membrane-shaping host proteins. So, we're studying this process, a part of our laboratory is studying this process of RNA virus genome replication using a couple of model viruses, and I'll just briefly introduce two of these model viruses in the discussion today. The first is something that we call bromovirus. And if we're looking here in a cell that's replicating the genome of this virus and there's some very novel structures going on right here. So, the nucleus is normally separated from the surrounding cytoplasm by a double membrane, and here we have a fairly normal section of that double membrane. But you can see in this cell because of the presence of the virus, this space between those double membranes has expanded and filled up with vesicles that are about 60 nanometers in diameter. And we have several ways, including the isolation of these nuclei free from the cell, to further expand that space, and when we do that we can see that these vesicles are associated with the inner surface of the outer membrane, and our further published studies show that what's going on there is that the virus is folding in the membrane, away from the cytoplasm, to create these vesicles. 

And I'll show you in a moment that their interiors remained connected to the cytoplasm by a channel. Further work that we've published shows that the vesicle is coated with viral proteins and contains a viral RNA and is synthesizing new RNA copies and exporting them out into the cytoplasm. This is a little RNA factory that synthesizes RNA and spews it out into the cytoplasm. This is one of our model systems. Another model system that we're working with, something called a nodavirus, does a very similar thing. These two viruses have many differences, but it turns out that they have many similarities in their replication machineries. One of the differences is that this nodavirus does its replication in a different site of the cell. It does it on mitochondria. These are the energy factories of the cell. Mitochondria also are normally bounded by a double membrane. 

And you see here in this nodavirus-infected cell that something has happened here that's rather similar to what we saw in the nucleus in the other case. That is that the space between these inner and outer membranes has expanded and filled up with vesicles which, in this case, are about 50 nanometers in diameter. And our further work with electron microscopy, which we're using to produce these images, shows that the interior of these vesicles are the sites of accumulation for the viral replication factors and also the sites of new viral RNA synthesis. So this is where the replication is happening. Now, using more advanced electron microscopy, we've been able to visualize these structures in three dimensions, and you're looking at the top of a three-dimensional electron density map of one of these modified mitochondria. And I'm just going to start a moving now that syncs through that mitochondria, and you can get a better sense of these new vesicles, these new replication compartments that the virus has induced. And now we're rotating that three-dimensional structure up, and we've modeled in, in blue, the outer mitochondrial membrane and now, in white, these modified vesicles. And for good measure now, in yellow, the inner mitochondrial membrane. And the student who did this, Ben Kopek, modeled many such modified mitochondria in cells and was able to answer many questions we had about the structure and connectivity of these compartments. And you can zoom in on these and see, for example, the connections of each of these out to the cytoplasm. 

The cytoplasm would be up here, and you can see the interior of the vesicle is connected to that cytoplasm by these, approximately, 10 nanometer necks which have a rather constant diameter. Further work that we did showed that, as in the other case, the interior of these compartments are lined by viral proteins, and the remaining space is filled by viral RNA. So, we have these two different viruses that are replicating on different sites in the cell but producing rather similar structures, again, membrane-bounded compartments, that are collecting the replication factors and the RNA templates that are being replicated and serve as the sites of synthesis for new RNAs that are exported out into the cytoplasm to be packaged into viral particles and infect new cells. Well, we've put a lot of effort into understanding how these structures assemble and function, and we've found, for example in the case of this bromovirus, the first system I indicated, that a single viral protein, a very important protein, highly multi-functional protein with the uninspiring name of 1A, is responsible for making these compartments, for inducing their formation. It goes to the appropriate membrane, it self-interacts and induces the formation of the membrane. It's also able to recognize the viral RNA and recruit it into these compartments and also to recognize and recruit another viral protein, which we call a polymerase, which actually then copies the RNA to make new copies of the RNA and, again, to send those out into the cytoplasm. So, going forward to understand this better, we're asking basic questions like, how does this 1A protein grab the membrane and reshape it to make this compartment? We actually understand the grabbing part pretty well. We've been able to map a particular domain in this protein and show that it makes a structure, a helical structure that inserts in the outer face of the membrane and is principally responsible for binding that protein to the membrane. But, going beyond that grabbing the membrane, after the protein has grabbed the membrane, how does it bend the membrane? And the point I want to make now is that is a consequence both of direct functions of this protein and also its ability to recruit at least two key forms of host proteins that are involved in reshaping membranes. And so, again, this virus protein, this 1A protein binds to the membrane, it self-interacts, and it does form an arced scaffold that appears to be important in imparting curvature, but our recent work, some published and some unpublished, show that forming these vesicles absolutely depends on the ability of this protein to recruit two different classes of host proteins. 

So, integrating all of the information that is available to us now, the working model that we're testing further is that this viral protein only initiates the deformation of the membrane and then recruits another set of host factors, the so-called escort machinery, and that escort machinery has key roles in squeezing the membrane to push more membrane into this growing vesicle, and these factors are essential for actually forming the vesicle. A second set of host proteins, the reticulons, is also recruited, and these proteins appear to have major contributions in several ways. One of those we believe is in stabilizing the necks of these structures. The first class of proteins, the escort proteins, are recycled off of these structures, so their role is essential but it's transitory. These reticulon proteins stably associate with these compartments. And, again, the key point is that both of these host machineries are essential to forming these vesicles and to the replication of the viral genome. If you knock out these machineries without going into the details now, the formation of the vesicles and the replication of the RNA crashes by ten to a hundredfold depending on which particular factors in these pathways you decide to knock out. And this is independent of the fact that the viral proteins involved remain normally expressed and normally localized to the right place in the cell. So again, the implication is that these two sets of host proteins are required to form these replication vesicles. And the bigger point is if we inhibit either their expression or their function, that blocks viral replication. 

That's actually how we found these things in the first place is by inhibiting their function, genetically or chemically, and, in this way, finding that they were required. So, this is true of all of the host genes that are involved in viral replication. And let me just expand. In this case, the two membrane-shaping proteins families that I just mentioned are only a small subset of the proteins from the host that are involved in these processes. So, using classical genetics and more modern systematic genetics we've been able to identify over 150 host genes that are involved in this process of building and functioning the replication compartments. And many of these we've been able to map to specific different steps in this process. So there are many, many potential targets that we can manipulate in the host to control these processes. This is a very attractive direction to go in to control the virus for a variety of reasons. One is that when we try to control viruses by targeting the virus genes themselves, what we almost inevitably find, with HIV, with flu, with other viruses, is that the virus again mutates so rapidly that it escapes out from under the antiviral strategies that we're putting onto it. We apply a drug, the drug initially knocks out the function of 99.99% of the viral genomes that are present, but because of this tremendous mutation rate and diversity, there are preexisting mutants, rare mutants initially, but preexisting mutants that are already resistant to the drug even though the virus has never seen the drug. 

And those resistant mutants grow up and resume infection and the treatment fails. Host genes are not subject to that ferocious mutation rate, those escape mechanisms. So, this is potentially a way to get much more stable, longer lasting resistance against viruses. Another very attractive feature of this is that some of the proteins I've already mentioned appear to be utilized by multiple different viruses. So while antiviral drugs and other agents, vaccines, targeting against viral proteins are highly specific, targeting some of these appropriate selected host pathways could knock out the replication of multiple viruses in one go. So, we and others are very interested in this strategy, and we've extended it beyond these laboratory models to other situations. A few years ago, our laboratory cooperated with, collaborated with Yoshi Kawaoka's group on campus to carry out the first genome-wide screen of host genes involved in influenza virus replication, and we identified a similar result here. Over a hundred genes involved in replication of influenza virus. In similar studies, we've identified host functions that are crucial for and we think targetable to control hepatitis B virus and human papillomavirus, these two critical tumor viruses that we mentioned earlier. And we've done similar work with HIV where we've identified host genes and new replication steps and new control targets at multiple phases of HIV replication. 

So, to summarize this, viruses are amazingly poor in genetic terms. That's a weakness. It causes them to depend absolutely on exploiting host functions for essentially all of their infection steps, and that dependence on host genes offers us valuable new opportunities for improving virus control to get longer lasting, broader spectrum control possibilities. I want to conclude by acknowledging a number of current and recent members in the lab, and for the work I discussed today on RNA viruses, particularly Arturo Diaz and Xiaofeng Wang and Ben Kopek. Linhui Hao in particular for the influenza virus work. For our HIV work, principally James Bruce, and for papillomavirus work, Johan den Boon and Mark Horswill. Thank you very much. [APPLAUSE]