Hello. I’m Britt Glaunsinger. I’m a Professor of Virology at the University of California, Berkeley and an Investigator at the Howard Hughes Medical Institute. And over the next couple of lectures, I’m gonna be sharing with you why it is that viruses have been invaluable teachers of biology. Now, when most people think about viruses, they conjure up images of those that are associated with serious human diseases, like this one: Ebola virus. It’s got a genome of RNA that encodes only seven genes. Yet those seven genes can devastate the human body. Or this one: influenza virus. It has eight segments of RNA in its genome, and last year alone it sickened 48 million people and killed nearly 80,000, just in the United States. Or herpesviruses: these are viruses that have double-stranded DNA genomes and are among the most successful pathogens on the planet. Nearly every human is infected with at least one herpes virus, whether or not they know it. Or HIV. This is a virus whose RNA genome reverses the central dogma of molecular biology, and is reverse transcribed back into DNA before it’s integrated into the host’s chromosomes, and ultimately leads to depletion of cells critical for immune surveillance. But what I also want you to appreciate is that these viruses that are associated with human disease are in fact only the tip of the iceberg of viruses that are on us or around us on the planet. We’re coated with viruses on our skin, or in our mucous membranes, or in our gut, that cause no human disease. And in fact, our own genomes are about 8% virus. This is not virus that’s actively replicating. These are remnants of viruses that invaded our genomes throughout the history of human evolution. But it’s now understood that some of these ancient viral remnants in our genome have actually been exapted by our own cells, and functionalized for things like helping to control gene expression or mounting appropriate immune defenses. Viruses are also the most abundant biological entities on the planet. It’s thought that they outnumber all other forms of life by at least an order of magnitude. It’s been estimated that something like 10^24 infections are happening every second of every minute of every hour of every day. Now, if you also consider the fact that our ocean’s biomass is about 80% microbial, and probably 20% of that is turned over on a daily basis by viruses, it’s not hard to appreciate that they’re probably major players in the carbon and oxygen cycles that regulate our atmosphere, and many other processes on the planet. So, viruses in general are playing huge roles beyond that of just disease. And they have taught us an immeasurable amount about biology in general. In fact, much of what we know about how our own cells work has been figured out by studying how viruses interact with them. And a key feature about viruses that has been important in this line of discovery is the fact that they are masters of genetic economy. And I’m gonna highlight this point throughout this talk, but, simply put, what it means is they’ve got really small genomes. It’s not just their genomes that are small, but the capsids that house and protect their genomes are also small. Let’s take the cause of the common cold — rhinoviruses. These viruses are about 30 nanometers in diameter. So, to put that in perspective, what that means is that it takes about 500 million rhinoviruses in order to fit on the head of a pin. Think about that next time you sneeze on someone, or are sneezed on by someone, in the movie theater or the grocery store. Not all viruses are, of course, as tiny as rhinoviruses. In fact, there are some viruses that are relatively giant — the mimiviruses and the mamaviruses that infect amoeba — and whose sizes rival those of some of the smallest bacteria. But on average, viruses are many orders of magnitude smaller than the host cells that they infect. So, if you took an average-sized virus and set it next to a flea, for example, that’s about the same size differential as you or me standing next to a mountain that’s twice as high as Mount Everest. And it’s not just their size of their… capsids that are small. Their genomes are remarkably compact. So, most viruses have approximately a million times less genetic information than the hosts that they invade. And they have to do a remarkable amount with those snippets of genetic information — things like figuring out how to attach to an appropriate host cell that is going to allow that virus to replicate; how to get inside that host cell, past the cell’s plasma membrane; how to release the genome from its protective coating of an envelope or a protein capsid; and then, how to steal the host machinery in a way that allows the virus to express its own genes and to replicate its genome, all the while fighting off and evading an arsenal of host immune defenses that are in place in order to stop viruses from doing just that. The virus, once it’s replicated its genome, then has to figure out how to package its genome into a protective coat of protein and/or membrane, and then it has to figure out how to get out of the cell and disseminate to find a new host cell. An important point is that, outside of a host, viral particles are basically inert. They are intimately reliant on their host cells in order to perform pretty much all of these functions. And that’s an important point because what it means is that if you can understand how a virus is doing these things — how it’s interacting with its host cell — you’ll of course learn something important about what the virus needs to replicate, but you will also learn something fundamental about how the host functions, be that host a human or an insect or a mosquito, a crop plant, or another microbe. And that is why viruses told us so much information about the fundamentals of molecular biology, and viral research has led to a number of huge discoveries in biology. This spans the gamut from the discovery of tumor suppressors and oncogenes to the basic understanding of how our DNA is replicated and how our genes are expressed. All of these things are critical for… for things like cancer. To technological advances and things directly related to human health, like the development of vaccines and gene therapy vectors. And of course, CRISPR, which was originally in place, of course, as an immune defense against viruses that invade bacteria. And things like understanding evolution and immunology. And I think it’s important to note that many of these discoveries were made, and continue to be made, using viruses that themselves cause little to no human disease. And that really emphasizes the importance of basic discovery-based research for fueling clinical innovation. So, let me try and put this in perspective using gene regulation as an example. You can think about gene expression as being like a complicated circuit. It’s governed by many, many different regulators or inputs, which all have to coordinate to give a particular gene expression output. And we know that viruses interface with many of the central regulators of this pathway. And that’s because their gene expression strategies closely mimic those of our own, but because of this genetic economy — their small genomes — they don’t have the space to encode all of the factors that they need to accomplish these tasks, and that’s why they have to steal them from a host. And the other important point is that they evolve very rapidly, particularly in relation to the hosts that they infect. So, they can figure out quite quickly what are the really central regulators of a pathway. So, let’s say, in the context of gene expression, you’re looking at a pathway that’s got 10 or 20 or 30 components. Well, if you track what the virus is targeting, it will often lead you directly to the center of the most important, or crux, factors involved in this regulation. So, I’m just gonna give you a couple of examples of this. A classic example is the early studies that were done with viruses that really helped shape our understanding of events that lead to cancer. Now, this comes from the fact that most DNA viruses need to steal the host DNA replication machinery in order to get their own genes expressed. The problem for the virus is that this machinery is generally only expressed in a cell as it’s undergoing S phase, and not during all phases of the cell cycle. And that’s because transcription factors that are required to turn on these genes — for example, the E2 family of transcription factors — are held in an inactive state in the cytoplasm by a tumor suppressor protein called retinoblastoma, or Rb. When the cell wants to enter the S phase, or the… enter that phase of the cell cycle, cyclin-dependent kinases will phosphorylate Rb in a way that causes it to release E2F so that factor can go into the nucleus and transactivate those genes. But if a virus is infecting a cell that’s not in S phase, it needs to figure out how to get that cell to turn on the machinery it needs regardless of the cell cycle. And so, it turns out that all viruses that have been studied, that are DNA viruses that do this, target the pathway in the same way. And what they do is they each encode a protein that binds retinoblastoma in a way that forces it to release E2 so that that factor, or complex of factors, can go into the nucleus and turn on these cell cycle genes. Now, as an aside, E2 itself was originally discovered because it transactivates the early, or E, 2 region of the adenovirus genome. So, that also taught us something about cell cycle regulation. Alright, now, this is one feature, but it turns out that cells of course have ways of sensing unscheduled entry into S phase — an unscheduled DNA synthesis — and want to stop that, because that’s a hallmark of what will lead to cellular transformation. And so, when this happens, they activate a protein that’s known as the guardian of the genome: p53. And p53’s job is to stop entry into S phase, and if it can’t do that, to lead the cell to a path of apoptosis, or cell death. This of course would be detrimental to the virus, because the virus needs to keep the cell alive long enough to complete its replication cycle and produce progeny virions. So, not only do all of these viruses target retinoblastoma, but all of these viruses also have proteins that target and inactivate the p53 tumor suppressor. And in fact, p53 was originally discovered as a 53 kiloDalton protein that interacts with the large T antigen from polyomavirus. This is a protein that we now know… or, a gene that we now know is mutated in the majority of human cancers, and is one of the most heavily studied genes in cancer biology. And so, the fact that these small DNA tumor viruses, even if they’re viruses that themselves don’t cause human cancer, have taught us that inactivation of two key tumor suppressor proteins is of fundamental importance for cellular transformation has really shed important light into cancer biology. Another example is viral manipulation of translational control. So, every virus is absolutely dependent on the host translation machinery in order to get their genes translated into proteins. No virus encodes genes that will make ribosomes, which are key to protein synthesis. The problem, though, is that viruses have to compete with the cellular messenger RNA for access to these ribosomes. And figuring out how they compete has taught us a lot about plasticity in the translation complex, or the translation apparatus. So, they can do this for example by cleaving and inactivating cellular messenger RNAs to essentially eliminate the competition. Or they can selectively target factors that are part of the translation complex that might be necessary for translation of many host RNAs, but not necessary for translation of viral RNAs. And I’m just showing you, here, many examples of viral proteins from different viruses that target individual components of the translation machinery. Now, studying these inactivation events has shed light on how different types of RNA might be able to get targeted or translated by distinct mechanisms. For example, if an RNA has a relatively unstructured 5′ untranslated region, or UTR, you might not need one of the key helicases to unwind that RNA. Or an RNA can get translated without the 5′ cap structure if it has an internal ribosome entry site, or IRIS element, that allows direct recruitment of the ribosome subunits to the RNA in the absence of a cap. Or certain RNAs can be translated by alternative or specialized ribosomes in a cell. And it’s notable that these are not things that are happening only during viral infection. You should think of viruses as thieves, not inventors. That means these things are happening normally in a cell, and many of them have now been discovered to be operational under certain cell contexts like cell stress. Finally, I just want to point out that viruses and viral components are centerpieces of many of the tools that we use on a daily basis in molecular biology. Let’s just take a standard plasmid vector as an example. This is a generic vector that you might use, or scientists might use, in order to express a gene of interest in cells, by transfecting the vector into cells or using a virus to transduce the vector into cells to express their gene. Well, if you’ve ever studied plasmid vector maps, you might appreciate that they are chock full of things that people have gotten from viruses. For example, your gene of interest is probably going to be transcribed by a promoter that comes from a virus — a really popular one is the cytomegalovirus immediate-early promoter. Your vector might have promoter sequences that allow you to generate RNA by in vitro transcription as well — things like T7 and SP6 promoters. Well, these are derived from bacteriophages and are used in the lab to be transcribed by T7 and SP6 polymerases, which are also viral. If you want to purify your protein from a cell, or detect it, often you’re gonna append an epitope tag to that protein. Most of these epitope tags also come from viruses — the FLAG tag, the HA tag, these are from influenza; the V5 tag from a paramyxovirus. And if you want to then purify your protein and remove a tag, you’ll usually have a proteolytic cleavage site that allows you to specifically remove that tag from the protein. These tend to also come from viruses — the TEV protease site, for example, from tobacco etch virus. Even the multiple cloning sequence that you’re gonna use to put your gene of interest into the vector… well, restriction enzymes come from the immune system of bacteria, that’s used to fight off phage invaders. And as your gene is being transcribed, it’s going to need to get the features added on to it that are going to allow it to be recognized by the host translation machinery, and one of these features is a poly(A) tail, so you’re probably gonna have a polyadenylation signal sequence, and these were originally mapped in viruses as well. So, something like the SV40 poly(A) sequence is probably a component of these vectors. And then finally, do you ever wonder why 293T cells are such workhorses for protein expression in biology? It’s because these cells express the large T antigen from polyomavirus, and that T antigen, if your vector has an SV40 origin of replication, or ori, will recognize that component or that sequence on your vector and amplify the vector, and replicate it, so that you can have a dramatic expansion of the number of templates available in the cell for transcription. And an added bonuses is, then, as these 293T cells are dividing in culture, your plasmid isn’t getting diluted out, because it’s being continually amplified. So, these and many other viral discoveries have been recognized for their importance through the receipt of many Nobel Prizes, both for things that are directly related to human health and of translational importance, like vaccines, but also more broadly for the spectrum of discoveries that have been made using viruses that have greatly enhanced our understanding of things like gene expression and the immune system and cancer. So, I’m now going to transition into an aspect of virus host interactions that is really the focus of my lab’s research, and it’s gonna be something that I’m gonna be going into more detail on in the second lecture, and this is gene regulation, and in particular, controlling the abundance of messenger RNA in cells. Now, in this particular diagram, if you think of messenger RNA as a pool of water, and the amount of messenger RNA in this cell is the amount of water that’s being held in this person’s hands, well, transcription of course is the process, that faucet, that feeds that pool of messenger RNA. And for many years, people thought of degradation of RNA as being this unfortunate consequence of loss of the RNA — the spilling of the water out of the pool — which is why you needed to continually replenish it through the act of transcription. Now, however, it’s well appreciated that RNA degradation is a real focal point for gene expression control, and that as many as half of the gene expression changes that occur in a cell in response to particular stimuli can be driven by alterations in RNA stability. This is either at the individual transcript level or to the bulk pool at large. And for me as a virologist, one of the things that really helps hammer home this point is that many different viruses that are unrelated to each other — from herpesviruses, to poxviruses, to influenza viruses, to SARS and MERS coronavirus — all help reshape the gene expression landscape of a cell during infection by targeting the process of RNA decay. They do this by a number of mechanisms, but there are some overt similarities. So, let me just summarize for you some of the ways that the viruses do this. Well, herpesviruses, they encode factors that are able to internally cleave RNAs, so these are called endonucleases. And this inactivates the RNA, right away, for translation. Influenza viruses, shown here in the nucleus of the cell, also have an endonuclease that preferentially targets RNAs undergoing splicing. Poxviruses like vaccinia have factors called decapping enzymes that remove the protective cap from the 5′ end of an RNA. And coronaviruses, like SARS and MERS coronavirus, have factors that will cause a ribosome to stall on an RNA, leading to an internal, or endonucleolytic, cleavage, and inactivation of that RNA. Now, an important point for all of these is that you are rapidly inactivating the RNA by cleaving it internally or removing its 5′ protective cap. And so, in doing so, the RNA is immediately not able to be translated anymore. But also, what you’re doing is you’re exposing the ends of the RNA to rapidly being accessed by host RNA degradation enzymes. And these are called exonucleases because they will degrade an RNA from either end, rather than internally. And major exonucleases in mammalian cells are the XRN1 exonuclease that chews things up from the 5′ end and things like DIS3L2 and the exosome, which chew them up from the 3′ end. So, all of these viruses are relying on this basal RNA decay machinery to degrade the fragments that are being created by the viral enzymes. It’s important to note that this is reiterative targeting of the same pathway by multiple different viruses. And whenever you see this in virology, it tells you that something about the way that they’re doing it must be very efficient, because different viruses are solving the problem by hitting on the same thing. And why that must be efficient in an RNA degradation example becomes obvious if we compare how viruses are doing this — or RNA decay during a viral infection — to RNA degradation that’s happening sort of normally — basal RNA decay in our cells. So, messenger RNAs are protected at their ends. They have a 5′ cap on one end and a poly(A) tail on the other end. And RNA decay is a very ordered process that begins with gradual shortening of that poly(A) tail. This happens by a number of enzymes that are called deadenylases, and it’s a rate-limiting and heavily regulated step in the cell. Once the poly(A) tail has reached a certain shortness, this licenses removal of the 5′ cap from the RNA by a decapping complex. And now, only once you’ve got a headless, tailless RNA, can that RNA be accessed by these fast-acting exoribonucleases, like XRN1 and the exosome. So, that’s what happens in an uninfected cell. But in an infected cell, viral nucleases, by internally cleaving the RNA, are bypassing these rate-limiting, regulated steps of RNA decay — of deadenylation and decapping — and creating RNA substrates that can be directly accessed by these fast-acting exonucleases. And because they’re relying on the host machinery to degrade those cleaved fragments, the intermediates are going to look indistinguishable from those that are normally produced in the cell, and might not raise any red flags for the cell. I should note that this strategy that the virus is using, of using an endonuclease… well, there are of course examples of endonucleases that are used in the host cell in an uninfected context. Many of these are used in the context of quality control. If you’ve got an RNA with an error, you want to get rid of it quickly, before it could get translated into a protein that has a mutation that might have a dominant-negative phenotype. And so in these cases, the cell uses its own endonucleases to recognize and cleave and inactivate those RNAs, ultimately following through this endonucleolytic followed by XRN1-mediated decay pathway that the virus is using. The difference, though, is that during viral infection, cleavage is not limited to a few RNAs. The scope is much, much broader. So, I’ll just close this portion of the talk by emphasizing that this ability of viruses to accelerate RNA decay plays a variety of different roles in the context of viral infection. A few examples that are notable are that RNA degradation is known to help viruses escape immune detection. So, for example, in response to an infection, a cell has many ways of sensing that infection, and the cell is going to start to turn on factors that are immune stimulatory and antiviral. And so, as it produces these RNAs for translation into antiviral factors, by cleaving all of these RNAs in the cell, the virus can prevent production of those antiviral factors and help escape immune detection. Shown on the bottom, here, is an example of translational control that I already sort of touched on. If the virus is cleaving many cellular RNAs, then these RNAs are not competing with the virus for access to ribosomes. And this can, in a number of cases, enhance the ability of viral genes to get translated in an environment where cellular genes might normally be favored. In certain viral examples, this RNA degradation also helps with the ability of the virus to transition between different gene expression cascades that are needed in the course of infection. And in in vivo mouse models, it’s been shown that this can help the virus traffic to where it needs to go, and in… in the animal, and replicate in the appropriate set of cells. So, what I’m gonna talk about in the next lecture is more scientific detail on what we’ve learned about RNA targeting by a particular viral endonuclease, and how that’s informed our knowledge of some of the different connections between different stages of the gene expression cascade. So, I’d like to end this talk by acknowledging the many generous funders that have helped support our research over the years, as well as the fantastic scientists that are part of my lab. The current members are shown here in these photos, but we have a long history of fantastic scientists that have worked with us on this. So, thank you very much for listening.