Virology 2015 Lecture #18: Transformation and oncogenesis

today I want to talk about transformation in oncogenesis and I want to start with an experiment that illustrates what we’re talking about we start with in this case a hamster embryo but it could be any source of mammalian cells we mince it up make single cells in suspension and then we put them onto plastic dishes with culture medium on top of them the cells typically will adhere to the plastic and will grow and on the left you can see a plate of cells if you looked at them under a microscope you could see individual cells they would grow and you would split them periodically once they reached confluence but eventually they would die because they’re not immortal however if you took the same cells and treated them in a variety of ways with a chemical as shown here sometimes UV light or a virus you can make the cells immortal and you will get what we call transformed cells and they’re shown here as growing very strongly and at the bottom is a higher magnification of what these two conditions look like so on the left would be our normal monolayer of cells derived from this embryo they’re very well behaved they form a nice mono layer they touch each other they stop growing on the right is the transformed cell again it’s been treated with a variety of substances which we’ll explore a bit today and it makes these cells immortal this is what we mean by having transformed cells so the idea that you could change the basic properties of cells and culture and make them transformed has been around for quite a while and it’s been known for a long time that these transformed cells have a number of different properties compared with untransformed cells and when when scientists first discovered this they were puzzled they didn’t really know what this meant today by the end you’re going to understand what has gone on with these transformed cells and so here are just some of the differences between normal and transfer sells their immortal first of all they grow forever HeLa cells are a great example these were produced from a tumor from a woman in 1951 and they have been growing ever since and they will keep on growing so they’re immortal they have lost anchorage dependence most normal cells have to attach to the plastic in order to grow many transform cells don’t need to do that and they will pile up and form fossa it will grow in soft agar as well they have a loss of contact inhibition they pile up so a normal mono layer you can see here on the top and at the bottom part a the cells grow in a single cell sheet when they touch each other they stop growing a transform cells will continue to grow they’ll pile up and their disordered as I said they will form colonies and semi-solid media if you suspend these cells in agar it’ll make colonies and normal cells don’t do that and finally they have a decreased requirement for growth factors in the medium and this is typically provided by serum we add serum to cell culture medium because it has all sorts of growth factors that cells need transform cells need a lower percentage of this than normal cells the other half of today’s discussion is on Co Genesis so the transformation now we’ve defined unko Genesis is the development of cancer most cancers are solid cancers so you have the formation of a tumor which is a swelling caused when cells continue to divide without stopping all the tissues in our body have the certain morphology because they stopped dividing the cells that make them up stop dividing at a certain point tumor cells or oncogenic cells do not do that they form a mass called a tumor and that tumor can be either benign or malignant it can be remain contained or it can spread to other tissues ie be metastatic so there’s some non solid cancers of course like leukemias but they are the minority most of them are solid tumors so that’s what uncle Genesis is the form nova cancer cancer is a genetic disease and we now know you need about a dozen mutations in different genes that encode signaling pathways that regulate cell proliferation survival and the determination of cell fate and finally maintenance of genome integrity so we have a variety of

protein proteins in the cell that find errors in our DNA when they arise and correct them they find breaks in the DNA and so forth and mutation mutations in those are part of what drives cancer to develop these mutations this dozen or so mutations they can be inherited can inherit it from your parents they may have had a variety of mutations each they pass them on to you they can be caused by DNA damage throughout and the Sun too much and get skin cancers because of the the UV light in the Sun is causing mutations in your DNA various chemicals in the environment can cause cancer as well and finally today’s topic viruses can cause cancer but they actually they actually as you will see do not cause cancer they cause cells to be transformed so that they’re on their way to becoming a cancer so here we have transformed cells on the Left we can make these in culture they may or may not be oncogenic these transformed cells are on the way to becoming cancer cells but they need to accumulate more mutations in order to do that so here on the right we have a mouse with a solid tumor which has been made by injecting cells into the mouse that tumor needs more mutations compared to the transformed cells so the reason this this has all been sorted out by studying how viruses transform cells that’s we’re going to talk about today so viruses transform cells and transformation remember makes them immortal they keep dividing forever and when that happens mutations accumulate and eventually if you get the right 12 or so mutations that’s transform sell will become a cancer so this is a very important distinction between transformation and and it cancer cell transformation you have altered cell properties but it’s not necessarily going to lead to tumors unless additional changes occur so we’re going to talk about how studying virus transform cells really helped us to understand the progression to cancer and one of the things you should remember is no virus can do it all that means no virus on its own can cause a cancer it needs help from the DNA of the cell what viruses do as you will see it makes cells divide and then they need to accumulate the mutations that lead to a cancer so the virus itself only thing the virus is doing is making the cells divide uncontrollably these are a list this is a list of both RNA and DNA viruses that are associated with cancers not just in people but also in other animals there we’ve talked about hepatitis C virus the retroviruses which we’ll talk about today and then the DNA viruses add no viruses hepatitis B viruses herpes viruses papilloma viruses polyomavirus and even poxviruses viruses are the contributing factor in about 20% of human cancers and we know of eight different viruses that are associated with human cancer and they’re listed at the bottom here we’ve talked about most of these epstein-barr virus hepatitis B virus hepatitis C virus htlv-1 human t-cell lymphoma Peck virus one HIV one human papilloma virus and the herpes human herpes virus eight and Merkel cell polyomavirus the way these viruses lead to cancer is slightly different today we’ll talk about these two human viruses and also a variety of viruses that cause cancer in animal models and one thing another thing you need to remember here is that transformation in oncogene this isn’t required for the replication of any virus it is I guess you could say an accident it’s a byproduct of what the viruses need to do as you will see now I have an asterisk here because for years I’ve made this statement because I think it’s true and and someone emailed me a few years ago about a virus that causes tumors of fish where it seems that actually the tumor is important for spreading the virus we’ll talk about this in a moment so I think there is some evidence that perhaps for that one retrovirus of fish it’s needed for replication but for all the others certainly the ones we’ll talk about today making a transform cell making a tumor is absolutely not needed for the virus to replicate so the story begins in 1909 Peyton Rous a virologist working

at the Rockefeller Medical Institute now Rockefeller University here in New York City he was brought a chicken by a farmer as the story goes it’s not clear if that farmer brought it from New Jersey or Long Island brought a chicken it had a tumor on it and rouse got interested in this and he did experiments where he would remove the tumor grind up the tumor filter it through a point to micron filter take the filtrate and inject that into other chickens now of course you recognize that he is trying to find out if a virus is involved because it’s a very small filter that would exclude bacteria well he found that these cell-free filtrates which presumably called contained viruses in fact caused tumors in healthy chickens so the first evidence that cancer could be caused by a virus infection took 50 years for people to believe this and to accept that this virus could cause cancer eventually he was given the Nobel Prize in 1966 I think this is the longest incubation period for a Nobel Prize after the initial discovery so he discovered a virus that caused solid tumors in chickens he called it Rous sarcoma virus we’ll talk about this today other people then picked up this virus and it led to two more Nobel Prizes so three Nobel Prizes for this cancer causing chicken virus really interesting now there is a wonderful book that describes the history of cancer called the emperor of all maladies I read this several years ago when it came out by Siddhartha Mukherjee who happens to be an oncologist at Columbia this book won the Pulitzer Prize and a movie was just released by PBS by Ken Burns which you can find online to think it’s free and you can watch it for a time and it’s a six hour three-part not made-for-tv movie this book is beautiful it’s so wonderfully written to have as anyone read it isn’t great yeah yeah he’s up at the Columbia you can go walk around and see him anyway I’ve taken some quotes from his book because he says it better than I can by the 1950s cancer researchers had split into three feuding camps the virologist led by rouse claimed that viruses cause cancer although no such virus had been found in humans epidemiologists argued that chemicals caused cancer although they couldn’t tell you how and the third camp had circumstantial evidence that genes internal to the cell might cause cancer I’m going to tell you today how virology basically unified these three different camps continuing with Mukerji in 1951 a young biologist named Howard Temin arrived at Caltech he was going to study fruit flies he got bored with it he switched fields and decided to study Rous sarcoma virus in renato dulbecco z’ laboratory tobeco was a guy who developed a plaque assay whenever something new came up he embraced it and when cell culture was first developed in the early 50s in immortal cells he jumped on it and developed a plaque assay and then he said to Taman yeah you can come in and study this virus now the problem at this point is that the virus had only been shown to cause tumors and chickens and taman said we can’t figure out what’s going on using chicken we have to use cells and culture so now cells and culture were just at the point where they could be used by a lot of people so he tried to get the virus to infect chicken cells in a dish and caused tumors essentially but they ruin really tumors of course as you already know he added the virus to a layer of normal cells the infection incited them to grow uncontrollably forcing them to form tiny heaps containing hundreds of cells called fossa the FO site time and reason represented cancer distilled into its essential elemental form cells growing uncontrollably so you recognize this now as transform cells here are some pictures of transformed cells here is they can have different morphologies these are all avian cells transformed by Ralph’s sarcoma virus you have these spindly looking cells here you can see that the transform cells are are piling up and growing in fossa and not behaving well so this is what teman figured out that you could take the virus infect cells and it would have a permanent effect on the cell it would transform them permanently alright it’s not cancer as I’ve told you but it’s on the way to cancer so he wanted to understand how that worked and I’ll tell you what happened in a moment at the same time

about ten years later DNA viruses were shown to have transformative properties as well in 1962 polyomavirus was shown to transform baby hamster kidney cells in 1964 sv40 was shown to transform a mouse cell line called 3 T 3 so rouses virus was an RNA virus now we have DNA viruses shown to transform cells and culture in these cases most of the cells died but there were a few rare ones left with these DNA viruses and they were transformed so really the first question I want to address is how can a virus infection transform a cell we have been telling you a lot now that viruses kill cells but a transform cell lives forever so how can that happen well first of all you have to modulate the cytopathic effects you can’t kill the cell obviously you have to reduce virus replication in fact these transform cells don’t produce typically infected virus poor infectious virus particles and the cell has to keep dividing it has to be immortal these are the properties of transform cells so virus infections have to do this and this should ring a bell a transformed cell is a form of persistent infection right a persistently infected cell the cell doesn’t die the lytic potential of the virus is modulated and that’s exactly what transformed cell is we’re the first question is which of the following is not a property of transform cells one increased requirements for growth factors to immortality 3 loss of Anchorage dependence for loss of contact inhibition and 5 colony formation in semi-solid media which is not a property of transform cells which of those oops sorry screwed up I totally screwed up yep I already have an activity in progress damn can I get to it okay let’s see how do we do no this is the wrong question where’s here we go thank you how did we do all right 81% said increased requirements for gross fact where’d it go you know they just changed the interface and I think every time someone answers it goes the graph goes away this is really bright guys good job anyway 82% of you ran today which is correct increased requirements for growth factors is not right some of you answered loss of Anchorage dependence the cell transform cells have lost Anchorage dependence they can grow in a semi-solid medium some of us said colony formation no that’s these are typical properties of transformed cells all right so let’s go through this story and see how Rous sarcoma virus and those DNA virus has sorted out what’s going on in transformation and oncogenesis this was a long road starting with rouses first observation in the 1900s so we have rouses story on the left here which is his study with RSV and we’re going to follow that to see how that was sorted out on the right we’re going to talk later about the studies with DNA tumor viruses and what they revealed and in the 60s and 70s people working on these two different kinds of viruses their results came together to form a convergence and really make this unified theory of growth control because that’s what came out of this work we finally understood what controls the division of cells so let’s start with rouse and his study and his work with with his Rous sarcoma virus how does it cause tumors and chicken how does it cause transformation of cell in vitro let’s get a little background on this virus first rouse is part of a family of viruses called avian Lu kosis

retroviruses glucose is a different word for leukemia these are in all chickens around the world okay it’s a typical retrovirus with an envelope and an RNA genome plus polarity has reverse transcriptase in it so it can make a DNA copy and integrate this into the cell but of course raus didn’t know any of this in 1909 in 1908 Ellerman and bang actually discovered these AV and Lu kosis viruses because they cause leukemia in chickens they actually showed that these viruses cause leukemia and chicken but people at the time didn’t think leukemia was a cancer because it wasn’t a solid tumor forming cancer so it was kind of ignored for a while most chickens are infected with AV and Lou kosis viruses a few months after they hatch they get it from the other chickens in the hatchery leukemia develops in about 3% of these Birds over 14 weeks of age most birds have a transient viremia when they get infected they become immune and they don’t get not leukemia so a very few number of birds do get leukemia and of course they have to last longer than 14 weeks and in some situations the bird birds are slaughtered for food before that as the birds get older they develop other cancers as well so first 3% of the flock may develop leukemia the remainder have nothing but if you allow the flock to age they will develop other cancers and that was the bird brought to rouse with a solid tumor these birds develop various tissue tumors or sarcomas and if you get virus from these tumors and yuri inject them into new chickens they cause sarcomas not leukemia all right so if you wait and get solid tumors from these birds not the leukemia you get the virus from the solid tumor it will cause a solid tumor not a leukemia and a fresh chicken and whereas isolated one of these viruses that’s what Rous sarcoma virus is now after rousted his initial experiment he repeated it many more times and other people came in and started doing the same thing getting different chicken tumors from different flocks all over the world isolating viruses and they all got very different viruses as you will see and most of them are defective I’ll explain that in a moment so here we have a RNAi tumor virus that is causing tumors in chickens it’s causing transformation of cells and culture as you saw teman proof it’s a permanent change so Taman got really interested in this and he said this might this virus must make a DNA intermediate and integrated into the cell for that cell to be permanently changed and that’s what made him go after the enzyme that would make that DNA from the viral RNA and he discovered along with david baltimore reverse transcriptase and they received a Nobel Prize for that we talked about that some time ago so this is because teman was reasoning this RNA virus for it to permanently transform a cell it has to make a DNA that goes into the cell and that was the origin of reverse transcriptase now the question is how does it cause solid tumors and chickens and a key finding was that if you look at the viral genome from Rous sarcoma virus and all the other viruses isolated from chickens that cause solid tumors they were recombinant wasn’t just the viral genome was present but rather a piece of the viral genome was replaced with a segment of host DNA we now call this DNA an oncogene because it has oncogenic potential so the fact that Rous sarcoma virus had within its genome a cell gene called an oncogene which was essential for its ability to transform cells and culture that was discovered by these two viral ageist Michael Bishop and Harold Varmus and they got the Nobel Prize in 1989 so we have rouses Nobel we have the Nobel Prize for reverse transcriptase and now the the Nobel Prize for discovering that these transforming retroviruses pick up an oncogene from the cel-3 3 no bells for Rous sarcoma virus so here’s what’s going on with these cellular genes remember the birds as they age the birds that didn’t get leukemia as they age they develop solid tumors these tumors have retroviruses in them and they’re all derived from avian lue kosis virus that same virus that infects all the birds within shortly after birth but they’re all defective they’re defective viruses with one exception Rous sarcoma

virus the virus that he first discovered turns out to be not defective at all the uncle gene that has picked up from cells was added in addition to the viral genome and all the other isolates of these viruses causing solid tumors and chickens were defective because they lost portions of viral DNA we’ll look at this in a moment all of these other viruses had different uncle genes in them and raus had one uncle gene was isolated from a sarcoma the oncogene was called sark but as other people isolated viruses from chickens that caused a variety of solid tumors and then this moved into the mouse model and people could isolate viruses from mice that cause tumors they named the various oncogenes that were picked up according to the type of tumor and this is a gold mine for molecular oncology they isolated so many different oncogenes by just looking at the ones that were picked up by these viruses so here the pro viral DNA sequences of these transforming viruses so here at the top is AV and Lu kosis virus so that’s the virus that infects all the chicken in the world shortly after birth it can cause leukemia in about 3% of them this is a typical retrovirus it has a gag pol and on gene and the pro viral DNA integrated is integrated into the host genome of Rous sarcoma virus picked up the SARC gene in red there from the host cell and that allows this virus to transform cells in culture but you see this Rous sarcoma virus is not defective it has gag Paul and envelope genes and so this is a non defective virus all these other viruses these below rouse these are all isolated from different bird tumors myeloblast ouma’s milo saitama’s and other sarcoma erythroblastosis reticuloendothelial system sort of tumors and from them were isolated unique transforming retroviruses that picked up different cellular sequences mid let’s Mick mill yes Erb A or B and rel these are cellular genes that allow these viruses to transform cells so we call them oncogenes all these viruses are defective you see because they’re missing essential sequences many of them our mission Paul many of them are missing parts of envelope as well on the right are other mammalian transducing retroviruses from non bird species and here at the top is a murine leukemia virus very much like avian lou kosis virus the progenitor of all of them it’s got an intact genome you’re probably wondering now why is this virus able to cause leukemia we’ll find out in a moment and from this virus infection of mice you could derive a whole series of different transforming retroviruses for example Abelson murine leukemia virus where the cellular on krajina able is replacing most of the viral sequences there are also some retroviruses from cats you can see here a simian sarcoma virus etc and these all have the red sequences which are oncogenes stolen from the host cell which allow these viruses to transform so the defective viruses which most of these are require a helper to produce more virus they’re usually missing part of the envelope the glycoprotein in the membrane of the virus so they can’t replicate without that and for them to grow they need to have in the same cell a virus that will provide the envelope so that’s why we call them defector they need defective they need a helper virus to grow rouses virus was not defective he it grew on its own so think about how lucky he was because if he had isolated defect the virus he wouldn’t have been able to grow it and he might have given up at that point but his virus wasn’t missing any viral genes it just had an oncogene stuck into it and it grew and all these observations then followed so just to emphasize this idea about defectiveness here’s the parent AV and Lucas’s virus from which all these others are derived here’s Rous sarcoma virus just added the SARC gene and here’s an avian sarcoma virus that was isolated from someone else you can see it’s missing most of the viral genes so it’s defective it requires it has to be Co infected with a navy Lou kosis virus in order for this to replicate so how do these viruses pick up cellular sequences this is one model for that we’re not quite sure because they’re probably all different

but here we have a pro viral DNA so it’s a viral sequence that of course from an RNA tumor virus integrated into the cellular genome and you can see it’s it’s got the 2lt ours and just downstream of the integration site is a cellular gene which we’ve labeled UNK for oncogene so remember the retroviral pro viral DNA is integrate pretty much randomly all over the chromosome with certain sites in the chromatin that’s preferred but pretty randomly as far as Chrome chromosomes go so eventually it will sit down next to an oncogene now you know wild-type mRNA can be made initiating at the left-hand LTR and terminating at the right-hand LT are sometimes deletions occur in the viral genome which allow the mRNA to proceed past the termination signal in the right-hand LTR so now you get a longer mRNA which now picks up some of these Honka gene sequences this will get packaged into a virus particle and we think it needs to be packaged along with a wild type virus genome so that reverse transcription will generate the 2lt ours all right you’re gonna get non homologous recombination during reverse transcription so the reverse transcriptase will copy this ltr’ and then jump over to the other genome and pick up the oncogene this is a random event as well so all these are low frequency events and that gives you now a provirus a new virus and a pro virus with an oncogene in it so we have gagged Paul in this case we’ve lost some envelope sequence now a lot of these uncle genes that have been picked up are different they’ve have various rearrangements or mutations that make them more potent and you’ll understand what this means in a moment only a few of them are transforming simply because they’re over produced in a virus infected cell many of them have undergone additional rearrangements and point mutations that make these cell proteins these Anki oncogenes constitutively active so that’s one way we think that we will pick up an oncogene again by transcribing through to the oncogene sequence this is a very rare event because several low frequency events are needed to produce it nevertheless in the lifetime of a chicken with lots and lots of Av and lou kosis viruses produced the probability is reasonable that this will happen so so far we’ve identified over 60 different oncogenes by these kinds of studies by looking at retroviruses of various species of animals not just birds but mice and cats and so forth that have been picked up by the retroviruses we call these proto oncogenes because when when they’re in the cell they don’t cause cancer they don’t transform cells it’s only when they’re picked up in the virus and perhaps changed do they become actual oncogenes these over 60 genes are all involved in growth control of the cell and regulating the cells growth control as we said earlier the ability of cells to divide in us is highly regulated most of our cells at any given time is not are not dividing and that pathway is controlled by these 60 different genes that encode different components of this regulatory pathway the normal cellular genes are abbreviated C and some named C ox such as C Sark so the precursor of Rho sarcoma virus is Sarkis C Sark C mix CMOS etc and when a retrovirus picks up these cellular sequences when they’re present in the viral genome we put a V in front of it like V Sark V mech vemos and as I said most of these V unk genes are altered in some way to make them transforming only a few of them are transforming simply by virtue of their overproduction in the virus what are these proteins do the so this actually revealed the entire growth pathway that regulates mitosis in cells the study of these viruses these transforming viruses that picked up various oncogenes so on this slide are the different classes of functional classes into which these oncogenes fall again all of these genes and the encoded proteins were discovered in viruses that transform cells RNA tumor viruses so for example so this is the pathway by which cell division is regulated if there are growth factors in the medium they bind to a receptor a signal is then transduced into the nucleus and then transcription factors are made and they

turn on genes that are required for cell division all the components of this pathway were discovered as oncogenes in transforming viruses for example some transforming viruses have picked up a gene for growth factor and by simply over producing the growth factor they transform the cells it’s like having a lot of serum in the medium many of the oncogenes are growth the receptors these are plasma membrane proteins that serve as receptors for the growth factors so the oncogene is is simply nor its normal function is to be a growth factor receptor the virus picks up that gene either overproduces it or it’s altered in some way so it’s constitutively active so the point is every component in this pathway is going to be active so the cells are always dividing some of these oncogenes are membrane brown protein kinases that are involved in transducing the signal from the growth factor there are some G proteins like grass in the cytoplasm there’s some cytoplasmic protein kinases and then a whole bunch of nuclear uncle genes which are transcriptional regulators transcription factors as well as cell cycle regulators so in the normal untransformed cells each of these proteins has a role to regulate cell division in a way that maintains the function of the organism when they’re picked up by a retrovirus the the regulation is gone they’re now over produced or mutated and now the cells divide uncontrollably once that these genes are delivered by a retrovirus so to understand how they work we have to look at the cell cycle which you know all cells undergo this cycle we have a phase of mitosis where the cells actually divide and that’s surrounded by phases of DNA synthesis the S phase and a variety of gap phases and a major control point for the cell cycle is right here just before mitosis there’s a go signal that has to be sent to the cell to make the cell divide so DNA has already replicated in the S phase now the cell says is everything right for dividing that signal is provided by these proto oncogenes the pathway I just showed you growth fact our growth factors present in the medium if so we can go through this cycle so all of these control elements were discovered by these transforming retroviruses we call these dominant oncogenes because you just need one copy in a cell in order to transform it so you put these proto in a resting cell they will stimulate mitosis uncontrollably without stopping that’s why they transform the cells that’s why they make them immortal now further study of retrovirus mediated transformation revealed that there are three general classes of transforming retroviruses we have ones that cause rapid tumor formation that is about two weeks after you infect the animal with the virus you get a tumor and that’s Rous sarcoma is one of those and that’s because these viruses carry a dominant oncogene like Sark as soon as the virus infects the protein is produced it’s over produced it’s produced at the wrong time the cells begin to divide they’re transformed and eventually they go on to form a tumor as long as they accumulate the right mutations now we have retroviruses that have intermediate kinetics of tumor formation an example is avian Lu kosis virus the parent virus of Rous sarcoma virus it takes months for these viruses to cause leukemias they do not carry oncogenes remember when I showed you the picture of avian glucoses virus I used it to illustrate a wild-type genome with all the right genes and no oncogene it doesn’t these don’t transform by picking up an oncogene what they do is they transform by sitting into the genome next to an oncogene and turning on its transcription okay and then we have we Colette’s this activation and then finally we have retroviruses like htlv that are very slow at transforming cells and causing cancers takes years for tumors to develop these viruses do not carry an oncogene they do not sit down next to an oncogene as does a lv rather they produce a regulatory protein a transcriptional activator that turns on other cellular genes and over produces them and makes the cell divide uncontrollably so we call that trans activation so here’s an illustration of these three different types of transforming retroviruses we have the rapidly transforming retrovirus characterized by Sark Rous sarcoma virus these viruses have picked up a cellular

oncogene it’s now Avianca gene and the over production or the mutation of that gene leads to transformation we have viruses with intermediate kinetics these viruses do not pick up an oncogene rather they integrate in the genome right next to an oncogene and they turn it on uncontrollably remember these are all regulated very tightly and the virus infection is messing with that regulation and then we have the slowly transforming viruses like htlv these have no they don’t pick up an oncogene they do not insert next to an oncogene but rather they produce trans activating proteins that are needed to transcribe the viral genes and these happen to also activate cellular genes for example like il-2 and its receptor and this causes the cells to divide uncontrollably now I said earlier that none of this is needed for the replication of most retroviruses they don’t need the transform cells they don’t have to pick up an oncogene or integrate next to one they don’t need to induce a cancer but there may be one exception the virus that causes this tumor this is the walleye dermal sarcoma retrovirus that’s a walleye and this fish develops this tumor at a certain time of year it’s a retrovirus induced tumor and then at some point in the fall the tumor falls off and that’s how the virus spreads to other fish and the fish is fine this fish will go on living and never develop any more tumors so this is one example where it might be that the tumor is actually necessary for transmission not for replication of the virus but for transmission so there’s a part in the virus rep reductive cycle where tumor formation is needed that’s not the case for any of the other about tumor viruses that we know of alright the next question which of the following allows Rous sarcoma virus to transform cells presence of the envelope gene presence of a paul-jean press of a Sark jean presence of ltrs or maybe none of these mostly you answered presence of a Sark Jean that’s absolutely right that’s the cellular oncogene that this virus has picked up which allows it to transform cells the envelope the LT RS they’re not essential because in many of these defective retroviruses they’re not present so that is how studying RNA tumor virus has revealed the growth control pathway of the cell but they didn’t reveal all of it the next half was revealed by the study of DNA tumor viruses so let’s take a look at that this begins in 1933 when Richard Shope identified a DNA virus that caused papillomas at papillomas another name for a wart in rabbit and here are two rabbits with papillomas have any of you heard of the term Jackalope all right so people if you go online and search Jackalope you will see that people report this mythical animal which seems to be a cross between a rabbit and a jackrabbit and a antelope with it with antlers this is what they are it’s just rabbits with big papilloma so you can see they grow pretty big these are harmless they will fall off in the rabbits or fine but they’re caused by a DNA virus I love this one he looks really mad doesn’t he if you saw him in the woods you might get scared so they don’t hurt the rabbits they just fall off they’re caused by a DNA virus called the papilloma virus later Ludwik gross discovered similar viruses that cause tumors in mice and these caused rare tumors in mice the natural hosts of these viruses is the mouse they’re ubiquitous they don’t seem to cause any role in mouse cancer but in baby animals of different species of not the normal species for this virus hamsters rats or rabbits these viruses will cause tumors of different sorts and the fact that they caused many different kinds of tumors led to its name pali oma many tumors so this is a mouse virus in mice it doesn’t cause cancer but in other animals it does later on sv40 was discovered this is a monkey DNA virus it’s a polyomavirus as well it was discovered as a contaminant in the early batches of polio vaccine these vaccines vaccines were grown in monkey kidneys and those monkeys that were used there captured monkeys and their kidneys were removed and putting culture they had sv40 in them these viruses so our

natural viruses of monkeys they don’t cause tumors and monkeys but they will cause tumors in hamsters parenthetically many many millions of Americans got inoculated with infectious sv40 as a consequence of getting polio virus in the 50s and there’s a lot of controversy over whether this has caused any tumors and people I think the evidence indicates not but there is a box in the textbook that addresses that so the natural host for this virus is in the monkey it doesn’t cause tumors doesn’t transform monkey cells in culture but it makes it transforms cells of a different species so this is a pattern now which you should recognize these viruses are not transforming cells of their natural host they’re transforming cells of a different host so this is a summary of this response of different cells to infection here are here’s sv40 which is in monkeys is replication is permissive the virus infects and kill cells mouse polyomavirus is not from monkey cells are not permissive for Mouse polyomavirus so nothing happens there as we forty is Mouse cells are not permissive for it mouse cells Mouse polyomavirus Mouse cells are permissive for it that’s the natural source of that virus and then sv40 again being a monkey virus but hamster cells are semi permissive rat cells are semi permissive for Mouse polyomavirus again hamster and sells r7 semi permissive and it is in hamsters and rats where you see tumors caused by injection with either sv40 or mouse polyomavirus oh no not the permissive cells where the virus kills the cells not the non-permissive cells where no replication occurs but in semi permissive hosts and this transformation of these wrong hosts right it’s not the natural host of these viruses is pretty rare that one transform cell per hundred thousand cells infected why why is this so we’re going to try and understand why and this is going to make sense to you in terms of the mechanism of transformation now those were little DNA containing viruses a circular DNA’s like sv40 which we talked about so much earlier in this course as models for transcription and DNA replication so another family of DNA containing viruses that cost tumors and animals those are the add no viruses there many human adenovirus serotypes none of them cause tumors and people however add 12 to 18 serotypes 12 through 18 cause tumors and hamsters and 7 through 11 also although not as well just like the transformation of cells by polyomavirus and the papilloma viruses transformation by adenovirus is a very rare event and again it happens in the wrong host it doesn’t happen in the natural host of these viruses so what is in common to all these different viruses papilloma paloma and AD no viruses well they all make a t antigen like protein in the infect itself you may remember that sv40 encodes a protein that we called large T or T and this protein binds the origin of replication it’s needed to unwind the origin and recruit the DNA synthesis machinery similar proteins are found in cells with polyomavirus ‘as in papilloma viruses we give them a different name call them e v e6 and e7 proteins but they’re the same idea there needed to initiate DNA synthesis and even add no viruses have T remember the big T stands for tumor that’s how these were first discovered these proteins were found in tumors of animals caused by these viruses and also in transform cells in culture cells transformed by these viruses so one thing they all had in common was the presence of these proteins they were called tumor antigens in the name has stuck so for add no viruses they’re called a 1 a and a 1 B these are essential viral genes remember the sv40 T antigen is needed for DNA replication if you take it away the virus is dead in the water they’re needed for replication and transcription they’re needed for viral DNA synthesis and these genes are the only ones left in common in tumor cells or transform cells so when you infect we take sv40 and you infect a hamster or a rat and you get a rare transformed cell typically the only viral gene left is the gene encoding T antigen same for papilloma the same for ad no viruses and

in fact you can take the T the gene for T antigen from any of these viruses sv40 or poly Ouma or adenovirus just take that alone on a plasmid and put it into cells and that will transform those cells and culture it’s a very easy way to make immortal cells so if any of you ever want to take a cell a primary cell made from a tissue and make it immortal so it will grow forever and you can use it for your experiments all you need to do is get sv40 T antigen on a plasmid and put it in those cells and that’s enough to transform the cells why why so why this is so will become very obvious in a moment T antigens are encoded by viral genes that are essential for replication present in tumors and transformed cells encoded by viral genes that have been incorporated into the cell genome antagonists of cell cycle checkpoint proteins all of the above good so you picked all of the above which is correct 85% these are encoded by viral genes that are essential I told you that so those who pick that it’s wrong present did tumors and transform cells that’s absolutely true encoded by viral genes that have been incorporated into the genome so I didn’t actually tell you that these were incorporated in the Gina I said these are the only viral proteins that the only viral genes that are present in tumors and transform cells but the genes the DNA is encoding them are integrated and finally Part D we haven’t talked about yet that’s coming up they are antagonists of cell cycle checkpoint protein because now we’re going to talk about what these are doing so now so far we know that cells transformed by these different viruses have all T antigen in them what else do we need to figure out this problem so a number of different labs made the discoveries that these T antigen these viral T antigens are binding to different cell proteins and that’s how they’re working in particular sv40 is binding a Cell protein of 53,000 Dalton’s secondly transcription of the adenovirus early genes the e to gene cluster requires a cellular transcription protein called e to F there’s now it’s now known that we have a family of such proteins they’re called the e to F protein and finally this protein this family of transcription proteins was found to be bound to a cellular protein called the retinoblastoma protein RB so three things interaction with p53 the requirement for e to F and the fact that e to F is bound to a protein called RB all three of these cellular proteins worth subsequently found to be key regulators of the cell cycle and their interaction with these viral T proteins or T antigens modulates that property so let’s go back to the cell cycle and here’s at the bottom is the 24-hour cell cycle during mitosis of course the cells divide they’ve already accumulated DNA they’ve replicated the DNA so they’re ready to go into the cell cycle and their various phases around mitosis and the S phase so cells replicate their DNA they divide and then they replicate their DNA and and so forth and remember there was a crucial go signal at the top before mitosis begins there’s also a restriction point in g1 before replication of DNA occurs so whether or not the cell goes through this cycle is determined by these two checkpoints the proto-oncogenes that we’ve talked about discovered in RNA tumor viruses will sense whether growth factors are available and allow the cell to go through mitosis is the life outside in the outside world rich enough to replicate the cell and all of the components of that signaling pathway were discovered in retroviruses there is however a second restriction point down here in g1 so not only do the growth factors have to be present in order for the cell to go through mitosis but other conditions have to be met as well and that is determined at this second restriction point because if conditions aren’t right there is no DNA synthesis and no cell division and this restriction point is regulated by the RB protein which was found to interact with that e to F family of transcription proteins now RB retinoblastoma protein was discovered in young children who have retinal tumors this the gene encoding this protein and those kids is deleted both copies of the gene is gone and they develop retinal tumors and this protein was discovered in those tumors

and called RB but many years later it was found to be a checkpoint regulator right here at this restriction point huge huge finding way beyond the implications for retinal blastoma so we call our be a recessive oncogene because both copies have to be deleted in order for the cells to divide uncontrollably remember the dominant oncogenes the ones picked up by the retroviruses they’re recessive only one copy is enough to sari their dominant only one copy is enough to transform cells or be you have to delete both copies it’s recessive so how does this work let’s put all this information together on the Left we have a cell showing the pathway for control of the mitotic cycle so if there are say growth factors present in the extracellular media they will bind to a growth factor receptor which will initiate a phosphorylation cascade that eventually results in progression through mitosis the M phase of the cell cycle and this signaling pathway all the components are oncogenes identified in retroviruses so if the world outside is rich the cell will divide a major regulator of whether DNA synthesis will occur after cell division is the retinoblastoma protein which is shown here in a phosphorylated form and so this the state of this protein determines whether DNA synthesis will occur and on the right it shows you how retinoblastoma protein works so retinoblastoma is the mustard-colored protein in an unfolded state it is bound to this e to F family of transcription proteins so e to F SAR needed for e2 early gene adenovirus transcription they are also needed for transcription of the various cyclones that are needed for mitosis so when retinoblastoma is bound to e to F the e to F can’t turn on the synthesis of mRNAs encoding all these genes that are needed for mitosis and cell division it’s only when retinoblastoma is phosphorylated can it be popped off of e to F e to F now can stimulate the genes required for DNA synthesis and eventually mitosis occurs so remember the growth factors plug in at the top of the cell cycle they allow by ptosis to occur but you don’t get the next round of DNA CIN this is unless RB is phosphorylated which will occur in response to signals integrated from earlier in the cell alright and this is why RB needs to be phosphorylated to pop it off of e to F because e to F is essential for it’s a transcription protein and it turns on genes that are needed for DNA synthesis in the cell now remember that DNA viruses do not like to replicate in resting cells because they require the DNA synthesis machinery s before T and adenovirus require various components of the cell and most of our cells are resting so these viruses need to kick them into mitosis we talked about this weeks ago the need to activate the DNA synthetic machinery by pushing the cells through the DNA cell cycle so T antigens are the proteins that do that the T antigens of sv40 papilloma viruses and aDNA viruses these get made very early on and they make the cell go through mitosis and eventually DNA synthesis so normally in a little confectioners is doing this so that I can replicate at the genome how does this happen remember retinoblastoma is normally bound up to e to F that keeps the cells from going through DNA synthesis these t antigens II won a large t e7 they bind our be directly remember this is one of the observations made after the T antigens were discovered they interact with RB they pull RB off of the e to F proteins without it being phosphorylated e to F can then go on to turn on all the genes that are needed for DNA synthesis and eventually mitosis so that’s why T antigens kick the cells into dividing because they sequester RB RB would not normally be phosphorylated the cell is going to decide what to do that but the virus is not waiting for the cell the virus needs to get the cells dividing so they make T antigen which binds up RB and lets the cell go through the cell cycle the entry is also under more control we

haven’t brought in what p53 does there is @a there if the cell senses that there is either DNA damage double-stranded breaks in DNA or unscheduled DNA synthesis ie viral DNA synthesis p53 can sense that so here is p53 it can sense the presence of a DNA break or it can sense DNA being replicated when it shouldn’t be it’s a complicated story we don’t have to go we don’t have time to go into but it knows when the DNA of the cell is being replicated under the normal conditions when a viral genome gets in there and starts replicating p53 can detect that and when it does it puts a halt to DNA synthesis in the case of double-stranded DNA breaks the idea is not to duplicate damaged DNA so p53 says we’re stopping DNA synthesis until we can make repairs in the case of viral DNA synthesis well p53 is kind of an innate immune response or even intrinsic it’s got to prevent viral DNA synthesis from occurring and that’s what so here’s what happens p53 senses here double-stranded DNA damaged and together with a number of other proteins it will bind to promoters of genes that are needed for progressing through the cell cycle and it will induce a proptosis so p53 when it senses unscheduled DNA or DNA damage it will kick the cells it will stop DNA replication it will kick the cells into a path osis and that’s why these DNA viruses need to counter p53 I told you this a long time ago and I said wait you’ll understand why viruses have to counter p53 this is why because p53 is halting replication and pushing the cell into a pitocin so these viruses all have antagonists of apoptosis to get around this how do the viruses counter p53 they do it in all sorts of interesting ways which are shown here when viruses infect cells here is p53 getting two cents double strand breaks or unscheduled DNA synthesis and it is it is net negated in a variety of ways by the different viral large T so for example sv40 large t sequester’s p53 it binds it up in a multi merit complex so that it can’t bind promoters it can induce apoptosis it can’t halt DNA synthesis II one B also sequester’s p53 the e6 proteins of the papilloma viruses and the e 1b proteins of the aDNA viruses cause ubiquitination of p53 which directs it to the proteosome and it gets degraded okay so this is how the viral T antigens antagonize p53 so that the cell will continue to go through its replicative cycle providing the virus with the DNA reagents that it needs for replication alright we’re almost at the point of solving this mystery first question why are all the genes except the T antigen genes turned off those are the only ones that are left in transformed cells and why is transformation so rare so that reasons it is is because several low probability events are needed to suppress the lytic potential you have to delete the lethal eight genes of the virus so when you infect permissive cells by aDNA virus the virus goes through the cycle and kills the cells there’s no chance of transformation occurring but if you replicate in a semi permissive host late gene expression is blocked so the virus is not killing the cells or in other cells the deletion of the late gene may spontaneously occur so you have to somehow modulate the production of late genes which can happen either in a semi permissive host or if it’s the natural host deletion in the viral genome occurs so that the virus doesn’t kill the cells but this is very rare and secondly T antigen has to be on in every cell and transmitted to every daughter cell okay so the DNA has to be integrated into the host DNA and T antigen has to be produced so these are rare events they don’t happen in every cell so remember the transformation is going to end up with a transform cell in which only the T antigen DNA is present and for that to occur is very rare so here’s the story in the end the virus uses antagonists of RB and p53 to kick the cell into the cell cycle to use it for DNA replication if rare events occur to isolate the gene encoding those T antigen in the cell and

keep it there permanently the cell is going to become transformed so these events the transformation is not needed for the replication of the virus the virus requires these to kick the cell into division but remember that so eventually is going to die but if some circumstances intervene where all of our genes are gone except that encoding T antigen that antigen is going to keep that cell dividing forever and that’s why they transform cells so these are really accidents of viral biology they have nothing to do with the normal viral life cycle so DNA tumor viruses have to kick-start the DNA synthesis cycle and they use T antigens to do that they inactivate both RB and p53 RB allows the cell to go through the checkpoint into DNA synthesis and p53 antagonism prevents the shutdown of DNA synthesis and the induction of apoptosis so this is how you get transformation you block lytic events you get rid of the late genes that would normally kill the cell and what you’re left with is a t antigen that’s that’s transformed the cell so it’s a rare event it’s it doesn’t happen in a permissive cell very frequently and much perhaps slightly more frequently in a semi permissive cell so these cells are transformed they’re not yet tumorigenic they have to replicate until they’ve accumulated these dozen or so mutant in various genes that I mentioned earlier which will then make the cells able to cause a cancer so all the virus has done is to leave behind a few genes which make the cells immortal the cell itself really becomes a cancer cell on its own because DNA replication is error-prone and if a cell multiplies uncontrollably eventually you will have all the mutations you need in order to be a cancer alright so this is the summary you have two checkpoints in the cell cycle you have the the go point the proto-oncogenes identified in RNA tumor viruses ask whether there are sufficient nutrients in the medium to make the cell cycle go and if so the cell cycle proceeds and the oncogenes picked up by retroviruses transform cells by pushing them through this cycle and the DNA tumor virus has antagonized this checkpoint down here the tumor suppressor genes which regulate the passage through the DNA synthesis phase the induction of apoptosis by p53 those are targets of DNA tumor viruses so this is how the cell cycle is controlled and we understand it today at these two points entirely because of work done with RNA and DNA tumor viruses it’s a great example of how viral adji exposes normal cell biology and it all started with a tumor in a chicken it just goes to show that you have to study weird things sometimes to make good discoveries you