Virus Infection and the Biology of Kaposi's Sarcoma • iBiology

Part 1: Kaposi’s Sarcoma: The Disease

00:00:02.20 Hello, I'm Don Ganem, I'm a professor of microbiology and infectious disease at the University of California in San Francisco
00:00:09.22 and investigator of the Howard Hughes Medical Institute.
00:00:12.03 I'd like to tell you today about one of the great detective stories
00:00:16.10 of contemporary biomedical research.
00:00:18.29 It's a story about a tumor called Kaposi's sarcoma. It was discovered over 130 years ago
00:00:24.08 and it was once thought to be very rare, but in the late twentieth century, with the supervention of the AIDS epidemic,
00:00:30.14 this tumor became one of the important public health problems in America and Western Europe
00:00:36.07 and it continues to be a public health problem in Africa and the Mediterranean basin.
00:00:42.21 It's a story that involves many different lines of work, clinical medicine, epidemiology, virology, molecular genetics, cell biology.
00:00:52.17 And for that reason, it's a fun story to talk about in a venue like this.
00:00:56.19 One of the most interesting things about KS is that it reveals how much physicians and scientists can learn from one another
00:01:06.16 if they would only learn how to listen to one another.
00:01:08.29 So, I'm going to tell you this story in a historical way, and we're going to begin at the beginning.
00:01:13.05 So, let's start with considering a little bit about KS as a disease.
00:01:18.02 So, our story begins back in 1872 with this man, Moritz Kaposi.
00:01:24.07 Kaposi was a dermatologist in Hungary and he described initially 5 patients with what he called "multiple idiopathic,"
00:01:32.08 idiopathic means we don't know what causes it,
00:01:34.14 "pigmented nodules of the skin."
00:01:37.00 Now, we recognize that this tumor, which predominantly involves the skin,
00:01:43.00 comes in several different forms. So, the classical form, this is the form that is generally attributed to Kaposi,
00:01:52.08 is an indolent tumor. This is a tumor that patients can live with for many years.
00:01:58.01 It's often said in medicine that KS is a tumor that patients die with, but not of.
00:02:02.20 It's an indolent tumor principally involving the skin. Kaposi described it in elderly men of Mediterranean extraction,
00:02:09.19 but now we know that other groups can be affected as well.
00:02:13.14 The tumor is typically localized to the lower extremities, and very rarely disseminates,
00:02:19.06 fewer than 10 percent of cases have dissemination outside the skin.
00:02:23.13 Many years after Kaposi died, it was recognized that KS also occurs in Africa
00:02:30.09 and is in fact more frequent in Africa than it is in the Mediterranean basin.
00:02:33.23 In fact, before the AIDS epidemic occurred in Africa, in a cancer hospital in Kampala, Uganda,
00:02:41.18 10 percent of all the surgery that was done for cancer was done for Kaposi's sarcoma.
00:02:45.24 So, this is an extraordinarily common tumor in Africa.
00:02:50.09 Clinically the endemic form resembles the classical form,
00:02:54.09 except that the disease a) occurs in younger people and b) can be locally invasive, invading underlying bone and other structures.
00:03:03.03 It is also common for the disease to involve lymph nodes in young children.
00:03:07.29 Now, in the mid-20th century with the advent of organ transplantation,
00:03:14.13 another form of KS was soon discovered.
00:03:17.23 And this is a form that is histologically very similar to the traditional form, but is much more aggressive in the clinic.
00:03:23.29 There's widespread dissemination to many sites in the skin and dissemination to organs
00:03:30.05 and this is attributed to the immunosuppressed nature of a transplant recipient.
00:03:34.28 And finally, as I think everyone knows, with the advent of the AIDS epidemic,
00:03:39.10 the most aggressive form of KS was described, KS became an extraordinarily common tumor in patients with AIDS
00:03:46.15 and in AIDS, as in the transplant recipient, it is a very aggressive disease.
00:03:51.22 The disease can be widespread, involving large regions of the skin, as I'll show you.
00:03:56.19 And can even involve major viscera, like the lungs.
00:04:02.03 At this level of the discussion, it's very important to emphasize that all these forms of KS have a common histology.
00:04:09.25 Under the microscope, they are virtually indistinguishable.
00:04:12.07 These differences have to do with their distribution, their tropisms for different organs,
00:04:17.01 in the whole organism, but under the microscope, it appears to be one process.
00:04:21.19 Now, let's talk a little bit about classical KS because this form of the disease undergoes a very slow evolution in the patient over time.
00:04:32.21 It begins, as you can see at the top of the slide,
00:04:35.11 with what's called a patch lesion. This is the earliest lesion that a dermatologist can recognize as being KS.
00:04:43.00 And as you can see, this lesion on the arch of the foot, is not even a mass, it's not even a nodule,
00:04:49.14 it's a flat region that's red and if you were to biopsy this lesion,
00:04:54.01 you'd see that in addition to the tumor cells of KS, there are many inflammatory cells,
00:04:59.11 T-cells, B-cells, monocytes and macrophages.
00:05:02.07 They're almost more prominent at this stage than the actual tumor itself,
00:05:07.01 so you can see, there isn't even a mass here yet.
00:05:09.11 With the passage of time, there is evolution to this so-called plaque-like form.
00:05:14.22 The lesion gets slightly elevated, becomes harder, indurated, and it's a little bit swollen, or edematous.
00:05:22.05 And with time, and this usually takes years, those plaque-like forms evolve into the nodular form,
00:05:29.09 shown below. This is an actual mass, it's in this case a dome shaped mass,
00:05:35.13 and as you can see, all of these lesions are discolored and they're discolored a reddish purple.
00:05:41.16 And the reason for that is that all of these lesions are displaying a tremendous number of new blood vessels.
00:05:46.22 I think you can see in the patch lesion at the top that the lesion is red.
00:05:51.11 If you were to press on that lesion, it would blanche, all the red cells, all that red is coming from red blood cells
00:05:57.22 inside of new blood vessels in the tumor. So, this is a highly angiogenic tumor.
00:06:03.00 As the lesion progresses, you can see it becomes darker and darker, more purple,
00:06:07.22 patients often think they have a bruise that isn't healing,
00:06:10.13 and the reason for that is that these new blood vessels that are formed in this tumor
00:06:15.21 are not normal and they're fragile and they rupture and red cells leak out into the tissue and undergo degradation
00:06:22.11 and as they do, their heme pigments get discolored and oxidized and that's what's responsible for the purplish hue of the lesion.
00:06:31.08 But you can tell from the fact that the lesions are red or purple to the naked eye
00:06:34.28 that these are loaded with new blood vessels, highly abnormal new blood vessels.
00:06:39.08 So, now we're going to move from classical KS to the form of KS that's much more familiar to Westerners
00:06:45.01 and that is KS in the context of HIV infection.
00:06:48.21 And shown here is an example of an AIDS patient with dermal KS, KS of the skin.
00:06:55.22 And you can see, unlike those classical lesions where there might be one or two or three spots on the lower extremity,
00:07:02.00 here, we see dozens of spots all over the torso. But again, if you were to biopsy each one of these spots,
00:07:09.11 it would look, under the microscope, just like the lesions in the classical case.
00:07:13.24 There are just more of them and they are widely spread over the entire body.
00:07:17.09 These lesions can be very disfiguring when they occur on the face,
00:07:23.22 first of all, they cannot be hidden and they become obvious stigmata of the underlying immunodeficiency.
00:07:29.13 They're very socially stigmatizing.
00:07:31.22 You can see in this patient, virtually the entire bulb of the nose has been replaced,
00:07:36.15 the ears extensively replaced, and that is actually a feature of KS lesions,
00:07:40.21 that they tend to occur at the tips of extremities, at places where the temperature is slightly lower than the core body temperature,
00:07:48.11 for reasons that are still unexplained, the nose, the ears, the tip of the penis, the toes,
00:07:54.12 these are all places where KS lesions tend to get very bad.
00:07:58.11 The other feature of KS in patients with AIDS is that large, confluent areas can be seen,
00:08:06.08 as in this patient, and interestingly, these confluent areas are often symmetric.
00:08:10.26 You can see in this case, both thighs are very extensively involved with nearly confluent regions of KS
00:08:17.18 in addition to the patches of KS over the torso.
00:08:20.22 So, again very potentially disfiguring.
00:08:24.27 But even though all of those skin lesions are stigmatizing and disfiguring,
00:08:29.21 the skin disease, as bad as it can be, and it often requires X-ray therapy for palliation,
00:08:35.15 the skin disease, no matter how bad it is, is not life threatening.
00:08:39.18 So, what kills patients with AIDS related KS?
00:08:42.25 What kills them is involvement of vital organs
00:08:47.04 and the most commonly involved vital organ in AIDS related KS is the lung.
00:08:51.27 So, shown at the top here is a chest X-ray of an AIDS patient with pulmonary KS
00:08:58.06 and you can see that both lungs are heavily infiltrated with abnormal tissue.
00:09:03.28 And this tissue, if you biopsied it, would prove to be KS.
00:09:07.12 At the bottom, there's an example of an autopsy specimen of the stomach of a patient who had developed KS in the stomach.
00:09:16.00 KS frequently involves the gastrointestinal tract,
00:09:18.19 less commonly in the stomach than it does in the colon.
00:09:22.21 But as you can see again, the mucosal surface of the stomach is studded with red spots that, on biopsy, proved to be KS.
00:09:29.25 Patients die of these complications, especially pulmonary KS leads to respiratory failure.
00:09:35.14 Intestinal, or a GI KS, can lead to abdominal pain and occasionally bleeding.
00:09:42.13 But the most common cause of death from KS is pulmonary KS and lung failure.
00:09:48.03 Ok, now, I've been using the word sarcoma to describe KS and that would imply that KS is a traditional form of cancer.
00:09:58.02 So, a sarcoma, for those of you who don't know, is a cancer of the connective tissue,
00:10:02.26 of the mesenchymal tissues, muscle, fibrocytes, et cetera.
00:10:07.06 KS is named a sarcoma but in fact, it is a very atypical lesion and differs from classical cancer in many ways.
00:10:17.10 And the first and most important way is at the level of the light microscope.
00:10:22.06 So, if you look under the microscope of most tumors, they are monotonous,
00:10:25.29 they are the product of the clonal outgrowth of a single cell and a slice through that nodule
00:10:31.09 would show that, you know, a fairly monotonous collection of tumor cells, which are all derived from the same cell
00:10:37.20 and are therefore all of the same cell type.
00:10:40.02 When you biopsy KS, it's quite a different story.
00:10:43.12 There are many different cell types in the lesion, as I'll show you in a second.
00:10:46.29 When the lesion becomes nodular, the main cell in the mass is something called a spindle cell.
00:10:52.13 And it's called a spindle cell because it is shaped in an elongated, it is an elongated shape, structured like a spindle.
00:10:59.11 For many years, pathologists discussed what cells gave rise to spindle cells,
00:11:05.15 now we know that they derive from endothelial cells.
00:11:08.23 Endothelial cells are the cells that line blood vessels and lymphatics.
00:11:12.06 But in addition to those spindle cells, the tumor cells, there are many inflammatory cells, as I've mentioned.
00:11:17.28 T-cells and B-cells, monocytes and macrophages, and then there are these abundant abnormal new blood vessels, or neovascular spaces,
00:11:26.12 oftentimes with extravasated red cells, red cells that have leaked out of them into the tissue.
00:11:32.07 And then had their globin pigments degraded to something called hemosiderin.
00:11:37.02 So, here's a light microscopic section through a skin lesion of KS
00:11:43.25 and you can see at the top, the epithelial cells of the skin, they're normal, and they're not involved,
00:11:52.24 the process of KS involves the deeper regions of the soft, of connective tissue underneath the skin.
00:11:58.10 This is the so-called dermis, so, the epidermis, the epithelial cells are normal,
00:12:02.13 the process involves the dermis and right off, you can see even without being a pathologist,
00:12:07.03 that there's a lot of red stuff in this section.
00:12:09.17 All that red stuff are red blood cells within new, abnormal vascular spaces.
00:12:14.17 So, you can see right away the tremendous angiogenic component here.
00:12:18.11 It's harder to see at this magnification, but there are scattered throughout the dermis, a lot of inflammatory cells,
00:12:25.12 B-cells, T-cells, et cetera, monocytes.
00:12:28.04 And, in addition, weaving in and out of the plane of the section are the spindle cells.
00:12:33.08 You don't see them very well in this image because they are weaving in and out of the plane of the section,
00:12:37.09 when the microtome goes through the section, you can't see them well.
00:12:40.18 But every once in a while, just by chance, the microtome will cleave them along the correct sectional plane
00:12:47.04 and you can see them. So, here's a nice example from a case of KS in the GI tract,
00:12:52.17 and here, you can see the spindle cells lined up as they've been cut along their long axis.
00:13:02.05 And you can see now why they're called spindle cells, because they are very elongated.
00:13:05.25 Now, I like to think of KS, so, KS is clearly an extremely complicated process under the light microscope.
00:13:11.21 I like to think of KS as being derived from three components that are not independent of one another,
00:13:18.06 but that can be thought of slightly separately.
00:13:20.27 Of course, like any cancer, there's a proliferative component.
00:13:23.25 And in the case of KS, the cells doing the proliferation are these spindle cells.
00:13:29.04 They're the cells that are going to give rise to the mass, or the nodule at the end of the day.
00:13:32.29 In addition, there's a process of inflammation and on top of that, is a process of angiogenesis.
00:13:39.05 These things are related to one another, but we're going to think and talk about each of them separately
00:13:44.13 because the viral infection that causes KS makes separate and different contributions to each one of these things.
00:13:50.18 Ok, so what is the origin of the spindle cell? I've mentioned to you already that everyone's confident that they are derived from endothelial cells,
00:13:59.15 the cells that line blood vessels and lymphatics,
00:14:01.23 and that is derived from the fact that they stain for a variety of endothelial markers,
00:14:05.21 CD34, CD31, are present in most spindle cells, but spindle cells are themselves a heterogeneous collection.
00:14:13.26 Most of them don't stain for classical endothelial markers, like Factor VIII or Von Willebrand's factor,
00:14:19.09 but a few percent of them do.
00:14:20.15 So, that's another feature of spindle cells, they're not monotonous, they're heterogeneous.
00:14:24.25 They're not uniform. A big question has been whether they're derived from vascular endothelium,
00:14:31.07 the cells that line blood vessels, or lymphatic endothelium, the cells that line lymphatic tubes,
00:14:36.18 structures that drain fluid from tissues.
00:14:40.03 And for many years, there was a conviction that KS derives from lymphatic endothelium,
00:14:47.03 and that may be so, what drove that conviction was the fact that markers like podoplanin, VEGF receptor-3 and LYVE-1,
00:14:55.04 which are classical markers of lymphatic endothelium, regularly stain a KS lesion.
00:15:01.08 But all of this conventional wisdom was overturned in 2004 when it was described by several groups,
00:15:07.23 Chris Boshoff, Mike Lagunoff, Michael Detmar, that when KSHV infects vascular endothelial cells in culture,
00:15:15.27 it causes them to express many lymphatic markers.
00:15:18.22 Conversely, if you infect lymphatic endothelium in culture with the KS virus, they often express many vascular endothelial markers.
00:15:27.27 And this reprogramming of endothelium may explain why KS cells display markers of both lineages.
00:15:35.19 So, I think we're probably never going to know for sure whether KS derives from a lymphatic or a vascular endothelium,
00:15:42.09 because the viral infection that's linked to this tumor changes the expression program.
00:15:47.08 So, at the moment, we have simply to say that they're of endothelial origin and leave it at that.
00:15:52.03 Now, I want to emphasize a bunch of other extremely unusual features of KS.
00:15:58.07 Things that strongly differentiate KS from ordinary, garden variety cancer.
00:16:02.28 First of all, the lesions are multi-centric, I think you're already seen that, that these lesions occur all over the body
00:16:08.21 and interestingly, they don't occur in a pattern of a primary tumor followed by a bunch of metastasis.
00:16:14.22 Most patients will come to medical attention, yeah, they have three or four spots that appeared more or less simultaneously,
00:16:22.10 and they're partner noticed three or four additional spots on the back.
00:16:27.00 Not a story of one mass followed by, later in time, a lot of other distant metastases.
00:16:33.10 And in fact, if you biopsy these different lesions, you can often show that they are not related to one another.
00:16:40.03 KS lesions are frequently oligoclonal or polyclonal.
00:16:44.29 Now, there are examples in the literature of clonal KS.
00:16:48.24 And I think this is consistent with the idea, that like any polyclonal process in evolution,
00:16:54.06 over time, some clones will do better than others and there will be cases where all the lesions are related to one another.
00:17:01.24 But biopsies and clonality studies tend to show that many of these lesions are not related to one another
00:17:08.00 and represent independent occurrences. One's always taught in classical cancer biology that tumors are genetically unstable,
00:17:16.08 this is what drives aneuploidy and chromosomal rearrangements in tumors.
00:17:20.04 That kind of genetic instability is not a feature of KS.
00:17:23.21 KS spindle cells, if micro dissected out and examined, don't have any chromosomal rearrangements.
00:17:28.20 Typically, they're diploid. Now, there are rare exceptions to that,
00:17:32.14 but by and large these tumors are not nearly as genetically unstable as classical cancer.
00:17:37.18 And another feature that's quite striking, is that, and this was well described in the classical era,
00:17:44.17 even before the AIDS epidemic, that rare cases of spontaneous remission of KS are known.
00:17:50.03 It's not especially common, but it does happen, and it happens much more frequently in immunodeficient patients
00:17:58.05 if immune function is partially restored.
00:18:01.04 So, transplant patients who have their dose of cyclosporine lowered will often have a remission,
00:18:06.07 without any chemotherapy and this is also frequent in AIDS patients whose underlying HIV disease
00:18:12.16 is arrested with antiretroviral therapy. So, that does not happen in patients with lymphoma or lung cancer,
00:18:19.21 who have these similar ministrations. So, again, we're talking about a disease that's really on the cusp between the benign and the malignant.
00:18:28.03 One particularly interesting feature of KS that I want to spend quite a bit of time on in this lecture
00:18:35.01 is the fact that there seems to be a relationship with other inflammatory states.
00:18:39.21 Back in the 80's and early 90's, before the advent of good therapies for HIV,
00:18:44.15 doctors who saw patients with KS often remarked on the following sort of occurrence.
00:18:50.20 Patients with stable dermal KS, who had a few lesions of KS but were otherwise not seriously afflicted,
00:18:57.26 if they came into the hospital with an AIDS related infection, pneumocystis pneumonia,
00:19:02.26 cryptococcal meningitis, something like that, a severe inflammatory state, and if they survived that infection,
00:19:09.22 in the weeks that followed, sometimes their KS would rage out of control and have widespread dissemination of dermal KS
00:19:17.28 or develop visceral KS. Again, this wasn't a uniform occurrence,
00:19:21.28 but it was frequent enough to be noticed by many practicing AIDS clinicians.
00:19:25.21 Another example of that is shown here.
00:19:28.14 Patients with KS at a distant site, if they undergo surgery, may develop a local lesion of KS right at the site of the incision.
00:19:37.15 So, this is a slide illustrating a patient who had gum surgery
00:19:41.28 and six days after the surgery on the gums,
00:19:45.00 developed a florid lesion of KS right at the site of the incision
00:19:49.04 and this tumor was so bad that it ultimately had to be treated with X-ray therapy in order to be resolved.
00:19:55.09 And this is an image from a paper by Jennifer Webster-Cyriaque, a very talented AIDS clinician in North Carolina.
00:20:02.15 So, this is a well-known phenomenon in clinical medicine.
00:20:07.17 So, there appears to be some very strong relationship between local inflammation
00:20:11.24 and the development of KS.
00:20:14.14 Now, KS was a difficult process to study in the laboratory for many years
00:20:21.12 because it was very, very difficult, it turned out, to grow KS cells from a clinical lesion.
00:20:27.04 This problem was solved by Bob Gallo at the NIH in the late 80's and early 90's.
00:20:33.12 He developed methods for growing KS cells reproducibly in culture
00:20:38.22 and the trick was something that was very counterintuitive for a cancer biologist.
00:20:43.17 In cancer biology, we always tell students that cancer cells lose their dependence on exogenous growth factors.
00:20:51.00 This is why, for example, many cancer cells will grow in low serum.
00:20:54.18 That's considered a classical sign of transformation.
00:20:58.16 With KS cells, it's just the opposite. These cells have an exaggerated dependence on extracellular growth factors.
00:21:04.21 Gallo showed that the only method he could develop that would reproducibly allow spindle cells to grow from a KS biopsy
00:21:11.09 was to grow them in the conditioned medium of activated T-cells.
00:21:14.08 A cytokine and growth factor rich environment.
00:21:17.09 Even when cultivated like that, and they could be maintained indefinitely in such a medium,
00:21:22.26 but they didn't display the properties of morphologic transformation.
00:21:27.05 They didn't undergo, they wouldn't grow in soft agar, they wouldn't form tumors in nude mice.
00:21:33.03 In fact, Gallo showed, with Barbara Ensoli that if these cells were implanted into a nude mouse,
00:21:39.11 they would survive for ten days, maybe two weeks, and then involute.
00:21:43.04 During the time that they survived in the mouse,
00:21:46.03 they provoked a transient angiogenic response in the surrounding murine tissue.
00:21:51.00 And then when the human implant disappeared, or apoptosed, that angiogenic process involuted.
00:21:58.20 So, these experiments, and the clinical behaviour of KS and the histology of KS,
00:22:04.26 gave rise to a general understanding that what KS is really about is that it's a disorder of paracrine signaling.
00:22:12.24 It's spindle cells produce pro-inflammatory and pro-angiogenic factors
00:22:17.22 that recruit these inflammatory T- and B-cells and monocytes into the lesion.
00:22:22.09 Those inflammatory cells in turn recruit more inflammatory cells through the production of cytokines and chemokines,
00:22:28.02 but also, cross-feed the spindle cell component, supplying it with survival factors
00:22:34.22 that are necessary for the prevention of apoptosis and possibly growth factors that stimulate their proliferation.
00:22:40.14 So, all of KS could be understood now as a complicated paracrine minuet
00:22:45.27 where these different cell populations that are visible in the biopsy
00:22:50.03 are talking to one another, communicating with one another in a complicated way.
00:22:54.04 And that leaves us now with the question, how does this all begin?
00:22:57.16 What is the factor that initiates that process?
00:23:00.03 And this is where we'll pick up in part 2.

Part 2: The Virus

00:00:02.11 Alright, so let's move on now to the second part of the talk,
00:00:05.28 which is going to concern the discovery of the virus of KS itself.
00:00:10.26 Where did the notion that KS might have an infectious precipitate come from?
00:00:15.25 Interestingly, it didn't come from any microbiologist or virologist,
00:00:19.13 it came, rather, from clinicians and epidemiologists who were studying the natural history of KS.
00:00:25.18 And that natural history revolved around several striking facts.
00:00:31.09 Of course, when the AIDS epidemic happened and KS went from being an extraordinarily rare tumor
00:00:39.16 to a tumor that affected perhaps 20 to 25 percent of young gay men with AIDS.
00:00:45.21 Everyone initially presumed KS itself might be the cause of AIDS.
00:00:51.06 But this idea soon collapsed,
00:00:52.29 of course we know that KS occurred in Africa and the Mediterranean prior to the onset and spread of HIV through the human population
00:01:01.20 and it could be shown that in classical KS, there was no evidence of HIV in the lesion.
00:01:06.17 Also, it was soon shown that in the AIDS related form, there was no HIV DNA or RNA in the spindle cells themselves.
00:01:14.22 Most KS biopsies didn't have any HIV positive cells in them.
00:01:18.25 So, right away it became clear that although HIV was a very important cofactor,
00:01:23.16 that put people at risk for KS, it probably wasn't itself the cause, the proximate cause of the tumor.
00:01:31.05 Another particularly striking feature that supported that idea
00:01:35.24 was that if you look among HIV positive patients themselves,
00:01:40.14 KS was not uniformly distributed among AIDS patients. So, even among people who had contracted HIV,
00:01:45.28 and had gone all the way to a severe immunodeficiency,
00:01:48.17 they were not equally at risk for developing this tumor.
00:01:51.11 The KS was largely the province of gay men with AIDS, 20 to 25 percent of whom, in the early days of the epidemic,
00:01:58.29 would develop KS. Whereas, if you looked at hemophiliacs,
00:02:02.15 who had acquired HIV from blood transfusions, KS was relatively uncommon in the, 2 percent.
00:02:09.03 KS was also relatively uncommon in women compared to men,
00:02:13.12 most women acquired HIV either through drug use, needle sharing, or sexual, heterosexual contact
00:02:22.15 with HIV positive individuals, and women had a very low incidence of KS,
00:02:26.15 even when they went on to overt immunodeficiency.
00:02:29.00 And most impressively, children who acquired HIV by vertical transmission,
00:02:34.05 from the infected mom to the newborn baby, by transplacental or perinatal spread of virus,
00:02:40.23 almost never got KS, despite the fact that they had a very accelerated course and rapidly succumbed to the immunodeficiency.
00:02:47.18 So, these things all put the lie to the simple notion that HIV itself was the proximate cause of KS.
00:02:53.07 And when epidemiologists looked at this data, one thing struck them.
00:02:57.10 Which is of course, the striking prevalence of infection among gay men
00:03:02.02 suggested that sexual transmission of some other agent in the context of an immunodeficiency
00:03:08.03 might account for this result, particularly if the agent was more likely to be transmitted homosexually,
00:03:13.07 than by heterosexual transmission.
00:03:15.05 So, those facts swirled around the epidemiology community for a long time
00:03:24.06 before they caught the attention of microbiologists and infectious disease specialists
00:03:28.13 and virologists. Most people weren't sure what to make of these facts
00:03:34.02 and because of the strong bias that many molecular biologists have
00:03:36.27 that information from epidemiology is simply correlative and not powerful in any direct way,
00:03:41.24 many people in AIDS research discounted these results, or simply ignored them.
00:03:46.02 But two people who did not are shown here.
00:03:48.19 These are my friends Pat Moore and Yuan Chang who were then at Columbia,
00:03:52.29 now at the University of Pittsburgh. And they, as we did, took these results very seriously
00:04:00.18 and began a search for viruses in KS.
00:04:04.17 And, now, of course, simple attempts to cultivate viruses from KS didn't work
00:04:09.13 and had been tried before, so their approach was to use a very advanced genomic method
00:04:16.13 that had been recently described by Michael Wigler of Cold Spring Harbor,
00:04:19.16 a method called representational difference analysis, or RDA.
00:04:23.11 RDA is essentially a form of subtractive hybridization, a souped up form of subtractive hybridization
00:04:31.06 married to PCR amplification and it can be used to explore two different populations of cells,
00:04:36.21 the DNA of two different populations of cells, looking for segments of DNA that are present in one, but not in the other.
00:04:43.03 That's the fundamental notion behind subtractive hybridization.
00:04:46.05 And Pat and Yuan did this on KS tumors and normal cells, normal skin or other tissues from unaffected patients.
00:04:56.27 And after much to-do, they discovered from this procedure,
00:05:02.13 from a single KS tumor, two small segments of DNA of about 100 to 200 base pairs in length
00:05:08.01 which, when sequenced, which were unique to KS,
00:05:11.04 not present in the surrounding normal skin, and when sequenced, had distant evolutionary relationships
00:05:18.05 to genomes of known human herpesviruses like EBV, Epstein-Barr virus.
00:05:23.25 They were also weakly homologous to a simian virus called herpesvirus saimiri.
00:05:29.20 So, these were not EBV genomes, but they were related to EBV genomes
00:05:35.07 and they correctly surmised that must indicate the presence of a new herpesvirus
00:05:39.24 in these KS tumors and they rapidly went on to show that these small sequences
00:05:45.10 could be used as markers of KS and they were found not only in the AIDS-related form of the disease,
00:05:51.16 from which they were originally isolated, but all forms, including the classical forms, endemic forms,
00:05:56.19 whether KS was HIV associated or HIV unassociated from any part of the world,
00:06:03.25 KS tumors always had these DNA sequences in them.
00:06:07.29 And this result was rapidly affirmed by many other labs including our own.
00:06:12.13 So, now it's clear that these sequences define the existence of a new virus,
00:06:19.05 a new herpesvirus, a human herpesvirus.
00:06:21.21 Prior to this, there were seven known herpesviruses, now there are eight.
00:06:26.17 The formal name of the KS virus is human herpesvirus-8,
00:06:31.08 but most of us use the more informative name, KS associated herpes virus
00:06:35.25 because it indicates the major disease state with which the virus is associated.
00:06:41.17 Now, when the KS genome, KSHV genome was completely sequenced, as it was by 1996,
00:06:50.18 it became clear where it stood phylogenically.
00:06:54.16 So, all of the human herpesviruses cluster into three families:
00:07:00.02 the alpha herpesviruses, the beta herpesviruses, and the gamma herpesviruses.
00:07:06.14 And these, this segregation is now defined by DNA sequences, but initially these groups were defined biologically.
00:07:14.27 Alpha herpesviruses are viruses that reside, or establish a latent infection in neuronal cells;
00:07:21.05 gamma herpesviruses are viruses that establish latency in lymphoid cells.
00:07:28.09 So, that was a huge surprise, that this virus that was derived from a predominantly endothelial neoplasm
00:07:37.23 actually sorted with the lymphotropic herpesviruses.
00:07:41.02 And for a while there was confusion about this, people wondered this was not the right virus,
00:07:47.24 just some sort of passenger virus, but I'm going to show you very compelling evidence
00:07:52.25 that it is linked to KS, but this lymphotropism set off a search that turned out to be productive.
00:08:03.12 Once it became clear that the normal reservoir of KSHV is in fact the B-cell,
00:08:08.25 students of AIDS and especially AIDS-related lymphomas
00:08:13.22 began to look for the presence of KSHV in lymphoproliferative disease.
00:08:18.18 And Ethel Cesarman and her colleagues and others rapidly established that there are two lymphoproliferative diseases that are linked to KS.
00:08:29.20 One of them is a rare lymphoma called primary effusion lymphoma.
00:08:34.01 So, some of you may know that because of their immunodeficiency state,
00:08:37.23 AIDS patients get many forms of cancer, KS being the most common,
00:08:41.12 second to that is lymphoma, predominantly B-cell lymphomas.
00:08:44.28 Many of these B-cell lymphomas turn out to be EBV related, but one of them
00:08:49.24 is KSHV related.
00:08:51.04 It's a rare tumor called primary effusion lymphoma.
00:08:53.29 Now, unlike KS, this is a classical lymphoma. This is a clonal B-cell tumor. It's an unusual B-cell tumor
00:09:00.17 because the bone marrow and lymph nodes are generally normal and the tumor arises in serosal spaces,
00:09:12.09 spaces that are outside of solid organs.
00:09:15.03 The plural space, outside of the lung, the pericardial space, outside of the heart,
00:09:19.00 the perineal space, outside of the intestinal tract.
00:09:22.23 These essentially form ascites tumors in these cases
00:09:26.08 and they occur in patients who have very advanced AIDS,
00:09:31.17 they carry a very poor prognosis. And these cells that are growing already in liquid culture in these spaces
00:09:37.20 are adaptable readily into tissue culture and grow readily in culture,
00:09:41.22 unlike KS cells. The other lesion that was found to be linked to KSHV
00:09:48.17 is a disorder called Multicentric Castleman's disease.
00:09:52.10 The initial paper on this was written by a man named Soulier, in France,
00:09:55.24 who discovered that AIDS related multicentric Castleman's disease is very tightly linked to KSHV.
00:10:05.00 These lesions have KSHV DNA in them.
00:10:07.02 Multicentric Castleman's disease, or MCD, is an interesting disease that's right, again,
00:10:12.03 at the cusp between the benign and the malignant.
00:10:14.12 It is a polyclonal process in which B-cells proliferate, patients develop very high fevers,
00:10:20.12 big spleens, lymph node involvement, they have sky-high levels of IL-6 in the peripheral blood,
00:10:26.06 so in those regards, they look like they have some sort of acute disease.
00:10:31.18 But these patients have a proliferative syndrome and they require chemotherapy for its control.
00:10:37.13 So, again, a rare disease, but these two diseases are B-cell diseases linked to KSHV.
00:10:43.12 I'm not going to say anything more about them today,
00:10:45.18 but just to point out that they are further support for the idea that the principle reservoir of KSHV is in the B-cell
00:10:53.17 and there are pathologic consequences to the B-cell infection.
00:10:57.08 An important aspect of this was cultured primary effusion lymphoma, or PEL cells,
00:11:03.29 were central to advancing the virology of KSHV.
00:11:08.08 With our colleagues Mike McGrath and Brian Herndier in San Francisco,
00:11:12.06 who had isolated several PEL cell lines from patients at San Francisco General Hospital,
00:11:18.29 we showed that these cells lines, which retained KSHV DNA,
00:11:24.02 could be coaxed to produce the virus particles themselves.
00:11:28.06 So, the B-cell lymphoma lines are latently infected, they have cryptic infection of KSHV,
00:11:35.15 but if treated with phorbol esters or other chemicals, we showed that they could undergo lytic reactivation,
00:11:40.23 and produce infectious virus particles, electron micrographs of which are shown in this slide.
00:11:45.04 And that provided a great source of early antigens and virus for future work,
00:11:51.18 so finally, we had a system for cultivation, in which virus particles could be collected and used to infect other cells,
00:11:58.21 but also, these PEL cells, because they were infected, could be used as a source of antigen
00:12:05.17 to develop a serologic test.
00:12:07.28 I want to tell you about the serologic test,
00:12:10.23 but I wanted to emphasize why we were interested in making a serologic test for KSHV.
00:12:15.15 Because remember, at the time, the question was, was KSHV really the agent predicted by the epidemiologists?
00:12:22.14 Was it really causally linked to KS?
00:12:25.19 We didn't want to spend a decade working on the molecular biology of a virus that was not the causative agent of KS,
00:12:32.18 so we and others, many others, including Pat Moore, Thomas Schulz, Chris Boshoff, many other groups, Denise Whitby,
00:12:41.26 began to try to develop, based on PEL cells in culture, serologic tests.
00:12:48.02 And the one that we developed is schematized here.
00:12:51.03 It's an immunofluorescence test in which the antigen from latently affected primary effusion lymphoma cells,
00:12:57.21 we incubate these with a patient's serum, wash and then come in with a second antibody,
00:13:02.16 a FITC conjugated, a fluorescinated anti-human IgG.
00:13:07.02 And this is what it looks like, the nuclei of the cells light up,
00:13:10.22 with an antigen that we called latency-associated nuclear antigen, or LANA.
00:13:15.19 And it's a very crude test, those of you who do immunofluorescence know that it's an insensitive method.
00:13:24.11 The sensitivity of this method was about 75 percent, 65-75 percent. But it is highly specific.
00:13:31.04 The pattern of staining, nuclear, punctate nuclear staining, was highly specific.
00:13:37.07 The test is laborious, it all has to be done manually,
00:13:39.23 but it was our first entree into developing a test that would diagnose KSHV infection,
00:13:47.02 or exposure to KSHV in a human serum.
00:13:50.06 Nowadays, this kind of testing has gotten much more advanced, it's typically done by enzyme immunoassay,
00:13:57.09 often for LANA with one or more lytic antigens thrown into the mix
00:14:01.28 and the tests have improved to a sensitivity of 90 percent and specificity of 97 percent,
00:14:07.18 although there are still no commercially available tests for routine diagnosis.
00:14:11.21 But using our first order, homebrewed immunofluorescence test, Dean Kedes, an infectious disease fellow in my lab,
00:14:19.05 studied over, in the aggregate, 5,000 patients
00:14:23.04 from various groups. And his work and the work of others established a striking fact,
00:14:30.16 which is that KSHV prevalence in the population strikingly mirrored KS risk.
00:14:37.14 So, if you look, as Dean did, at the blood donor population, a proxy for the whole population,
00:14:43.14 only 2 or 3 percent were positive in this test.
00:14:47.20 Similarly low rates were found among HIV positive hemophiliacs,
00:14:51.05 and only slightly higher rates were shown in HIV positive women,
00:14:55.05 groups that we knew were at very low risk for KS.
00:14:59.00 But, when he looked at HIV positive gay men, seropositivity rates rose to anywhere from 30 to 60 percent,
00:15:08.15 depending on the population and the test.
00:15:10.10 And this is a group where we know roughly 20 percent of these folks had or would develop Kaposi's sarcoma.
00:15:19.19 So, there was a striking segregation of KSHV infection, or exposure, with known risk for KS.
00:15:26.05 And this was a very powerful fact that strongly advanced the argument that the genome discovered by Moore and Chang
00:15:33.27 really was the agent that had been predicted by the epidemiologists.
00:15:38.17 There's a lot more in the decade that has followed,
00:15:43.12 many groups now have examined populations all over the world
00:15:47.04 and what was true in the United States is true globally. Where KS is uncommon, the seroprevalence of KSHV is low,
00:15:55.04 so in the US and Western Europe, it's somewhere between 2 and 7 percent,
00:15:58.27 whereas in endemic zones of the Mediterranean basin, southern Italy, Sicily, Sardinia,
00:16:09.14 between 20 and 25 percent of the whole population is seropositive.
00:16:14.07 And in central and southern Africa, where KS has long been known to be a highly prevalent tumor,
00:16:22.02 the seroprevalence is between 50 and 70 percent.
00:16:24.24 So, again, not only in our domestic population, but globally, where KSHV infection is frequent, KS is common,
00:16:33.27 where infection is rare, KS is rare.
00:16:37.14 The other thing about these low prevalence and high prevalence zones
00:16:41.27 is they define two different epidemiologic patterns of KSHV spread.
00:16:45.17 And the mechanisms here are still not fully understood,
00:16:48.19 but in Western Europe and America, where seroprevalence is low,
00:16:51.17 infection is principally derived by sexual transmission in adulthood,
00:16:56.19 and especially among gay men,
00:16:58.10 something about homosexual sexual congress appears to be a particularly efficient way of spreading KSHV
00:17:07.13 and this is unusual in sexually transmitted diseases,
00:17:09.20 most of them have relatively equal rates of exchange among the different sexes.
00:17:14.13 But in western Europe and America, that doesn't seem to be the pattern.
00:17:18.01 In the endemic zones, however, in Sicily and Sardinia,
00:17:21.24 and especially in Africa, there is very efficient spread by some non-sexual route.
00:17:29.08 We know this because many children, many pre-pubital children in these cultures
00:17:33.11 seroconvert in the first decade or two of life, before the onset of sexual activity.
00:17:39.07 And then of course, after the onset of sexual activity, rates of infection continue to rise,
00:17:44.22 so there is sexual transmission in those societies, but there's also another form of transmission,
00:17:49.17 which we believe is due to salivary exchange.
00:17:53.04 There's lots of KSHV in saliva, saliva is the principle body fluid that has virus in it,
00:17:58.21 and it's thought that in those cultures, salivary exchange is an important route of transmission.
00:18:03.26 Why these patterns are what they are, however, we really don't know,
00:18:08.24 why some regions display one pattern of spread and others another,
00:18:12.16 is something that remains to be understood.
00:18:16.08 However, we did show, with Jeff Martin, a very gifted epidemiologist here at UCSF,
00:18:22.13 that among gay men, sexual transmission is very strikingly efficient
00:18:29.22 and is strongly correlated with the number of sex partners.
00:18:32.27 So, here you can see the rate of antibody positivity rising as the number of sexual partners increased,
00:18:41.01 and this was the first evidence for sexual transmission in the United States.
00:18:44.15 Alright, so let me sum up the epidemiologic argument that links KSHV infection to KS,
00:18:51.14 because I think there's a nuance here that's very important to understand.
00:18:55.05 So, here are the points.
00:18:57.11 First of all, KSHV DNA is present in all KS lesions,
00:19:01.07 irrespective of whether they're HIV positive or HIV negative.
00:19:04.20 Second, as I showed you, both locally and globally, KSHV seroprevalence tracks strikingly with KS risk.
00:19:11.24 I haven't shown you this, but it's true, that infection with KSHV precedes KS development
00:19:18.14 and predicts an increased risk of KS.
00:19:20.26 So, in prospective studies that we did with Jeff Martin,
00:19:24.04 we showed that if you were a young gay man living in San Francisco
00:19:28.21 and you were dually positive for KSHV and HIV, and you received no effective treatment for either virus,
00:19:36.01 over the next ten years, 30 to 50 percent of those men developed Kaposiâ€™s sarcoma.
00:19:41.23 Finally, we note that KS is never observed in the absence of markers for KSHV.
00:19:49.11 So, if you do not have KSHV infection, you will not get KS.
00:19:53.21 So, those are the hard epidemiologic facts that link KSHV to the tumor,
00:19:58.25 but, it's important to realize that what they establish is that KSHV is necessary for the tumor.
00:20:05.17 There's good reason to believe that KSHV infection is not sufficient for the tumor.
00:20:10.07 I told you that somewhere between 3 and 7 percent of the US population is infected with KSHV
00:20:15.14 but these subjects in general have a very low risk.
00:20:19.04 The actual magnitude of this risk hasn't been measured because KS is a relatively rare tumor outside of the AIDS population in the United States.
00:20:27.04 But in Sicily and Sardinia, island populations where there's very good epidemiology,
00:20:32.18 and where seroprevalence is 20 to 25 percent,
00:20:36.07 there are pretty accurate numbers that allow us to estimate that if you're KSHV seropositive
00:20:42.00 and don't have HIV, your risk of developing, only 1 in 3,000 to 1 in 10,000 of those people
00:20:48.27 will develop KS.
00:20:51.21 Therefore, it's very clear that in addition to KSHV, some other cofactor must be required.
00:20:59.06 Now, we don't know what that cofactor is in the HIV negative form of the disease,
00:21:04.16 but in the AIDS related form, we know that that cofactor is HIV.
00:21:09.27 Here's one illustration of that fact and this is derived from epidemiologic studies of Jeff Martin back in the 90's,
00:21:17.25 before there was effective treatment for HIV.
00:21:21.12 It was shown that your risk of developing KS, prospectively, was strongly linked to the viral load.
00:21:28.25 So, if you had greater than 100,000 copies of HIV per milliliter of plasma,
00:21:35.01 people rapidly and efficiently developed KS,
00:21:38.08 whereas, if you had an intermediate level, 10,000 to 100,000, your risk was slightly lower,
00:21:43.21 and if you had less than 10,000 copies of HIV per milliliter of blood,
00:21:50.05 spontaneously, now, without treatment, you had a lower still risk of KS.
00:21:55.19 So, that was one thing, there was a clear relationship, a dose relationship,
00:21:59.07 between the activity of the HIV infection and the risk for developing KS over ten years.
00:22:04.23 And a particularly striking feature has been what's happened to the epidemiology of KS
00:22:11.17 since the introduction of highly active anti-retroviral therapy, or HAART.
00:22:16.03 Highly active therapy became widely available in mid-90's, around '95, '96,
00:22:23.00 and since then has been progressively adopted. And as HAART has become more prevalent among HIV patients in the United States,
00:22:33.24 the incidence of new cases of Kaposi's has plummeted, has decreased about 90 percent.
00:22:39.03 So, the data I'm showing on this slide are from Washington State, but the same thing has been seen all over the country,
00:22:44.25 and in fact everywhere where HAART has been widely adopted.
00:22:49.27 So, not only is there a correlation between spontaneous HIV replication and KS risk,
00:22:54.24 but the interdiction of HIV replication has led to a striking reduction in KS risk.
00:23:00.20 So, all of this goes to say that KSHV is the essential driver of the process,
00:23:07.28 but it requires an amplifier like HIV in order to produce clinical disease.
00:23:14.25 Alright. So, how does KSHV infection predispose to KS risk?
00:23:20.03 That's the question now. Nobody doubts that KSHV infection is the central element of pathogenesis here,
00:23:26.00 but how does it predispose to KS?
00:23:29.08 Of course, to a virologist, this question boils down to one thing:
00:23:33.08 what viral genes are expressed in the tumor and what do they do?
00:23:37.06 And this is where, and we'll explore this question in the next part of the lecture.

Part 3: KSHV Latency and KS Pathogenesis

00:00:02.18 So, let's move on now to consider how KSHV mechanistically is linked to KS pathogenesis.
00:00:08.17 And the beginning of wisdom here is to understand a fundamental aspect of herpesvirus biology,
00:00:17.05 which is that like all herpesviruses, KSHV has two transcriptional programs:
00:00:23.03 a latent program and a lytic program.
00:00:26.02 So, what does that mean? Latency is a state in which the viral genome persists in the nucleus
00:00:32.28 in an episomal form. There's no integration in this form of the life cycle.
00:00:38.21 So, there's a circular plasmid maintained in the nucleoplasm.
00:00:43.04 And the important thing is that gene expression from this plasmid is highly restricted.
00:00:48.06 So, the KSHV genome is 165 kilobases, it has over a hundred open reading frames,
00:00:53.17 but during latent infection, only a handful of these open reading frames are expressed.
00:00:57.27 In fact, the main open reading frames that are expressed are shown in the boxes here,
00:01:02.10 there's one other gene down here that's expressed in latency,
00:01:05.05 but fundamentally, it's a highly restricted program,
00:01:08.10 less than 5 percent of the genome is actually transcriptionally active during latency.
00:01:13.12 As a result, there's no virus production and the cell survives.
00:01:17.13 Now, in that sense, it's a cryptic infection, but it's not irreversibly cryptic.
00:01:23.28 Because the whole genome is still retained in the nucleus,
00:01:28.00 there is the possibility to re-awaken gene expression from the rest of the genome.
00:01:32.02 And when that happens, the virus enters its so-called lytic cycle.
00:01:37.18 So, the lytic cycle is the cycle that is perhaps more familiar to students
00:01:41.02 who've had microbiology, this is the phase in which all the open reading frames of the virus are expressed.
00:01:48.04 They're expressed in a developmentally and temporally regulated fashion,
00:01:52.17 immediate early genes to late early genes, DNA replication, late genes.
00:01:56.16 So, there's this sequential, or cascade of gene expression, but the net result of that is that all the viral open reading frames are expressed,
00:02:04.27 viral DNA is replicated, progeny, virions, are assembled, the cell is killed and viruses released.
00:02:11.27 Ok, so, these are the two cycles
00:02:16.12 and we're going to consider in this segment what latent infection contributes to KS development
00:02:24.04 and in the final section, what the lytic infection might contribute.
00:02:28.06 It's important to know that in KS tumors, advanced KS tumors,
00:02:35.15 most of the spindle cells, all of the infection in a KS tumor is trained on the spindle cell compartment.
00:02:42.11 The infiltrating inflammatory cells are by and large not infected.
00:02:46.24 So, it's really the endothelial cells of the spindle cell compartment that harbor the virus
00:02:50.29 and in fact, 95 percent of those cells are latently infected, or even more, sometimes.
00:02:57.28 There is lytic infection going on in the tumor, it's limited to a very small subset of spindle cells,
00:03:02.16 and we'll come back to that at the end of the lecture because I think it is important,
00:03:06.04 it's not a negligible aspect of this, and we'll come back to that.
00:03:10.17 So, because the latency program is so restricted, involving only a handful of genes,
00:03:16.00 there's been a lot, and because it's on in every tumor cell in the tumor,
00:03:19.23 people have been interested in understanding what the latency program is all about,
00:03:25.20 what are the genes that are expressed and what do they do?
00:03:28.04 So, here I'm showing a schematic of the major latency locus and you can see that there are four open reading frames here
00:03:37.20 and these include a gene down here called kaposin, that we'll have a great deal more to say about it.
00:03:44.11 And another gene over here called FLIP.
00:03:49.03 These two genes are pro-inflammatory genes that I'll be telling you a great deal more about.
00:03:53.20 There's also a cyclin D homologue expressed in the latency program.
00:03:57.25 A fully active cyclin D homologue. And then of course the protein LANA, that I mentioned earlier.
00:04:04.00 LANA is the antigen in our original immunofluorescence test for infection.
00:04:10.15 Now we know that LANA plays a very important role in the maintenance of the latent episome
00:04:15.07 and I'll be discussing that in brief later on.
00:04:18.11 A more recent discovery is that the latency program also includes a dozen pre-microRNAs
00:04:28.14 that are processed to engender 17 mature microRNAs.
00:04:34.15 These are all products of a latent transcription unit.
00:04:39.00 Ten of the 12 pre-microRNAs are found in the intron of that unit.
00:04:42.16 I won't have more to say about these microRNAs in today's talk,
00:04:46.15 they're a subject, as you might imagine, of very active investigation in our lab and others.
00:04:50.24 And suffice it to say we really don't know yet what all of these do,
00:04:56.02 but it's virtually certain they're playing an important role in either the maintenance or the elaboration of latency.
00:05:02.12 Alright, now one of the dirty little secrets of KSHV virology is that latent infection of many cells in culture
00:05:15.08 doesn't produce a very striking phenotype.
00:05:18.17 So, it turns out that once you learn the tricks of how to grow KSHV,
00:05:22.01 it's possible to infect practically any adherent cell in culture with the virus.
00:05:26.09 The default pathway is latency. And generally, if you're working with established or already immortalized cell lines,
00:05:34.13 there's no particular phenotype that's linked to that latent infection.
00:05:37.27 And when latent infection is carried out on a lot of primary cells, again, we don't typically see immortalization.
00:05:46.02 So, this is strikingly different from the situation in Epstein-Barr virus.
00:05:50.10 Some of you may know that EBV is a very powerfully immortalizing virus,
00:05:55.08 the latency program of EBV is very strongly immortalizing.
00:05:59.01 After, to this day, EBV is still the only reproducible method for immortalizing human B-cells from peripheral blood.
00:06:07.20 B-cells have a very short lifespan in culture unless you infect them with EBV,
00:06:13.06 but every time you do, an immortalized line will grow out.
00:06:16.18 So, the EBV latency program is powerfully immortalizing.
00:06:19.20 KSHV's latency program doesn't seem to be that way.
00:06:23.16 And for a while, we were quite despondent that the latency program had virtually no phenotype that we could dissect in the lab.
00:06:30.13 But that turns out not to be true.
00:06:32.16 It was initially shown by Gary Hayward and his colleagues and his colleagues at Johns Hopkins
00:06:37.09 that primary endothelial cells that are infected with KSHV do have a phenotype,
00:06:42.11 and I'm going to show you an example of that on the next slide.
00:06:44.23 This is the work of Claudia Grossmann in our lab,
00:06:47.03 but I want to emphasize the primary discovery here was by Gary Hayward at his group at Hopkins.
00:06:53.29 If you take primary endothelial cells, in this case, dermal microvascular endothelium,
00:06:59.22 and do either a mock infection, above, or a KSHV infection, below, and wait a week,
00:07:07.12 what you discover is that after a week, so, in the first few days of infection, there's a little bit of lytic replication
00:07:13.19 and spread throughout the culture.
00:07:15.10 That's ultimately extinguished and all the remaining cells are latently infected.
00:07:20.28 And what you can see is that the normal endothelial cells have a sort of cobblestone morphology,
00:07:27.27 but when they're, each of the cells is sort of more or less cuboidal,
00:07:31.20 but when the cells are infected with KSHV, within a week, of the latency program being established,
00:07:37.22 the cells undergo this very exuberant, or dramatic, spindling phenotype.
00:07:43.10 They become extremely elongated, just like the spindle cells I showed you earlier in the KS biopsy.
00:07:49.26 It's important to realize, though, that these cells are not immortalized.
00:07:54.18 In our hands, and I think in the hands of most people in the field,
00:07:58.12 these cells will hang around for a week or two in culture, and then they'll die.
00:08:03.23 We have never observed outgrowth, long-term outgrowth, of these cells in culture.
00:08:09.21 So, the phenotype is one of cell shape, a cell shape change.
00:08:16.23 That shape change is due to a massive reorganization of the actin cytoskeleton.
00:08:22.14 So, here is a phalloidin staining of uninfected and infected cells,
00:08:26.09 and you can see that the actin cytoskeleton has undergone a rearrangement into these long tubules,
00:08:31.09 or cables, of actin. But again, I want to emphasize, the cells are not immortalized.
00:08:36.21 Sometimes, they survive a little longer than their uninfected counterparts, sometimes not.
00:08:41.21 I don't know for sure whether there's a survival phenotype, but there's certainly not immortalization.
00:08:47.08 Now, one of the interesting things about having a cell culture phenotype
00:08:51.14 that is so strikingly reminiscent of one aspect of the in vivo phenotype
00:08:57.15 is that because there's very limited gene expression in latency,
00:09:00.23 we can hope to identify which viral genes are linked to this process by cloning the individual latent genes,
00:09:07.07 one by one, into retroviral vectors and using them to infect primary endothelial cells
00:09:12.13 and then waiting to see if the cells infected by these individual retroviruses containing individual KSHV genes
00:09:22.12 will undergo a shape change.
00:09:24.16 And this is an experiment that was conducted by Claudia Grossmann in my lab
00:09:28.10 in the, about 2004, 2005, and the result is very gratifying.
00:09:34.26 It shows that a single KSHV gene, that encoding vFLIP, is responsible for the spindling phenotype.
00:09:43.02 So, you can see here that the cells infected with the empty vector
00:09:47.25 or vectors containing any of the other KSHV genes, failed to change the shape of the endothelium,
00:09:53.23 but cells infected with the vFLIP retrovirus strongly undergo the spindling phenotype.
00:09:59.13 That spindling phenotype, by the way, is cell autonomous,
00:10:03.16 conditioned medium from these cells, if put on fresh endothelial cells do not cause them to spindle,
00:10:08.00 and it's due to expression of the FLIP gene.
00:10:10.12 So, let's review the NF-kappaB activation pathway.
00:10:14.12 NF-kappaB, as you may know, is a transcription, a heterodimeric transcription factor,
00:10:19.26 which exists in the ground state in the cytoplasm
00:10:23.05 and it is retained in the cytoplasm by its binding to an inhibitor called IkappaB.
00:10:28.14 So, when IkB binds to the two subunits of NFkappaB, that subunit is retained in the cytoplasm
00:10:38.14 because of masking of its nuclear import signals.
00:10:41.20 But that process is regulated. It's regulated by a kinase called IKK,
00:10:46.08 which is a heterotrimer of three subunits, alpha, beta and gamma.
00:10:50.07 IKK phosphorylates IkB and that phosphorylated form of IkB undergoes degradation in the proteasome
00:10:59.14 and the loss of IkB allows the release of the active transcription factor,
00:11:05.12 whose nuclear import signals are now available to the cellular machinery.
00:11:09.08 It can be imported into the nucleus and turn on the promoters of a large number of cellular genes.
00:11:17.14 Interestingly, the genes that NFkappaB controls are largely genes that involve pro-inflammatory signaling,
00:11:27.09 so a huge number of cytokines and chemokines.
00:11:30.06 Also, certain growth factors, VEGF being among them.
00:11:34.26 And many factors involved in cell survival and the prevention of apoptosis.
00:11:39.01 So, NFkappaB is a pro-inflammatory and anti-apoptotic, or pro-survival,
00:11:44.25 transcriptional program that involves hundreds of genes.
00:11:48.09 It turns out that FLIP, and this is not our work again, is an inhibitor of IKK, an activator of IKK.
00:11:58.13 It binds to the NEMO subunit and triggers constitutive phosphorylation of IkB
00:12:05.01 and constitutive activation of NFkappaB.
00:12:08.01 That results in a pro-inflammatory and anti-apoptotic signal cascade.
00:12:14.17 Now, how do we know that that activity of FLIP is responsible for the shape change that I just showed you?
00:12:21.01 We know that because it's possible to develop antagonists of the IKK/IkB signaling pathway.
00:12:30.22 And one such antagonist is something called the IkB super-repressor, or IkB-SR.
00:12:37.17 This is a variant, a mutant, of IkB, in which the two serines that are phosphorylated by IKK
00:12:47.18 are mutated such that the molecule cannot be phosphorylated
00:12:53.08 and therefore, if overexpressed, all the NFkappaB in the cell is retained in the cytoplasm
00:12:59.12 in a form that can never be delivered to the nucleus.
00:13:02.23 So, in order to ask whether the NFkappaB inducing activity of FLIP is responsible for the shape change,
00:13:11.29 Claudia did a simple experiment.
00:13:13.24 She transfected HUVEC cells, infected HUVEC cells with a retrovirus that expresses the IkB super-repressor
00:13:20.28 and then superinfected them with either a control virus or the FLIP virus
00:13:25.15 and asked about the morphology.
00:13:27.03 And the results were very gratifying.
00:13:29.25 In cells that did not get the IkB super-repressor, FLIP expression led to the characteristic spindling that I showed you earlier.
00:13:36.22 But in cells that did get the super-repressor, that spindling morphology change was totally ablated.
00:13:44.20 So, clearly, it's the NFkappaB induction by FLIP that is responsible for the spindling phenotype.
00:13:53.20 So, let's summarize the FLIP effects. Latent FLIP expression triggers a morphologic change
00:14:01.02 in endothelial cells that makes them look just like a KS spindle cell.
00:14:05.18 The shape change is cell autonomous and is due to the induction of NFkappaB.
00:14:10.15 Now, of course, the induction of NFkappaB in these endothelial cells not only induces a cell autonomous shape change,
00:14:16.21 whose biochemistry we're still trying to understand,
00:14:19.10 but also triggers the release of a large number of paracrine signaling molecules,
00:14:23.27 cytokines, chemokines, VEGF, et cetera.
00:14:26.23 Pro-inflammatory and pro-angiogenic molecules.
00:14:31.15 And I think the relationship of this to the biology of KS that I've mentioned earlier
00:14:36.14 is now obvious, this is one of the things that links viral infection
00:14:41.29 to the production of a pro-angiogenic and pro-inflammatory microenvironment.
00:14:47.05 But I want to emphasize that neither authentic KSHV latency nor FLIP expression itself
00:14:52.03 will immortalize cells in vitro.
00:14:54.10 They may confer survival benefit on endothelium
00:14:57.19 and it's known from the work of Ethel Cesarman and others that in primary effusion lymphoma,
00:15:02.27 the survival of the lymphoma cells is absolutely dependent on FLIP,
00:15:06.20 so there is certainly, in the B-cell compartment, FLIP is very important in extending cell survival.
00:15:11.22 We haven't seen such a dramatic effect in the endothelial cell.
00:15:14.27 So, that's just worth keeping in mind, that we don't see immortalization here,
00:15:18.25 and I'm going to come back to that in a minute.
00:15:20.25 Alright, so, FLIP is a pro-inflammatory gene embedded in the latency program
00:15:27.25 and it turns out it's not the only one.
00:15:30.05 A few years ago, we discovered a second gene in the KSHV latency program
00:15:35.24 that is also a pro-inflammatory gene.
00:15:38.17 So, this locus was discovered by Rob Sadler, who was then a post-doc in my lab in the early to mid-90's,
00:15:47.09 and Rob discovered that latency, that one of the regions that was transcriptionally active in latency
00:15:55.20 encoded a 1.4 kilobase transcript that included the K12 open reading frame, shown here,
00:16:06.14 but in fact, upstream of the K12 open reading frame, a short 60 amino-acid open reading frame,
00:16:12.15 were many, were two sets of GC-rich 23 nucleotide repeats.
00:16:21.08 So, that, these clusters of repeats were called DR1, direct repeat 1 and direct repeat 2.
00:16:26.19 And initially they were annotated by the computer as being non-coding.
00:16:32.02 Because there were no AUG codons anywhere in this region.
00:16:35.23 There were neither AUG codons nor stop codons anywhere,
00:16:38.15 so the computer said that the only thing that was being expressed as a protein was open reading frame K12,
00:16:43.25 a sixty amino acid, highly hydrophobic membrane protein.
00:16:48.23 But it turned out that that wasn't true, that Rob showed that these direct repeats
00:16:55.18 themselves could also be translated in infected cells
00:16:58.21 and the translation was initiated not at AUG codons, but at variant CUG codons,
00:17:03.12 which of course, the mammalian ribosome is capable of using as initiation codons at low efficiency.
00:17:08.24 In fact, there are several products from this locus now. The product of open reading frame K12 itself
00:17:15.15 is called, we now call kaposin A, but two other proteins can be made.
00:17:21.05 What we call kaposin B, which is the product of just translating DR2 and DR1,
00:17:26.29 followed by a stop codon, and then, if you initiate translation in a different frame,
00:17:31.16 you can make something called kaposin C, which translates the repeats of DR2 and DR1
00:17:36.24 fused in frame to the membrane segment of K12.
00:17:41.23 We call that protein kaposin C.
00:17:44.08 We don't understand much about kaposin C yet, but kaposin B has been taken up by Craig McCormick in the lab
00:17:52.15 and Craig showed that kaposin B is an adapter molecule in signal transduction.
00:17:58.17 And he showed that by exploring its interaction partners in a yeast two hybrid screen,
00:18:06.22 from which he recovered a very interesting kinase called MAP kinase associated protein kinase 2, or MK2.
00:18:14.23 So, it turns out that the interaction between kaposin B and MK2 is direct.
00:18:20.24 If we make purified kaposin B in E. coli and purified MK2 and mix them together,
00:18:28.18 in this case the kaposin B is fused to GST, and in this GST pull-down format,
00:18:34.20 we see very efficient pull-down of MK2 by the GST kaposin B protein
00:18:43.20 and this binding is referable to the DR2 segment, not the DR1 segment.
00:18:49.27 So, what is MK2? MK2, it turns out, is an effector of a signaling pathway called the p38 MAP-kinase pathway.
00:19:00.11 So, let's talk a little bit about that pathway.
00:19:03.05 The p38 MAP kinase pathway is a signaling pathway
00:19:07.22 that is devoted to sensing inflammation and other stresses in the microenvironment.
00:19:12.10 So, some of those stresses include oxidative stress or osmotic stress,
00:19:17.16 but also a bunch of cytokines in the microenvironment, like IL-1 or TNF, IL-6.
00:19:26.20 Signaling through that pathway can activate a cascade of kinases called the MAP kinases
00:19:33.16 that converge on a p38 MAP kinase in the cytoplasm.
00:19:38.07 Phosphorylation of that kinase leads to its import into the nucleus,
00:19:42.08 where it can associate with the protein of interest for us, which is MK2, the target of kaposin B.
00:19:48.05 So, MK2 is itself a kinase, it resides in the nucleus in an inactive state.
00:19:54.03 When p38 is activated it binds to it in the nucleus, phosphorylates it.
00:19:58.20 That complex has some nuclear substrates, which I won't talk about today,
00:20:03.28 but it is also very efficiency exported back into the cytoplasm,
00:20:08.00 where it can phosphorylate other cytoplasmic targets that I will talk about.
00:20:15.07 So, one thing you can see is that signaling through this pathway, including the things that activate inflammatory signals,
00:20:23.25 like IL-1 and TNF, lead to the cytoplasmic export of MK2.
00:20:28.06 And phosphorylation of cytoplasmic targets.
00:20:31.03 And an early clue that the phosphorylation of MK, that the interaction of kaposin and MK2 was real
00:20:39.10 came from this localization experiment.
00:20:42.07 If you look at cells that are transfected with kaposin B, and just ask where it resides in the ground state,
00:20:48.19 it resides in the nucleus, as does MK2, here.
00:20:53.18 And if you apply TNF or LPS, inflammatory signaling molecules that lead to MK2 exiting the nucleus,
00:21:02.23 you can see that, of course that process isn't a hundred percent efficient,
00:21:07.04 there's still nuclear MK2, but you can see this obvious cytoplasmic blush of staining
00:21:12.23 that is not present in the ground state. Well, when you co-express kaposin B,
00:21:18.02 the same thing happens, that same cytoplasmic blush occurs with LPS or TNF,
00:21:23.19 just as it does for MK2.
00:21:27.10 So, the two proteins colocalize in the nucleus and they are co-transported under these pro-inflammatory signaling conditions.
00:21:33.27 And in fact, even more interesting, because when you examine the sequence of kaposin B,
00:21:38.27 it doesn't appear to have any classical nuclear localization signals
00:21:42.28 for import or export from the nucleus.
00:21:45.03 And we believe that it's shuttling is being governed by its interaction with MK2.
00:21:50.24 Now, how does kaposin B affect MK2's kinase activity?
00:21:55.19 In principle, it could either be an inhibitor or an activator,
00:21:58.16 but when we do an immune complex kinase assay, that is to say we transfect cells with kaposin B,
00:22:05.25 and now immunoprecipitate MAP-kinase associated protein kinase 2, and give it a substrate,
00:22:12.05 in this case, HSP27, we can see that cells that are expressing kaposin B have enhanced MK2 kinase activity
00:22:20.03 compared to the controls that don't express kaposin B.
00:22:23.12 So, kaposin B's interaction is an activating event, kaposin B activates MK2 kinase activity.
00:22:29.29 So, what does that mean? Well, I haven't told you yet what MK2's cytoplasmic kinase activity is for.
00:22:37.18 It's probably for many things, but one thing that it is clearly for is to govern the half-lives of cytokine messenger RNAs.
00:22:46.15 So, some of you may know that cytokine messenger RNAs were discovered back in the 80's
00:22:51.21 by Bob Kamen to have AU-rich elements in their 3 prime UTRs
00:22:59.06 that make their messages extremely unstable.
00:23:02.10 And for many years it's been known that certain growth factors, like Myc, and oncogenes,
00:23:07.04 but also many cytokines, have these AU-rich elements, or AREs, in their 3' UTR
00:23:13.19 and these confer great instability on the messages so that at the ground state, they're present at very low levels.
00:23:20.01 The new information, in the last 5 to 7 years, is that that instability is regulated.
00:23:26.17 And it's regulated by a cytoplasmic machinery whose members are still being enumerated,
00:23:32.25 but it turns out that one of the components of that regulation is MK2.
00:23:39.06 MK2's cytoplasmic kinase activity blocks the degradative machinery
00:23:45.05 that normally chews on ARE containing messages
00:23:49.08 and leads to their post-transcriptional stabilization in the cytoplasm,
00:23:53.21 and that in turn, since many of these ARE-containing messages are cytokine encoding mRNAs,
00:24:00.29 should lead to an enhanced release of secreted cytokines.
00:24:05.00 So, before I show you the experiment, let's look at the logic here.
00:24:08.13 What this pathway really is is an amplification scheme
00:24:12.15 in which inflammatory signals from the microenvironment, proceeding through this pathway,
00:24:18.24 lead to more inflammatory cytokine production.
00:24:21.19 This has made the whole p38/MK2 pathway an interesting target for the pharmaceutical industry
00:24:27.20 which has been trying to develop inhibitors of this pathway as potential anti-inflammatory agents.
00:24:32.12 Many such compounds have been developed. Interestingly, in animal testing,
00:24:36.25 they seem to predispose to staphylococcal and other infections,
00:24:40.20 which is why they haven't yet become successful drugs in the clinic.
00:24:43.19 But it speaks to the role of this pathway in inflammation and host defense.
00:24:48.18 Ok, so does kaposin B really have the activities that would be predicted from this?
00:24:56.10 Well, let's first think about how this might relate to KS pathogenesis.
00:25:01.00 It turns out that AREs, many ARE containing molecules have roles in KS pathogenesis.
00:25:06.20 IL-6 is known to affect spindle cell survival,
00:25:10.05 TNF-alpha, very abundant in KS lesions. A particularly interesting one is gamma-interferon.
00:25:15.13 Gamma-interferon, we showed a few years ago, is a weak inducer of the lytic cycle of KS,
00:25:21.02 so can promote KSHV growth.
00:25:23.27 But, the most interesting thing about gamma-interferon is that about a decade ago,
00:25:28.07 the biotechnology industry cloned this gene and expressed it
00:25:31.27 and was interested in exploring its use as a therapeutic.
00:25:34.22 And one of the things they were interested in was would gamma-interferon be a therapeutically useful drug for KS?
00:25:40.25 The drug, I think was given to 3 patients, two of whom had a florid exacerbation of KS
00:25:46.13 and that aspect of drug development was dropped.
00:25:50.12 So, up regulation of gamma-interferon is linked to the progression of KS
00:25:55.13 and of course, a final gene that's very important in KS pathogenesis is VEGF.
00:26:00.24 VEGF is controlled by an ARE and VEGF is a very potent pro-angiogenic factor.
00:26:07.21 So, I think there's lots of reasons to believe that things that manipulate this pathway
00:26:11.07 could have very dramatic effects on KS.
00:26:15.04 But all of this is only relevant if kaposin B expression really does stabilize ARE containing messages.
00:26:22.21 So, how can go about looking at that?
00:26:24.12 And Craig McCormick in the lab developed, adopted this assay that had been developed elsewhere,
00:26:31.01 in which an AU-rich element is cloned downstream of a globin gene that's under the control of a tet-operator.
00:26:37.03 That gene is then co-expressed with kaposin B and transfected into HeLa cells
00:26:43.24 that have a tet-off configuration of the tet activator.
00:26:48.25 Tetracycline or doxycycline is then added, the gene is turned on, I'm sorry, the gene for the globin gene is turned off
00:27:00.15 by the addition of doxycycline, and then RNA is harvested at various points
00:27:05.19 and probed on a Northern for beta globin.
00:27:08.20 So, this is a nice system because you can assess the stability of a message
00:27:11.27 without toxic drugs like actinomycin D.
00:27:14.08 The globin gene is turned off by the addition of doxycycline
00:27:18.19 and the half-life of the RNA is measured.
00:27:21.05 So, here's the experiment in the top panel. You can see that beta-globin itself,
00:27:27.28 with no ARE is a stable RNA and that stability is not regulated by kaposin B.
00:27:33.06 When you put an ARE from, in this case GM-CSF, but we can do this for other AREs,
00:27:39.24 you can see that as Kamen showed years ago, RNA stability is greatly impeded.
00:27:45.11 RNA is turned over very rapidly. But, that turnover can be partially inhibited
00:27:52.13 by co-expression of kaposin B. And as you would surmise from that,
00:27:57.07 cells expressing kaposin B have exaggerated production of GM-CSF and IL-6 and several other cytokines.
00:28:06.22 Now, we don't yet know exactly how kaposin B's interaction with MK2 triggers MK2 up regulation,
00:28:16.00 but here's a simple model.
00:28:17.06 Craig mapped the binding site for kaposin B to the central domain of the molecule.
00:28:22.15 It turns out that MK2, which exists in the ground state in an inactive form,
00:28:27.27 is inactive because the molecule folds back over on itself
00:28:31.20 and its C-terminal inhibitory domain inhibits the catalytic domain.
00:28:37.19 We know that kaposin B binds to the catalytic domain,
00:28:40.25 so we proposed that its action here is by displacing that inhibitory loop and leading to up regulation of kaposin B.
00:28:48.05 The story is more complicated than that
00:28:50.03 and to date, we don't have any direct biochemical evidence in vitro that this is the sequence of events
00:28:55.12 but it's our working model as we move forward.
00:28:58.01 Alright, so I think we can understand that the expression of FLIP and kaposin
00:29:06.00 can help us understand the pro-inflammatory phenotype of a KS lesion
00:29:09.29 and as students of the disease, that's very satisfying.
00:29:12.22 But as virologists, there's a deeper paradox here.
00:29:16.01 And the question involves why does KSHV want to do this?
00:29:21.14 We teach our students in graduate school that viruses usually want to evade
00:29:27.25 or block inflammatory and immune responses
00:29:30.21 and if you look elsewhere in the world of virology, particularly among the pox viruses and other large, DNA viruses,
00:29:37.03 there are many factors that are devoted specifically to blocking these things.
00:29:42.07 Soluble TNF receptors, soluble secreted cytokine binding proteins,
00:29:47.21 things that impede cytokine and pro-inflammatory signaling.
00:29:51.28 And the reason for that is clear: that viral evolution is principally driven by one thing,
00:29:58.19 which is spread; it's about enhancing viral replication, release and spread,
00:30:04.12 from one cell to another, one tissue to another, one individual to another, one population to another.
00:30:09.10 It hurts us to admit this as physicians and as human beings,
00:30:13.02 but disease, virally associated diseases, which is why we study these viruses,
00:30:17.09 is a sideshow in viral evolution. Disease usually only afflicts a minority of infected subjects,
00:30:26.00 there are examples of diseases that promote spread, respiratory infection
00:30:30.03 is very efficiently spread by causing disease,
00:30:33.07 but for most viral diseases, disease is a side show.
00:30:37.10 And that is certainly true of KS. KSHV is not, is spread in the population,
00:30:42.10 is not materially enhanced by the 1 in 10,000 people who's going to develop KS.
00:30:46.14 So, here we have a paradox and we have to presume that somehow,
00:30:52.08 promoting a pro-inflammatory environment is somehow beneficial to KSHV spread.
00:30:58.14 How might that operate? Well, one idea is that having lots of B-cells in the microenvironment
00:31:04.26 of an infected cell might provide a favorable microenvironment, or milieu, for viral dissemination.
00:31:13.23 That's a provocative and sort of attractive idea,
00:31:17.05 but at the moment it's totally conjectural and we don't have an experimental system that yet validates the idea.
00:31:25.00 So, I think it's important to remember that these pro-inflammatory molecules are playing some as-yet-undermined role
00:31:31.28 in the natural history of KSHV infection,
00:31:34.00 despite the fact that they are playing a role we can understand in the pathogenesis of the disease.

Part 4: Is KSHV Latency the Whole story?

00:00:01.17 Alright, so, we've considered in the last section the contributions that the latency program might make
00:00:08.14 to at least some aspects of the tumor.
00:00:12.26 And that is to say the pro-inflammatory and pro-angiogenic components of the tumor.
00:00:17.25 But we're now going to turn our attention to the question of,
00:00:21.16 is latency the whole story?
00:00:23.22 And why are we interested in this question?
00:00:25.28 We're interested in it because I've already told you that the latency program of KSHV is not powerfully immortalizing.
00:00:33.19 In fact, we can barely show that it's immortalizing at all.
00:00:36.24 And we don't observe prolongation of cell survival in culture,
00:00:41.05 that could be a laboratory shortcoming, that we haven't found the right conditions under which to assay it,
00:00:47.08 but, you know, it's clearly a very different situation from EBV,
00:00:52.21 where the latency program is very powerfully immortalizing.
00:00:56.09 And that led us to wonder whether the latency program was really sufficient
00:01:01.27 to engender all the pathogenesis in KS.
00:01:07.05 And has led us to consider some other lines of investigation.
00:01:11.09 Now, I'm going to take a somewhat of a detour on my way to that question
00:01:15.12 through one other aspect of latent cell biology.
00:01:19.21 And this concerns our old friend LANA, the latent protein that I told you was the major antigen in our initial serologic work.
00:01:28.01 But that others have shown plays a very important role in maintaining the nuclear episome
00:01:33.28 during latency. So, you'll recall that I said that in latency, there's no integration.
00:01:39.26 The viral genome, which is linear, in the virus particle, undergoes circularization in the nucleus
00:01:46.04 and is then stably maintained in the nucleus at a low copy number,
00:01:49.14 about twenty plasmids per cell.
00:01:52.06 It turns out that KSHV, as might be predicted, encodes machinery that is involved in that replication.
00:02:01.17 And this is the work largely of Ken Kaye, Rolf Renne, Erle Robertson,
00:02:06.07 Adam Grundhoff in my lab contributed some to this as well.
00:02:11.15 But these groups showed that LANA protein can specifically bind to sequences in the terminal repeats of the genome,
00:02:19.11 and that those repeats are very important in promoting episomal replication,
00:02:26.16 so that they function as a latent origin of replication.
00:02:30.28 But also, these groups have shown that KSHV can bind several components of host chromatin,
00:02:37.22 including histones 2A and B, Brd4. And that's led to a model
00:02:42.20 that suggests that LANA protein is not only involved in episomal replication
00:02:47.29 but also in the segregation of KSHV genomes to daughter cells,
00:02:54.17 the so-called tethering model, in which one domain binds to the KSHV episome
00:03:00.27 and another domain at the other extremity of the molecule
00:03:03.10 binds to chromatin and as a result during metaphase and during mitosis,
00:03:11.18 these KSHV episomes can piggyback onto the host chromosomes,
00:03:18.21 whose spindle apparatus is ensuring their proper segregation to both daughter cells.
00:03:23.25 So, in this fashion, any latently infected cell that undergoes cell division can assure that statistically,
00:03:29.29 with a pool of twenty, it's very likely that both daughter cells will be infected.
00:03:35.18 So, this is the received wisdom about KSHV replication as derived by studying primary effusion lymphoma cells in culture
00:03:43.27 and other cells that have been selected to maintain the episome.
00:03:47.14 But when we began to look at authentic KSHV infection, not transfection and selection of isolated plasmids,
00:03:56.18 we discovered that this mechanism must function very inefficiently in authentic infected cells
00:04:04.02 such that KSHV latency, at least as studied in cell culture, is extremely unstable.
00:04:09.04 This came to light when Adam Grundhoff infected a whole series of different cells in culture with KSHV
00:04:16.29 and then looked at, over time, how well they retained the episome
00:04:23.15 by looking periodically at what percent of the cells were still positive for LANA.
00:04:28.26 And what he found is that most cell lines behave like this,
00:04:32.28 that within a couple of weeks, if the cells are growing in culture, they actively lose the episome.
00:04:39.02 So, this was true for immortalized endothelial cells, this was true for HeLa cells
00:04:46.21 and a whole bunch of other cells, would lose the episome altogether.
00:04:50.02 There were a couple of cell lines, like SLK and 293, that lost their plasmids very rapidly in the beginning
00:04:59.26 and then out a few weeks later, a sub-population of about 10 percent of the cells
00:05:05.12 would stably maintain the episome, ten to fifteen percent of the cells.
00:05:09.02 And when Adam went to these SLK cells, so SLK is an endothelial cell line,
00:05:16.18 and he made single cell clones out at 3 or 4 weeks and found that 90 percent of these clones
00:05:24.25 were negative for the genome, had no LANA staining and no KSHV DNA.
00:05:29.05 So, they hadn't simply extinguished LANA protein expression,
00:05:31.23 they'd actually lost the episome.
00:05:33.23 While about ten percent of the clones actually retained KSHV DNA and continued to stain for LANA.
00:05:39.16 In fact, the brightness of LANA was correlated with the copy number of episomes.
00:05:43.29 So, faintly LANA positives had lower copy numbers than brightly LANA positives.
00:05:48.27 So, from these SLK negative segregants, or so-called SLKn,
00:05:54.25 he established some cell lines and we similarly established lines from SLK positive lines, which he called SLKp.
00:06:02.05 Now here's the interesting thing about SLKp cells.
00:06:05.18 So, these cells have maintained the stable episome,
00:06:08.08 so you would have thought if they'd undergone some genetic change
00:06:11.12 that allowed them to stabilize the episome, that if you superinfected them
00:06:15.17 with another plasmid or episome from KSHV, that they would maintain such a plasmid.
00:06:22.22 But that is not true. When you look at SLK positive, p-cells,
00:06:28.11 they lose the episome, the incoming episome, very rapidly, just like the parental SLK cells,
00:06:33.28 even though they continue to harbor their own stable episome.
00:06:37.11 So, what that suggests is that episome stabilization is a property that acts in cis,
00:06:46.09 it's not the result of some trans-acting factor that's been turned on or modified.
00:06:50.06 And in fact, we know from other studies that the rate at which this happens
00:06:55.13 is very strongly suggestive that that change is epigenetic.
00:06:58.23 In fact, we've now been able to prove that by taking genomes from a PEL cell line,
00:07:04.17 like BCBL-1, which is very stably latently infected and therefore has, whatever its genome has,
00:07:11.10 all the appropriate modifications that are necessary.
00:07:14.29 If we induce that virus and infect SLK cells, it is rapidly lost.
00:07:20.03 So, that says that it's not a genetic change, it must be an epigenetic change.
00:07:24.00 So, stabilization is epigenetic and acts in cis.
00:07:27.13 Ok, so here's the, let me just re-state those things.
00:07:31.28 Newly established latent episomes are unstable, and if cells are proliferating,
00:07:36.07 episome loss is going to happen. Now, this is phenomenon that we observed in culture,
00:07:41.01 but we have very strong reasons to believe that it's also true in human beings.
00:07:44.11 And this has to do with early experiments of Bob Gallo's.
00:07:47.29 You'll recall that I mentioned in the beginning that Gallo showed that you could grow spindle cells from a KS biopsy
00:07:55.03 if you put them in circumstances where they had conditioned medium from activated T-cells.
00:08:00.12 So, under those conditions, spindle cells will actually divide.
00:08:04.08 What was subsequently shown is that under those very same conditions,
00:08:09.24 episomes of KSHV are lost, such that routinely all the cells that are derived by that protocol
00:08:15.16 after a month or two in culture, are KSHV negative,
00:08:18.25 even though they were KSHV positive when they went in.
00:08:21.24 So, in fact, KS spindle cells in a human host are not stably immortalized.
00:08:30.21 And that differentiates them from PEL cells, that are stably, latently infected.
00:08:36.06 In the spindle cells, KSHV hasn't undergone whatever those epigenetic changes are.
00:08:43.01 Or can't propagate them.
00:08:45.13 Now, rare cells in culture can undergo episome stabilization
00:08:49.01 and the in vivo counterpart of that is the PEL cell.
00:08:53.05 These tumors that are growing in the body cavities of patients.
00:08:57.09 Those cells have been very strongly selected in the patient
00:09:00.14 and that selection must have included these epigenetic modifications that are required for stabilization.
00:09:06.27 Alright, so I want you to keep that in the back of your mind
00:09:09.26 as we ponder this question.
00:09:11.22 Can latency alone account for tumorigenesis?
00:09:18.26 Now, here we inherited a certain amount of dogma in tumor virology
00:09:25.00 that was derived largely from EBV. And this dogma says that in herpes viral oncogenesis,
00:09:31.27 the latency program does all the heavy lifting.
00:09:34.08 It does the work of tumorigenesis by stimulating proliferation and extending cell survival
00:09:39.26 and the latency program plays no role in cancer, because after all,
00:09:44.16 if a cell enters the, and the lytic program plays no role in cancer,
00:09:49.20 because after all, if a lytically infected cell, if a cell flips into the lytic cycle, that cell is going to die, ok.
00:09:57.05 So, how could a lytic infection contribute to a proliferative disease.
00:10:01.19 Those cells can't contribute to the mass.
00:10:04.02 Now, everyone has always acknowledged that early in the natural history of infection,
00:10:08.22 the lytic cycle must play an important role because it's involved in disseminating the infection throughout the host.
00:10:14.24 And in the case of EBV, that results in infecting a lot of B-cells,
00:10:21.10 the default pathway in the B-cell, as it is for KSHV, is latency.
00:10:25.14 So, it's always been acknowledged that the lytic program is necessary early in infection
00:10:29.27 to spread throughout the body and put a lot of B-cells into the latency program.
00:10:34.10 But then it is imagined that once in the latency program,
00:10:37.20 they begin their slow but inexorable mutational march to cancer,
00:10:42.16 rearranging Myc genes and other genes that are necessary for the final development of the tumor.
00:10:48.07 So, in that dogma, the lytic infection plays this adjunctive role early,
00:10:54.21 but no ongoing role in the process of tumorigenesis.
00:10:58.05 And that model was the model that we inherited as tumor virologists
00:11:06.07 and tried to adapt to this paradigm, but very clearly, things were amiss with the model.
00:11:15.04 First of all, we could never show that the latency program was strongly immortalizing,
00:11:19.07 second, we had this problem that the latency program, at least in KS cells,
00:11:24.07 appears to be very unstable.
00:11:26.08 And that led us to wonder whether the lytic cycle might play some role in KS pathogenesis.
00:11:34.10 But because we had no animal model of KS, we couldn't put that to a direct experimental test.
00:11:39.15 However, once again, clinical medicine provided a very strong clue,
00:11:47.01 at just the time that we were considering this.
00:11:50.04 And this was a clinical experiment that was designed to ask no question about KS.
00:11:55.03 It was a clinical experiment that was designed about, to answer a question about
00:12:00.07 the treatment of another herpesviral infection, cytomegalovirus.
00:12:03.27 So, some of you may know that AIDS patients are very susceptible
00:12:07.16 to the reactivation of cytomegalovirus, or CMV,
00:12:10.25 a particularly devastating complication for them is reactivation in the retina.
00:12:15.21 So, ocular, or retinal CMV infection, was a leading cause of blindness in patients with untreated AIDS
00:12:25.29 and during those horrible early days of the AIDS epidemic, those of us who practice medicine
00:12:30.23 saw many patients who after becoming thoroughly debilitated from their, and wasted, from their HIV infection,
00:12:37.14 went blind from reactivation of CMV in the retina.
00:12:40.22 Now, in the 90's, treatment with ganciclovir was developed
00:12:45.02 and ganciclovir is a drug that was not very well absorbed orally,
00:12:49.01 so ultimately the standard of care became the development of implantable reservoirs
00:12:54.29 of ganciclovir that could be implanted right into the eye.
00:12:58.08 And the treatment was to implant these right into the affected eye
00:13:02.11 and the release of ganciclovir there controlled CMV in that eye.
00:13:07.05 And pretty effectively controlled it for a period of many months.
00:13:10.15 The problem was that that treatment does not give rise to bloodstream levels of ganciclovir,
00:13:17.22 and there was the concern, would patients develop retinitis in the opposite eye that wasn't treated?
00:13:22.14 So, in order to address that, a clinical trial was started
00:13:25.19 in which AIDS patients with CMV retinitis got the standard treatment, which was the implant into the affected eye,
00:13:32.18 and then they were randomized to either receive nothing, a placebo,
00:13:36.18 or oral ganciclovir or intravenous ganciclovir.
00:13:39.19 And very large numbers of patients were studied in a trial that was orchestrated by Dan Martin,
00:13:44.11 a very gifted ophthalmologist at Emory, then at Emory.
00:13:48.02 This was a multi-center trial, it involved many centers and the results of this trial were truly remarkable.
00:13:54.23 And when I read this paper in the New England Journal of Medicine, I was just shocked,
00:14:00.06 because when the paper came out,
00:14:01.14 well, first of all the results showed that systemic ganciclovir did protect the contralateral eye,
00:14:07.19 but incidentally, the clinicians noticed that there was a striking decrease in new cases Kaposi's sarcoma
00:14:16.00 in the patients that got randomized to oral or especially intravenous ganciclovir.
00:14:21.18 A six-fold drop, or more, in incident KS.
00:14:25.28 Now, when this paper came out, it was greeted with a giant ho-hum,
00:14:29.21 oh yes, more evidence that herpesviruses have something to do with KS.
00:14:35.16 But to a virologist reading this paper, and one steeped in the dogma of tumor virology,
00:14:41.07 this was a profoundly important result, deeply subversive of the conventional wisdom.
00:14:46.08 Why do I say that?
00:14:47.20 I say that for two reasons. There are two things you need to know about
00:14:50.18 in order to correctly interpret this experiment.
00:14:54.07 The first is, who gets CMV retinitis?
00:14:58.26 We know that CMV retinitis is a late complication of AIDS.
00:15:02.13 Most of these patients have had HIV and KSHV infection for over a decade
00:15:08.21 and that they have less than 50 CD4 cells left,
00:15:12.22 that most of them will die within 6 months.
00:15:15.03 So, these are people who in the long natural history of HIV
00:15:18.18 are at five minutes to midnight.
00:15:21.02 So, that's fact number one.
00:15:22.29 They've been carrying HIV for a long time, if they've got KSHV, they've been carrying it for a long time.
00:15:31.11 The second thing you need to know is that ganciclovir is absolutely specific for the lytic cycle
00:15:37.01 and does nothing whatsoever to latent infection.
00:15:39.19 We often grow latently infected cells in ganciclovir to prevent lytic reactivation.
00:15:44.22 So, it has no impact on latency, it's specific for the lytic cycle.
00:15:48.23 So, if you put those two facts together, you can see that this is a profoundly subversive result
00:15:54.06 of the conventional wisdom because what it really says is that the continuous operation of the lytic cycle
00:16:02.23 day in and day out, over the full ten years of the co-infection
00:16:07.20 is absolutely necessary for the development of KS. So, what these numbers reflect the six month incidence of KS
00:16:15.14 at, you know, in the last six months, after carrying these viruses for ten or fifteen years.
00:16:20.23 So, really what the experiment says is not only is lytic infection important
00:16:27.12 early in the natural history, to disseminate to lots of endothelium,
00:16:30.27 it's necessary continuously, day after day, week after week, month after month,
00:16:35.26 throughout the entire natural history of the infection.
00:16:38.25 And that was really a shock, but really, began to make us consider
00:16:46.28 that the lytic cycle must be playing some important role here.
00:16:50.23 Ok, alright, so how might lytic replication contribute to KS?
00:16:57.25 And here's where two of the things that I've already mentioned, and emphasized, might come into play.
00:17:03.10 First of all, if latency isn't immortalizing,
00:17:06.14 then if latently infected cells are destined to die,
00:17:12.02 then how can a mass ever expand? The only way a mass can expand is if
00:17:17.21 newly, cells newly infected and recruited to latency outnumber the number that are dying at any given time.
00:17:27.22 So, and, that is a function of the lytic cycle, lytic infection releases virus
00:17:32.06 and allows new cells to become infected. The default pathway is latency.
00:17:37.04 So, in order to constantly be recruiting new cells to latency,
00:17:40.05 you need the operation of the lytic cycle at some level to generate enough virus to do that,
00:17:45.12 to replace dying cells.
00:17:47.19 Second, I've shown you that latency isn't very stable,
00:17:50.15 that latently infected cells, if they begin to proliferate, will segregate uninfected cells.
00:17:55.27 And those uninfected cells, if they lack whatever KSHV confers as a survival advantage
00:18:01.26 are not going to contribute to the mass.
00:18:05.14 So, again, if latency isn't stable, lytic infection allows, again, the recruitment of new, latently infected cells
00:18:13.03 that can replace these uninfected segregates.
00:18:16.10 Finally, there's another possibility that I haven't discussed yet.
00:18:21.04 But you'll recall that I emphasized when we discussed the histology of KS
00:18:26.14 that KS is really a pastiche of three processes:
00:18:29.21 proliferation, inflammation and angiogenesis.
00:18:32.24 Now, we've already discussed that the lytic cycle is not likely to contribute to the proliferative component of the tumor
00:18:40.15 because lytically infected cells die, they lyse.
00:18:43.07 So, they can't really become a part of the mass.
00:18:46.18 But inflammation and angiogenesis are process that are driven by paracrine factors.
00:18:52.23 So, if the lytic cycle could produce paracrine factors,
00:18:57.19 cytokines, chemokines, growth factors, that would operate in trans,
00:19:01.28 they could stimulate a) the proliferation of other cells in the culture
00:19:07.05 and b) the influx of inflammatory cells and new blood vessels
00:19:11.21 that would contribute to this environment that we've already shown is a big part of the mass.
00:19:15.23 So, here's a third as yet-hypothetical way. Now, interestingly, when you look at the viral genome,
00:19:27.07 it turns out that KSHV has captured a number of paracrine factors.
00:19:34.25 There are three CC chemokines in the genome that could contribute to inflammation and angiogenesis.
00:19:41.28 There's a viral homologue of IL-6, a very potent cytokine that we've already earlier observed
00:19:49.06 is important in the natural history of multicentric Castleman's disease.
00:19:52.25 It's a very strong promoter of B-cell survival, and others have shown, is an up regulator of angiogenesis.
00:19:59.17 And I want to emphasize here that the identification of these factors is not our work
00:20:03.14 but is the work of Pat Moore, John Nicholas and many other groups that have contributed to this.
00:20:10.09 But the point is that these genes are not expressed during latency,
00:20:14.21 they are only expressed during the lytic cycle.
00:20:17.11 In addition, Enrique Mesri and Silvia Montaner and others have shown that a virally encoded G-protein coupled receptor
00:20:24.21 is a signaling molecule that is not itself secreted from the cell,
00:20:28.20 but that can trigger a signaling pathway that up regulates the release of other pro-angiogenic factors
00:20:35.28 and especially prominently is VEGF among them.
00:20:39.05 Now, it's a little controversial about the role of the GPCR in pathogenesis
00:20:44.06 because the GPCR, as a lytic cycle gene, is not secreted from cells
00:20:49.18 and we showed a few years ago that lytically infected cells shut off many host genes
00:20:56.20 so that there's only a rather narrow window in which expression of the GPCR
00:21:01.09 can up regulate genes and lead to secretion, but this is almost without a doubt a part of the pathogenesis of KS.
00:21:09.18 So, whether these secreted factors are host derived or virally derived,
00:21:14.08 the lytic cycle can produce many powerful angiogenic and pro-inflammatory substances
00:21:19.05 that surely must play some role in the evolution of a KS lesion.
00:21:24.03 So, let's sum up. KSHV's latency program can provoke inflammation,
00:21:31.22 it can provoke cell survival, at least in the B-cell environment, still not clear whether it does that in an endothelial environment,
00:21:37.17 and possibly proliferation, although we've never observed that in culture.
00:21:41.11 But these effects are modest in comparison with other known tumor viruses.
00:21:46.00 And from clinical observations that suggest that the continuous operation of the lytic cycle is also required,
00:21:53.07 we believe that the lytic cycle is playing a special role in KS pathogenesis
00:21:58.25 by regenerating latently infected cells that are lost either to cell death or plasmid segregation.
00:22:04.05 And by providing paracrine signals for inflammation and angiogenesis.
00:22:08.10 Now, obviously, there's a lot we have left to learn.
00:22:11.25 We still don't know what the function of the cyclin gene is in latency,
00:22:14.21 we still don't know whether latency can provoke proliferation
00:22:18.17 under conditions that we simply haven't replicated yet in cell culture, so there's lot of unanswered questions.
00:22:23.23 But from what we already know, we can make some inferences that may have some clinical significance.
00:22:29.20 First, from the fact that the ongoing operation of the latency program,
00:22:35.08 of the lytic cycle is important in pathogenesis, we might surmise that drugs like Ganciclovir
00:22:40.20 might play some beneficial role in the therapy of an established KS lesion.
00:22:45.21 What I've shown you is that the administration of those drugs can prevent the development of KS,
00:22:51.02 but what hasn't been shown is whether the use of these drugs could be therapeutically valuable in the treatment of KS.
00:22:58.07 That's turned out to be a difficult question because we already know
00:23:04.02 that, you know, in the clinic, because Ganciclovir has toxicities,
00:23:07.28 it's a bone marrow suppressive drug and there's been some reluctance to treat people
00:23:11.09 for prolonged periods with Ganciclovir.
00:23:14.03 The other thing is we have other strategies for dealing with KS
00:23:17.14 including, in the AIDS related form, the treatment of HIV itself,
00:23:20.23 which is a very successful treatment. So, it's not clear to me that we're ever going to see
00:23:25.20 a clinical trial of drugs like Ganciclovir.
00:23:28.22 We don't know, furthermore, how long one would have to treat a patient
00:23:34.13 with a drug like this in order to see the clinical benefit.
00:23:37.16 That depends on the rate of plasmid segregation, the half-life of a latently infected cell
00:23:42.09 and many other things for which we don't have an accurate number.
00:23:44.24 But, I think it's still out there as a theoretical issue and may one day be the subject of clinical investigation.
00:23:51.16 Second, what is the role of HIV in amplifying the risk of KS?
00:23:58.12 There are many ideas about this, Gallo's group has advanced the argument
00:24:05.12 and Barbara Ensoli in particular, now independent many years in Italy,
00:24:09.19 has advanced the argument that HIV makes a specific genetic contribution to KS,
00:24:16.17 that soluble Tat released from infected cells can play a role as a growth factor on spindle cells.
00:24:23.07 Be that as it may, I think there are easier explanations for the linkage.
00:24:28.02 We know that immunodeficiency states in general predispose to amplifying KS risk,
00:24:34.06 cyclosporin and other anti-rejection drugs in the transplant setting are a good example of that.
00:24:39.19 And I think now that we know the critical role of the lytic cycle in this process,
00:24:44.05 we can make some simple analogies. We know what happens to herpesviral infections
00:24:49.03 when people become immunodeficient. Typically, patients lose control of the lytic cycle,
00:24:55.02 and develop higher levels of viremia, high viral loads and this contributes to exacerbation of disease
00:25:01.21 in things like CMV and herpesimplex, it's very well established
00:25:07.18 that immunodeficiency states lead to loss of control of the lytic cycle.
00:25:12.27 So, all one has to really imagine is going on in KS is the same thing happening in KSHV.
00:25:18.16 If HIV infected the patients with immunodeficiency lose control of the lytic cycle,
00:25:23.19 we would expect that they would have higher viral loads and be at increased risk
00:25:27.22 for the development of KS.
00:25:29.13 So, I think the things that we've already established here suffice to explain most of the relationship with HIV.
00:25:38.01 But it's still possible that HIV does make a specific contribution
00:25:41.16 over and above the immunodeficiency and that's grist for future scientific investigation.
00:25:46.14 But I want to conclude on a note about the relationship between science and medicine
00:25:52.00 because it turns out that not only did medicine at every stage provide all the critical clues
00:25:57.03 for scientists who were listening to decode a lot of what's going on here,
00:26:02.00 but medicine continues to do so.
00:26:03.24 Just in the last year or two, clinical trials in post-transplant KS
00:26:07.27 have shown that rapamycin is an extremely effective treatment for post-transplant KS
00:26:12.17 and I think people are now beginning to experiment with rapamycin in other settings,
00:26:16.21 not related to transplant.
00:26:18.04 If it turns out to be as effective there as it is in the transplant form,
00:26:22.06 it'll suggest very strongly that mTOR, the target of rapamycin,
00:26:26.16 which is a known signaling molecule whose signaling pathway is becoming increasingly understood by cell biologists,
00:26:34.03 is somehow very influential in KS biology
00:26:36.29 and we're going to have to start to understand what mTOR signaling does
00:26:40.19 and how viral infection influences mTOR signaling,
00:26:44.17 but I think this is pointing already to a very potentially fruitful line of new research
00:26:50.05 for people interested in the molecular and cellular biology of KS.
00:26:54.28 So, I'll conclude by reminding you that, as the Talmud says,
00:26:59.20 "the work of the righteous is done by others,"
00:27:01.20 nothing I showed you was done with my own hands,
00:27:04.04 and I want to pay particular tribute to two groups in closing.
00:27:08.02 First are my students and post-docs over the past 15 years
00:27:11.06 who have advanced all this work, none of this would have been possible without them.
00:27:15.15 And finally, a nod to my many colleagues and competitors in the field,
00:27:20.08 they have set the bar extremely high for us and for each other,
00:27:23.15 it's been a lot of fun to explore these things and I often say to my students
00:27:29.06 that we are blessed by having excellent competitors who keep us on our best game
00:27:33.11 and who set the bar high and advance the work.
00:27:36.23 So, with that, I'll close, I hope you've enjoyed the things I've had to say,
00:27:41.21 it's really a remarkable story that large numbers of people have contributed to
00:27:47.04 and we've been blessed to be a part of it. Thank you.

Videos in this Talk

Part 1: Kaposi’s Sarcoma: The Disease
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad
Part 2: The Virus
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad
Part 3: KSHV Latency and KS Pathogenesis
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad
Part 4: Is KSHV Latency the Whole story?
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad

Speaker: Don Ganem

Total Duration: 1:46:22

Recorded: March 2009

All Talks in Microbiology

Talk Overview

In 1872, Moritz Kaposi first described a disease that included pigmented skin tumors and in some cases tumors of the viscera. While KS has been endemic in some areas of the world for many years, a highly aggressive and deadly form of the disease emerged concurrently with the HIV/AIDS epidemic. KS is a very unusual cancer including multiple cell types, including cells of a “spindle” morphology, in a single tumor. In this section of the talk, the atypical biology of this cancer it will be discussed.

Epidemiological studies suggested that HIV was not the causative agent of KS. In the mid-1990s, a new human herpes virus, Kaposi’s sarcoma associated herpes virus (KSHV), was identified as the likely cause of KS. Development of a serological test for the presence of KSHV in patients showed that KS seroprevalence strikingly mirrored KS risk and, additionally, KS was never found in KSHV negative patients. This data strongly supported KSHV as the causative agent of KS. However, epidemiology also showed that additional cofactors are required for disease development and in the case of patients with AIDS, HIV is this cofactor.

In this section of the lecture, we will look at which viral genes are expressed in KS and how they cause disease. KSHV has both a latent and lytic cycle. In the latency cycle, very few genes are expressed and no new virus is produced allowing the host cell to survive. In the lytic cycle, all open reading frames are expressed, new virus produced and the host cell killed. Greater than 95% of KSHV infected cells contain virus is in the latency cycle. A single KSHV gene, vFLIP, expressed during the latency cycle is sufficient to cause a change of cell morphology to a “spindle” shape. Additionally, vFLIP and a second gene kaposin B promote the expression of cytokines and the inflammatory phenotype seen in KS lesions. How this increase in the inflammatory response benefits KSHV is yet to be determined.

Can the latency cycle alone account for tumorigenesis? Experiments showing that infection of latent cells is quite unstable together with the fact that latent infection with KSHV does not immortalize cells suggested that it does not. An epidemiological study in which patients were given a drug that is specific for virus in the lytic cycle (ganciclovir) resulted in a significant decrease in new KS tumors suggesting that the lytic cycle is also key to disease development. How this might happen and what this means for further understanding and treatment of KS is discussed.

Speaker Bio

Don Ganem

Don Ganem joined the Novartis Institute for Biomedical Research in Emeryville, California as Vice President and Global Head of Infectious Diseases in 2011. His research group focuses on understanding and developing better treatments for viral, bacterial and fungal infections in normal and immunocompromised individuals. While an undergraduate student at Harvard, Ganem did research in bacterial… Continue Reading