A historical personal perspective on human retroviruses and their infection of T cells

Field: Medicine (Health Sciences)

For more than 50 years I have been interested in several aspects of the biology of blood cells—slowly I also became involved with viruses that infect them. The former goes as far back as my college days when I carried out some primitive experiments on the thymus gland on mice, while in medical school on erythropoietin, and at the University of Chicago (as an intern and resident in medicine) with a bit on heme synthesis.1 After joining the National Cancer Institute (NCI) I began to perform a comparison of some enzymes of leukemic cells (and sometimes nucleic acids) versus normal lymphocytes.2-6 This became possible after the introduction of phytohemagglutinin by Peter Nowell.7 Phytohemagglutinin could make blastlike cells from normal lymphocytes, which seemed to resemble the leukemic blast cells of patients with acute lymphoblastic leukemia. To study these cells, I needed to grow them so I became interested in the new field of growth factors, which had been pioneered by Leo Sachs at the Weitzmann Institute in Israel, Rita Levi-Montalcini in Rome, and Stanley Cohen at Vanderbilt University. This led me to factors from human embryos, which temporarily sustain the growth of human acute myeloid leukemic cells,8 which led to the establishment of the first human myeloid leukemic cell line known as HL-60.9, 10 Ultimately, this interest in growth factors led us to the discovery of an important new molecule which we called T-cell mitogenic factor—then T-cell growth factor—which enabled the first growth of human T cells in culture,11, 12 in contrast to just a blastogenic effect.13 This molecule was eventually renamed interleukin-2 (IL-2) and became very important to subsequent research in immunology and, as will soon be apparent, for the discovery of human retroviruses. It was in the beginning of the 1970s that my interest also turned to viruses based on an understanding that many leukemias in animals were caused by a certain type of virus, then called leukemia viruses or RNA tumor viruses. Eventually such viruses would be called retroviruses. I was able to utilize my experiences with one particular set of enzymes, namely DNA polymerases of normal and leukemic cells, and enter the field of retroviruses of animals because a DNA polymerase in these viruses had just been discovered by the late Howard Temin and Satoshi Mizutani14 and by David Baltimore.15 This very special DNA polymerase soon became known as reverse transcriptase and would play a key part of our research throughout the 1970s. It would ultimately be used as a sensitive technique for, and as a surrogate marker of, human retroviruses. Yet as I became interested in these viruses it was at an inopportune time, one ripe with biases. In this context, it seems to me that humans have a very short memory span of roughly 25 to 30 years. After the great influenza epidemic during World War I and by the time of polio in the 1950s, an interest in epidemic or pandemic viruses was declining, and there was an atmosphere that these things were relics of the past. Our focus needed to be on chronic degenerative diseases (including cancer), which ironically can often be caused by viruses. This was certainly true again by the time of AIDS. However, when I came to the NCI in 1965 there was still a memory of polio and of the importance and reality of possible epidemics, but by the 1970s departments of microbiology were fading and the US Centers for Disease Control and Prevention (CDC) was threatened with serious reduction. Viruses as a possible cause of cancer were deemed to be almost impossible. For instance, I recall that the Cold Spring Harbor meeting of 1974 concluded that cancer was never caused by a virus. Indeed, there was not even a role for a virus—even in a step in the multistages of the cause of any human cancer. To make matters worse, I was interested in looking for the presence of human retroviruses. Of all viruses, the feeling was rather emphatic that no human retroviruses existed, and many people felt that retroviruses could not even infect humans. As is now known, these biases were shattered by the early 1980s. Viruses were shown to be involved in approximately 20% of all human cancer and retroviruses were discovered in my laboratory, human T-lymphotropic or leukemic virus Type I (HTLV-I) reported in 1980 and HTLV-II reported in 1982. Soon to follow was the great AIDS epidemic caused by still another retrovirus, human immunodeficiency virus (HIV). It is this story that I will summarize in this article. In a letter from the Journal of Virology dated September 15, 1980, my submission on the discovery of the first known human retrovirus (which was later accepted in Proceedings of the National Academy of Sciences) was rejected and in a letter I have kept for the record said “… there is little point in perpetuating this controversy about the ‘presumed viral nature of this material.’ ” However, there were encouraging factors for us to stay with this pursuit—at least in my mind. The first was the discovery of bovine leukemia retrovirus by veterinary scientists, which was extremely difficult to find because of its very low replication in contrast to what was thought to be the case in animal retrovirology.16, 17 This was followed by the discovery of the first natural occurring leukemia-causing virus in primates, namely the gibbon ape leukemia virus (GALV), which was found by Kawakami and Buckley18 in cases of chronic myeloid leukemia very similar to the disease in man. A few years later my colleagues and I discovered another strain of GALV associated with a T-cell leukemia of these animals.19 Another encouraging factor came from discussions with Max Essex and colleagues at the Harvard School of Public Health regarding feline leukemia virus and my meeting the discoverer of that virus, William Jarrett from Glasgow, Scotland. Both encouraged my continuation of this pursuit in man. Still another factor had to do with an interspecies transmission of a retrovirus from one primate to another that occurred more or less before our eyes. Interspecies transmission of retroviruses was a known phenomenon, but it was thought that this occurred extremely rarely and many, many thousands of years ago as evidenced by studying the fossilized remnants of old retrovirus infections that left their remains in the DNA of normal vertebrates. Yet, we witnessed a situation where a virus from a pet gibbon ape infected a new world monkey, known as a woolly monkey. The virus caused a fibrosarcoma in the woolly monkey and when analyzed it was shown to be a combination of defective retroviruses which had appropriated a cell-derived oncogene (PDGF) into its genome and a helper retrovirus, which was simply another strain of GALV. We thought man could not be an exception, so we continued our pursuit. There were two advances in technology that were necessary for our discovery of the first human retrovirus: 1) development of reverse transcriptase assays and 2) discovery of IL-2. Electron microscopy is not very sensitive and HTLV-I would not have been discovered if we depended solely on it. Instead the discoveries by Temin and Mizutani14 and Baltimore15 of reverse transcriptase gave us an assay that could be used as a surrogate marker for a human retrovirus. This assay was quick, extremely sensitive (3 log better than electron microscopy), and rapid. Moreover, we could culture cells and make many assays over a short period of time, which would not be possible by electron microscopy. However, we had to spend considerable time and effort in the 1970s to develop this assay to be sufficiently sensitive and specific to distinguish it from the DNA polymerases (α, β, and γ) of human cells.20 With much effort and the great help of many postdoctorals and visiting scientists, we achieved this goal of having this assay readily available. Second, the IL-2 work was essential for the discovery of all human retroviruses. As mentioned, we discovered this substance in 1976.11, 12, 21 It was one of the first cytokines and was the first lymphokine. Without actually realizing it, the experience with the T-cell leukemia of gibbon apes as well as the availability to us of the molecule, IL-2, pushed us toward the study of T-cell malignancies in man. With the great dedication and work of a postdoctoral fellow, Bernard Poiesz, we isolated a retrovirus in samples of one such patient with a severe T-cell malignancy that had both a leukemia and lymphoma component.22, 23 Many others in my group contributed to this work.24-29 This was not limited to those whose work made the technology possible but also people who helped in the analyses of the virus to show it was unique and not a laboratory contaminant and others who contributed to the demonstration that it was not a one-time isolate but was present in other members of the family of this man as well as some other patients.28, 29 While developing a blood antibody test we detected seropositivity and isolated HTLV-I from other patients with the disease—the first few being African American, a Caribbean, and a Caucasian American (merchant marine).23, 30 All the isolates were from IL-2 initiated cultures of CD4+ T cells. We carried out seroepidemiologic studies, which showed that it was uncommon in the US population but more prevalent in African Americans. We quickly carried out tests in the Caribbean and found a higher frequency of seropositivity as well as disease, and we were informed by British colleagues that immigrants in the United Kingdom had a relatively high incidence of this disease (see below). These tests became the basis of a blood test that is required to be performed on donor blood samples before a blood transfusion and would be of great help to us in the soon to be coming AIDS epidemic. HTLV-I causes adult T-cell leukemia (ATL) in approximately 5% and a neurologic disease similar to multiple sclerosis in approximately 3% of infected persons and rarely autoimmune diseases giving rise to neuropathies, arthritis, uveitis, and other disorders. It also leads to a moderate immune suppression. The serious central nervous system disease called tropical spastic paraparesis is now referred to as HTLV-I–associated myeloencephalopathy. It is the only known leukemia virus to this date and one of the first documented human tumor viruses. The leukemia caused by HTLV-I is usually exceptionally aggressive, often associated with high blood calcium apparently due to an activation of the parathyroid hormone by one of the HTLV gene products, and is frequently also associated with skin manifestations resulting from tumor infiltrates. ATL was characterized as a distinct disease by Takatsuki and his colleagues in Japan in 1977 (before knowledge of the virus).31 The diagnosis does not require a molecular biologist, virologist, or clinician but a pathologic examination of the cells which show a starry-like or flower-like nucleus—namely, a nucleus that seems to radiate in multiple directions. Takatsuki and colleagues had found clusters of ATL in Kyushu Island of southern Japan, but they did not know there was an etiologic agent. Shortly after virus isolation, we developed a blood test for its presence based on finding HTLV-I–specific antibodies in sera. With a retrovirus the presence of antibodies is a clear indication of chronic infection. Because of the rapid integration of the virus genome antibodies does not mean simply exposure nor protection but rather an indication of some replicating virus in the individual. We used the conventional enzyme-linked immunosorbent assays (ELISAs) as well as competitive radioimmune precipitation assays and other immunologic methods for testing various populations. Later, derivatives of these studies led to the development of the HTLV-I blood test which was not required in the United States until after the later HIV blood test was implemented. The most conclusive evidence comes from the molecular biologic studies that are illustrated in Fig. 1 concerning the integration patterns. Unlike other human tumor viruses no known cofactor appears to be necessary—although it is likely that some factors may promote or reduce the HTLV leukemogenic effect. Retroviral integration patterns indicate causality. HTLV-I is endemic in Japan, among Australian aboriginals, parts of Southeast Asia (e.g., Indonesia), parts of South America and Iran, and among some tribes in Africa and in their descendants in the Caribbean Islands, as well as in the rest of the Americas and sporadic elsewhere.32 HTLV-I is transmitted by blood, sex, and mother to child via nursing, but it is a virus that is difficult to transmit, probably because it is almost always transmitted by the infected cells themselves. When the cells enter into the blood stream and make contact with the lymphocytes of the recipient activation of the lymphocytes is initiated and the virus is transmitted through this cell to cell contact. That does not mean that extracellular virions cannot be infectious because hemophiliacs in the past could be infected with contaminated plasma, but it is likely the dominant transmission is from infected cells. No one knows the number of people infected around the world because surprisingly many countries have not yet been evaluated. Some people estimate the number at 10 million and some estimate it at 20 million and higher.32 In 1981 we discovered HTLV-II and published these findings in 1982.33 This was of course occurring just as AIDS was being described in the United States. We isolated the virus from a young, white male with a T-cell variant of hairy cell leukemia. It is not certain how he acquired this infection but his girlfriend was an Inuit from Alaska. Because of the closer sequence hematology of HTLV-II to Asian monkeys, HTLV-II may have originated from Asian simian T-cell leukemia viruses (STLV-1 Asian). HTLV-II is very closely related to HTLV-I bearing more than 90% genomic identity, but it is far less pathogenic. Its importance from a 2014 perspective is learning the reason for the difference in the leukemogenic activity of HTLV-I versus HTLV-II, that is, why HTLV-I is far more pathogenic. Current research focuses around a relatively newly discovered gene known as HBZ, which is at the 3′ end of the genome of HTLV-I and HTLV-II and expresses itself in an antisense fashion. The discovery of HTLV-II also helped prompt the idea that AIDS was caused by a retrovirus. Our thinking after the discoveries of HTLV-I and HTLV-II was, why not more human retroviruses and one causing this new disease, which also involves CD4 T cells and seems to involve the same risk factors for transmission, and since HTLV-I gives minor immune suppression what about variants that might give more substantial immune suppression? HTLV-I is highly related to African STLV-I, especially STLV-I derived from chimps. This gives rise to the idea that there was more than one entry of primate to man of these viruses, some from African primates, and some coming from Asian primates. The kind of disease a virus causes is usually dependent on its tropism (the type of cell it infects), the strategy for its survival, and our immune response to the virus. In the case of HTLV-I its target cells are mature T lymphocytes particularly those that are CD4 positive. The leukemia it causes is a proliferative outgrowth of CD4+ T cells. The strategy for HTLV to survive depends on propagation of the integrated form of its genes (mainly the provirus). Because there is very little viral replication, HTLV drives proliferation of the T cells that contain its provirus. Therefore, survival of the virus is dependent on T-cell proliferation and we could regard ATL as an occasional and accidental consequence of infection, but how does that occur? Well, the mechanisms are quite complex. In addition to the usual genes that all retroviral genomes contain (gag, pol, and env), which encode the viral structural proteins as well as the viral enzymes such as reverse transcriptase and viral protease, the study of the genome of HTLV-I gave rise to the first discovery of other retrovirus genes. Near the 3′ end of the genome are genes that encode viral regulatory proteins known as and Both are essential to HTLV replication, but the also several genes and T-cell an antisense gene known as was discovered (see and there is evidence that it may be to the of the leukemic There are several other genes at the 3′ end of the HTLV-I their been more and is still less is the importance of HTLV-I and HTLV-II and as the leukemia in approximately 5% of infected people a and the neurologic disease approximately 3% in a The other due to as well as the immune may in another few of infected people roughly over a in parts of the world it is an important disease if are the of For in Iran, Japan, and even parts of and some of the Caribbean HTLV-I can surprisingly have more importance than A blood test is certainly needed in some countries and it is a required blood test before blood is used in transfusion in the United Japan, and a few other In Japan, there are to or but this cannot be in some of the Second, an understanding of the of the molecular of leukemia with the in and an in to study may be for leukemia in and even the Its of neurologic disease multiple which may be based on an autoimmune and could be for mechanisms involved in the more multiple sclerosis of the there are of HTLV-I that the idea that AIDS would be caused by a retrovirus. mature CD4 T to moderate immune African and the transmission by blood, sex, and mother to in the that AIDS was likely caused by a retrovirus. Max Essex at School of Public Health and I a new retrovirus as the cause of AIDS in 1982. We this and these the work in our as well as the laboratory in by to for this kind of virus in with However, there were many other as to its Some of the causes such as and autoimmune white blood cells transmitted by However, of these could the development of the disease of nor AIDS associated with of factor or blood infectious were such as of virus, and several A a as the cause of AIDS in the Journal of in form from the National Institute of and Disease in only a few before the of our on what we then called but this could not the infection of hemophiliacs from factor at this because the were and only viruses could through the Our with Max Essex that this was a retrovirus, but one closely related to the as it turned was the only that in its the was in its it was not since HIV is not a of HTLV as we but more a to another of retroviruses known as HIV many in with HTLV-I and HTLV-II such as the CD4 of transmission, presence of genes at the 3′ end of the genome not before in other and a to of the known retroviruses at the as well as a by the to cause immune It seems that there was of these two of distinct primate retroviruses. In the group led by at the Institute reported a new human It was derived from one patient with who had not yet developed AIDS. The importance is that it was the first a new retrovirus that would known as and it was distinct from HTLV-I and Its were that it was only one isolate and the virus could not be nor did the patient have AIDS. the made no as to the role of the virus in the cause of AIDS nor could colleagues and I had the for T-cell to them as well as their first of IL-2. I also the antibodies to distinguish the virus they had discovered from HTLV-I and the reverse transcriptase assay they was the one described in our HTLV-I 1 in their the virus was one but the was a and AIDS had some unique that were past viral or such as severe acute and so These unique the of some years or even and were not to what they did years the are to what one does days or or but not years A is that by the time AIDS was there were multiple and which one was was a I was to have several as they in particular then at of and We reported our in the and We did not these for until we had a number of isolates and the antibody test (the blood to of HIV as the cause of AIDS. Our findings would show that the virus was and that we could find the virus with isolates blood in cases of AIDS or people who were the of what was called at the A reported in one of these was the demonstration of of the virus in cell line We were able to do this with isolates of HIV and, had to which for of the virus to colleagues all over the This occurred in the late of to this were my as well as my I had felt that of the virus by virus would not be to show that HIV was the cause of because as most scientists, I is to a by virus isolates could be very difficult 1) were limited and not even in some 2) T-cell culture technology was not in at this and AIDS had to be and by that time the patients had very few T cells to The consequence was that very few were the blood test was able to because the blood test was to to work sensitive for virus and came almost it enabled of thousands of samples We were able to cell HIV both to the in to with to and some and other for blood testing all over the In our blood test we made the point that we could by but that a also be to the of the test as well as to such as only performed the because the was set in blood in much of the world we were the into for the first However, soon it became that we were in our that the would make the test more this new virus in cell culture not only made the blood test it also made for It was in my laboratory that who would soon after the NCI and his from and in to discovered that was an of the HIV reverse and into the and made the demonstration that a viral disease could be with evidence that a in virus with of the to was not an but it was the that the field and the into for a disease in a for the first Ultimately, this led to the in that this infection from a disease with high and to one that a normal we continued research on new because and is still a there is always a for new and new for We are out of viral but there are that can be because any replicating virus, to a great on factors for its some of these factors can be in that do not to serious The first of this our demonstration with my colleagues that at a relatively low could with the of the used for of viral DNA by reverse or CD4 to entry of the virus, and this is nor Second, there are discussions about the of through of cell after all virus This seems but the are likely through gene such as and such are There will still be considerable all cells. For cells in the are but the for some pursuit. of course are the to a we mean that the will no virus is and remains even there are still cells in the that contain integrated is or the infected cells and that those cells then while to any of virus that is However, this on two all cells will when are by T cells and that we all virus that is Both are likely to be new such as some of the The combination of may make This is being now and time will A is to a much higher of infected This is not only true in developing but it is also true in our because finding all the infected people remains a serious and of the known infected persons to is also We can only this with and and dedication to testing and which The is infection and the our to this is a of testing to the as soon as possible after HIV infection is some are I that be limited to such as with one infected and the other not yet This may to a in the epidemic. and be we to be certain that there are an number of of human virus and all human viral In my colleagues William of and of the for in at University School of and in the late former of the and the and of the at in and I the The of in that all of viruses. The or and and its among to virus the of medical and research at the possible of viruses in diseases of would have those of in that had on and transmitted viruses, and in after their made their availability known to the World Health and the would have had the to in the would have to from and of the In the would a of the kind that was not during the early days of by the being that a few with in retroviruses to involved but had no for The no of