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The discovery of the first human retrovirus: HTLV-1 and HTLV-2

The discovery of the first human retrovirus: HTLV-1 and HTLV-2 I describe here the history leading up to and including my laboratory's discovery of the first human retrovirus, HTLV-I, and its close relative, HTLV-II. My efforts were inspired by early work showing a retroviral etiology for leukemias in various animals, including non-human primates. My two main approaches were to develop criteria for and methods for detection of viral reverse transcriptase and to identify growth factors that could support the growth of hematopoietic cells. These efforts finally yielded success following the discovery of IL-2 and its use to culture adult T cell lymphoma/ leukemia cells. Discovery that human T cells made cytokines Background After arriving at NIH in 1965 I spent my first year as a ("lymphokines") and early hints of human retroviruses young physician caring and treating (mostly unsuccess- The main leukemia I worked on was acute lymphocytic fully) acute leukemias in children: a vivid experience and leukemias (ALL). After all, these were the most common one which made me absolute in a decision to be fully of the acute leukemias and gram quantities of these cells involved in laboratory research and not return to clinical were available from my clinical colleagues at NCI. Impor- medicine. My research interest almost from the very start tantly, these were the only leukemias for which reasona- was in the biology of blood cells, and I focused on com- bly similar normal control cells were available, namely, parisons of human leukemic cells with normal leukocytes. normal human lymphoblasts. Scientists in Philadelphia This was mainly limited to comparative biochemistry. had just discovered that a plant lectin, phytohemaggluti- Specifically, I studied enzymes of pyrimidine nucleoside nin (PHA), could induce human lymphocytes to become and nucleotide metabolism, tRNA species and their corre- activated and go through a mitotic cycle. These normal sponding amino acyl-tRNA synthetases, and finally DNA lymphoblasts looked like ALL cells, but these were days polymerases [see refs.[1-5] as examples] – though now before most of us would or could know of the great com- this approach would seem to be empirical in the extreme, plexity of subtypes of lumphocytes. Functional discrimi- because we have so many obvious rational things in can- natory assays were barely available and monoclonal cer research today. However, at that time "fishing expedi- antibodies with their capacity to provide surface markers tions" were "where things were at". I hoped to uncover were yet to come. Thus, we did not then sub-classify lym- clues that might help us better understand the nature of phocytic leukemias. Herb Cooper of NIH had learned leukemic cells and also their origin. how to purify lymphocytes from columns packed with nylon; myeloid cells would adhere, but lymphocytes passed through. Cooper generously provided this Page 1 of 7 (page number not for citation purposes) Retrovirology 2005, 2:17 http://www.retrovirology.com/content/2/1/17 technique to me. During this period (1968–1970) I the DNA of the target cell. He referred to this integrated became very impressed by the studies of Leo Sachs in form as provirus, the name given to his theory. In 1970 Israel and later also of Don Metcalf in Australia who Temin and his colleague Mizutani, and separately, David where showing that, like some lymphocytes, myeloid cells Baltimore, gave credence to the theory with their discov- could also be grown in the laboratory but not in liquid ery of the DNA polymerase carried by all retroviruses, culture. Instead, they used the technique previously reverse transcriptase (RT)[9,10]. For me it also meant a applied to virus transformed cells of cell growth on a convenient, inexpensive, and extremely sensitive assay for methylcellulose solid surface in the form of cell colonies. a retrovirus. (This would be one of two technologies that However, growth was transient and the amount of cells would be key for later discoveries of all human quite limited, precluding many types of biochemical, retroviruses). molecular biological, and virological experiments. None- theless, from this system, Sachs and his colleagues and RT forms in virions only upon budding from the cell. Metcalf and co-workers made seminal discoveries, includ- Consequently, finding this enzyme in media of cultured ing a growth/differentiation factor, granulocyte macro- cells implied release of retrovirus particles, and finding RT phage colony-stimulating factor (GM-CSF), which was from extracts of cells implied the presence of a cell associ- specific for the myeloid lineage. Sachs logically believed ated virus particles, as for example, virions associated with the main production of GM-CSF would be from myeloid the cell surface membrane. We found rare cases of leuke- cells, i.e., a feedback regulation – granulopoietic progeni- mia that scored positive in RT assays. The problem, how- tors proliferated and formed "dead end" granulocytes, ever, was that RT might be a product of a normal cellular which should produce their own granulopoietic factor gene. We needed to develop the assay not only as a very [see refs. [6,7] for reviews]. sensitive one but also one that would distinguish RT from all of the then known cellular DNA polymerases (alpha, Meanwhile, while comparing ALL cells to normal lym- beta, and gamma). This became a major objective [see phocytes, I decided to test the conditioned medium of the refs. [5] and [11-14] for examples]. PHA-stimulated normal cells for growth factor. The late Alan Wu had just joined me from the laboratories of Till Armed with these RT assays we did find a few cases of and McCulloch in Toronto shortly after the publication of adult lymphocytic leukemias with RT showing all the their famous paper describing hematopoietic stem cell characteristics of RT from a retrovirus (we had by then assays (in mice) for the first time. Alan and Joan Prival, a purified and characterized RT from many different animal post-doctoral fellow, joined me in reporting the then sur- retroviruses). We published on the one best characterized prising finding that lymphocytes (T cells) made GM-CSF in Nature New Biology in 1972 [15]. We believed this was [8]. This would be the start of my long involvement with a "footprint" of a human retrovirus, but we failed to iso- "conditioned medium" from PHA-stimulated lym- late virus from this patient. (Though we will never know, phocytes. Dane Boggs, F. Ruscetti, and co-workers in Pitts- it is interesting to speculate whether this young adult had burgh had described the same phenomena at almost ATL because of some clinical similarities to ATL). We also exactly the same time. These papers were likely among the thought it would attract wide interest and excitement in first to describe lymphokines (lymphocyte-derived the field. It did not. It was clear that we had to isolate a cytokines). replicating virus, one we could study, perpetuate, and give to others. In this period (early 1970s) I began to study animal retro- viruses because in several animals these kinds of viruses The obvious and easiest approach to virus isolation was caused leukemias. Thus, no matter whether human retro- by using cell lines. Cell culture technology had become viruses (leukemia-causing or otherwise) existed or not, a widely available by the 1960s, and many cell lines from study of animal retroviruses, especially focused on learn- different species were available. The approach is generally ing their mechanisms of leukemia causation, might pro- to co-culture the primary cells (in our case the leukemic vide insights into the mechanisms involved in human cells) with a wide variety of such lines and hope virus will leukemias. However, my co-workers and I also decided to take in one or more. This, of course, would be after scoring search for human retroviruses, an unpopular goal at this positive in the RT assay. However, by this period, there time, considering the decades of attempts and failures. I was increasing antagonism to research directed toward the was, nonetheless, encouraged by discussions with Wil- finding of human tumor viruses and especially of retrovi- liam Jarrett, the Scottish veterinarian who discovered ruses. The NCI had created the heavily funded Virus Can- feline leukemia virus, and by the work of the late Howard cer Program which was under attack for failing to find Temin. Temin, of course, had predicted that retroviruses clear evidence of tumor viruses. Moreover, by the mid- of animals replicated by having their RNA genome tran- 1970s there had been not only decades of failure to find scribed into a DNA form, which would then integrate into human retroviruses, there had been many false starts by Page 2 of 7 (page number not for citation purposes) Retrovirology 2005, 2:17 http://www.retrovirology.com/content/2/1/17 Table 1: Factors that led to consensus that human retroviruses did not exist 1. Failure to discover them after an extensive survey by many investigators in the 1950s, 1960s, and 1970s. 2. Ease of detection in animal models because of extensive virema. 3. Difficulties in growing primary human cells. 4. Results showing human sera with complement lysed animal retroviruses. many investigators utilizing the co-culture system that intensely nursing them daily for months. But they were involved cell lines, including one by me. The usual prob- lymphocytes, not myeloid cells. It was neither unique nor lem was a cross contamination with an animal retrovirus. interesting to grow human B cells. Even at this time For this reason I became convinced that we had to find Epstein-Barr virus (EBV) immortalized cell lines were well ways to grow primary blood cells, but not with the sys- known to grow often from normal blood or a bone mar- tems of Sachs and Metcalf. These methylcellulose colonies row mixed cell population. Indeed, they were the only of leukocytes provided too few cells and growth of these kind of blood cells that could be routinely grown in long- cells were limited in number and in time. When we had term culture, but analyses of the cells revealed that they our next hint of an RT positive leukemic sample it turned were T cells, which at that time had only recently been out to be from a patient with a myeloid leukemia, so we clearly delineated from B cells by certain functional assays searched for a growth factor that would maintain and pro- (the E rosette assay, for example). The factor we had found mote growth of human myeloid leukemic cells in liquid in the PHA-CM was a new growth factor. Francis Ruscetti suspension culture. This had not been achieved before. had then joined our group and carried out a set of experi- From an early-term (first few weeks) abortion, we ments that demonstrated this more fully, and we reported obtained some human embryonic cells that produced a these results in 1976–1977[18,19] and they were to be the factor that led to the first successful routine growth of first reports of what we termed a T cell mitogenic factor, these human leukemic cells in liquid suspension(16). We later called TCGF, and finally interleukin-2 (IL-2). The called these HL (human leukemia) cells, with a given purification was later [20]. IL-2 was among the first well- sequential number of the samples we had studied. One of defined cytokines. The combination of IL-2 growth of T these cultured cell populations became an immortalized cells with sensitive RT assays would be (and still is) the cell line, HL-60. It was the first human leukemic myeloid key to the discoveries of human retroviruses in T cell cell line [17] and like almost all the others, the HL-60 cells leukemias and AIDS. showed no evidence of virus. However, one growing human leukemic cell culture (not immortalized) did yield The debate about the possible existence of human virus, and it was anxiously propagated. Unfortunately, the retroviruses embryonic factor needed to keep these cells alive and In this same period the pressure against attempts to find growing was lost when the freezer in which they were human retroviruses intensified. It was not only the pre- stored broke down over a long holiday weekend, which vailing atmosphere of failure but also reasonable scientific was not recognized for some time. (My first lesson in arguments. For examples: (1) there was little evidence for never storing a divisible valuable all in one place!). This leukemia viruses in primates. (2) When retroviruses were led us to a frantic search to find another source. We found in animals they were not difficult to find. Extensive screened conditioned medium from a wide variety of cell viremia preceded disease, therefore, if they infected lines and cell strains, including many more fetal cells – all humans, they would be easy to find and would have been to no avail. discovered much earlier. (3) Human sera in the presence of complement lysed animal retroviruses, thereby provid- One approach was to culture many different types of cells ing a rational mechanism for the conclusion that humans from many different tissue sources (including human were protected. embryos) for several days, collect the media (conditioned media or CM), and add it to leukocytes from normal Finally, there were technical difficulties such as the ability human cord blood, samples of human bone marrow, and to culture primary human cells (see Table 1). myeloid leukemic cells. In this period (early mid-1970s), a post-doctoral fellow, Doris Morgan, joined our group We reasoned otherwise. Kawakami and colleagues had and took part in the search. As would be expected, CM just discovered gibbon ape leukemia virus, and linked it to from PHA-stimulated lymphocytes was one of the cell chronic myeloid leukemia in that species [21]. Later, we sources I asked to be screened. Doris was succeeding in discovered a variant of that virus which caused T cell growing cells from human bone marrow, and was leukemia [22]. Bovine leukemia virus (BLV) was discov- Page 3 of 7 (page number not for citation purposes) Retrovirology 2005, 2:17 http://www.retrovirology.com/content/2/1/17 Table 2: Factors encouraging us to continue searching for human retroviruses 1. The discovery of bovine leukemia virus (minimally replicates, difficult to find) 2. Technological advances – A. A sensitive specific assay for a footprint of a retrovirus, namely, reverse transcriptase. B. Capacity to grow significant numbers of primary human T cells in liquid suspension culture giving us access to virus detection and isolation, namely by using IL-2. 3. Discovery of a retrovirus causing leukemias in a species close to man, namely GaLV. 4. A documented example of a retrovirus transmission from one species of primates to another, namely GaLV from a gibbon ape to a wooly monkey [26]. 5. Purification and characterization of reverse transcriptase from a patient with an adult lymphocytic leukemia (type unknown) 1972 [15]. Discoveries of HTLV-1 and HTLV-2 ered [23,24], and it was noted that BLV replicated at very low levels thus putting to rest the notion of "extensive The first detection and isolation of HTLV-1 was in 1979, viremia always precedes animal retrovirus induced leuke- and the first detection came from the analysis of a T cell mias". The biased view came from the fact that the earlier line established by J. Minna and A. Gazdar from a patient small animal models were naturally selected for their util- these clinicians called a cutaneous T cell lymphoma. Alter- ity. Consequently, models in which virus is difficult to natively, such patients were also called mycosis fungoides detect would be selected against. As for human sera lysing or Sezary T cell leukemia depending upon clinical retroviruses, unfortunately those studies were limited to nuances. Though IL-2 was supplied by us for them to use tests of retroviruses from non-primates. Later, we would in their initial culturing of these cells, the cells rapidly learn that many primate retroviruses, including the retro- immortalized. An outstanding post-doctoral fellow, Ber- viruses of many, are not susceptible. nard Poiesz, carried out RT assays on these cells with pos- itive results, and we soon arranged for electron Our ultimate focus on T cell leukemias was dictated by microscopic analysis of concentrated RT plus cultures and several factors. First, most animal leukemias caused by ret- found retrovirus particles. Because putative human retro- roviruses are lymphocytic leukemias and of these T cell viruses viruses had been found many times before by sev- leukemias predominate. Second, the first and to this date eral investigators in established cell lines, only to be only leukemia of non-human primates is caused by a ret- subsequently shown to be accidental laboratory contami- rovirus [21], and a particular strain of this virus which we nants, by the late 1970s I was well aware that much more isolated caused T cell leukemia [22]. Third, fortune dic- had to be done before this work was presentable. For tated that we would end up focusing on human T cell instance, we had to (1) show that the same virus could be malignancies because of our discovery of IL-2 which isolated from primary tissue samples of the same patient allowed us to grow significant numbers of such cells in by culturing primary T cells with IL-2; (2) demonstrate many but not all instances (not all T cell leukemias or that the virus was novel, i.e., not any of the known animal lymphomas respond to IL-2). retroviruses; (3) show it could infect human T cells in vitro; (4) demonstrate specific antibodies to the virus in One other development also influenced our continuation the serum of the patient; (5) demonstrate that proviral of the pursuit of human retroviruses. This was a docu- DNA could be found integrated in the DNA of the cells mented interspecies transmission of a gibbon ape leuke- from which the virus was isolated; (6) provide evidence mia virus (GaLV) from a pet old world Gibbon ape to a that this was not a one-time affair by showing serological new world Wooly monkey. It was well known that retro- evidence of specific antibodies not only in the patient but viruses could move from one species to another, but in all in others as well. These results were successfully obtained cases these were very ancient events only discovered by in 1979–1980 and available by the time we submitted analyses of cellular DNA of many animals. But in this case and published our first report in 1980 [27], enabling us to the event occurred "right before our eyes", giving rise to follow quickly with several other essential reports [28-33], the virus from the Wooly monkey known as simian sar- also including independent isolates from other patients coma virus [25]. We felt humans could not be excluded, [29,34]. One of these patients was a black woman from and indeed later we would learn that the first human ret- the Caribbean, and the second was a white merchant rovirus discovered (HTLV-1) has close relatives among marine who acknowledged sexual contacts in southern many old world primates and may have arisen from an Japan and the Caribbean. These and all subsequent iso- ancient transmission from monkey to man. A more rele- lates of HTLV-1 in our laboratory were from primary cells vant example, of course, is HIV. There is much evidence cultured with IL-2. After an initial struggle to publish in that it came into humans as a much more recent infection the J. of Virology, fortunately, we were soon able to from African primates (see Table 2). publish the original report in PNAS, and this opened the door. It soon became clear that HTLV-1 was specifically associated with adult T cell malignancy (usually CD4+ Page 4 of 7 (page number not for citation purposes) Retrovirology 2005, 2:17 http://www.retrovirology.com/content/2/1/17 cells) in which the patients frequently had cutaneous forming capability of HTLV-l because the cell line that was abnormalities and hypercalcemia. Clinicians in the immortalized was from the normal donor [37]. Later, my United States had not at that time made any distinction of colleague M. Popovic was able to make this a routine, that HTLV-1-associated T cell malignancies from other neo- is, we would show that HTLV-1 could routinely immortal- plasms, and as noted above collectively referred to these ize normal human T cells [34]. It was obvious to all that patients with others (non-HTLV associated) as cutaneous the virus pictures shown by Hinuma were HTLV-1. By the T cell leukemia-lyumphomas. However, a few years earlier time of this meeting we had already published a few Kiyoshi Takatsuki and his co-workers Junji Yodoi and papers on HTLV-1. Hinuma called his isolate ATLV (adult Takashi Uchiyama defined clusters of leukemia in south- T cell leukemia virus), but argued against collaboration west Japan with special clinical features and cellular mor- claiming it was not possible to provide human sera from phology, which when coupled with the geographic Japan for "cultural reasons". In June 1982 Hinuma and clustering, led him to propose in 1977 that this was a dis- colleagues published on their isolate of ATLV [38]. After tinct form of leukemia. He named it adult T cell leukemia comparative analyses of isolates of ATLV and HTLV were (ATL) [35]. performed we published with Japanese colleagues M. Yoshida, T. Miyoshi and Y. Ito that HTLV-1 and ATLV were Two events significantly catalyzed the further develop- the same virus [39]. Consequently, we agreed that the ment of our work and of our understanding of HTLV-1 virus name should be HTLV to recognize the priority of and its role in T cell malignancies. The first of these (in the our virus work, and the disease would be referred to as summer of 1980) was information from Drs. Tom Wald- ATL in recognition of the Japanese priority in distinguish- mann and H. Uchiyama, who had come to NIH as a visit ing this malignancy as a specific identity which had been scientist. They brought to our attention the ATL cluster in "lumped" with other T cell leukemias/lymphomas in Japan so in the fall of 1980 I contacted two Japanese western countries and elsewhere as cutaneous T cell lym- friends, the late Yohei Ito, then Chair of Microbiology at phomas [40]. Yoshida was soon to make many of the Kyoto University and Tad Aoki for more information and major advances in the molecular biology of HTLV-1 but for sera from such patients to test for antibodies to HTLV. this is another story. This specific clinical entity had been described as early as 1977 by Takatsuki and his co-workers Yodoi and Uchi- The second meeting of considerable importance was in yama, and was called adult T cell leukemia by him. Aoki London chaired by the late hematologist Sir John Dacie and Ito sent sera from such patients to me in 1980, and and attended by Dacie, Drs. Daniel Catovsky, Robin these sera scored positive for antibodies to HTLV-1. Based Weiss, Mel Greaves, and William Jarrett among others on these results Ito organized a small meeting at Lake from Great Britain and by my collaborator in epidemio- Miwa outside of Kyoto attended by a few co-workers and logical studies, Dr. William Blattner, and myself. It was myself from the U.S. and Aoki, Ito, and several other Jap- Catvosky who called for this meeting because he noted anese scientists most notably Takatsuki, Y. Hinuma, and that we had found HTLV-1 mainly in African Americans T. Miyoshi. The meeting was held in March 1981. Several and black persons in the Caribbean and he had found an of my colleagues and I presented our results in detail. This unusual frequency of adult T cell malignancies in Carib- included description of several isolates of HTLV-1, charac- bean immigrants to England. He recognized the similari- teristics of purified HTLV-1 p24 as well as reverse tran- ties of their disease to Takatsuki's ATL. Thus, he postulated scriptase proteins, evidence of integrated HTLV-1 provirus they were one and the same disease and HTLV-1 would be T cell malignancies and healthy volunteers which pro- present in all. He was right. Promptly, Blattner accelerated vided clear evidence for the linkage of HTLV-1 to certain T his studies in the Caribbean and documented that HTLV- cell malignancies, and the positive serological results in 1 was endemic in some islands. He and Guy de Thé of Japanese ATL patients. In organizing this meeting the France would then show that this result depended upon intention of Ito was to foster wide collaboration in Japan the particular tribes in Africa from which the individuals with me and my co-workers on this disease. The meeting descended. summary was published in Cancer Research in November 1981 [36]. Some of these experiences would be a precursor of a per- sistent pattern, i.e., HTLVs are not easy to transmit, remain It was only at the end of the meeting when we were sum- within families and regions over long periods of time, and marizing and planning for this collaboration with the Jap- have old-world linkage. Ultimately, related viruses would anese investigators, that Dr. Yorio Hinuma "announced" be found in old-world primates and more distantly he too had a retrovirus. He presented EM pictures of virus related viruses in some ungulates. The modes of particles from a cell line established by Dr. Miyoshi by co- transmission would soon be forthcoming as sexual con- cultivation of ATL cells and normal human T cells. These tact, blood, and mother to child via breast feeding. Later results of Miyoshi were the first indication of the trans- in 1981 we isolated HTLV-2 from a leukemia described as Page 5 of 7 (page number not for citation purposes) Retrovirology 2005, 2:17 http://www.retrovirology.com/content/2/1/17 16. Gallagher RE, Salahuddin SZ, Hall WT, McCredie KB, Gallo RC: "a hairy cell T cell leukemia" [41], but this strain is far less Growth and differentiation in culture of leukemic leukocytes pathogenic that HTLV-1. Many of the features of these from a patient with acute myelogenous leukemia and re- viruses coupled with CD4 T cell tropism would prove to identification of type-C virus. Proc Natl Acad Sci U S A 1975, 72:4137-4141. be remarkably similar to those of the virus about to enter 17. Collins SJ, Gallo RC, Gallagher RE: Continuous growth and differ- our work, HIV. entiation of human myeloid leukaemic cells in suspension culture. Nature 1977, 270:347-349. 18. Ruscetti FW, Morgan DA, Gallo RC: Functional and morphologic A companion article in Retrovirology by Kiyoshi Takat- characterization of human T cells continuously grown in suki recounts the events surrounding the discovery of the vitro. J Immunol 1977, 119:131-138. 19. Morgan DA, Ruscetti FW, Gallo R: Selective in vitro growth of T disease, adult T-cell leukemia [41]. lymphocytes from normal human bone marrows. Science 1976, 193:1007-1008. Acknowledgements 20. Mier JW, Gallo RC: The purification and properties of human T cell growth factor. J Immunol 1982, 128:1122-1127. I would like to thank the past and present members of my laboratory, with- 21. Kawakami TG, Huff SD, Buckley PM, Dungworth DL, Synder SP, out whom the studies described in this article would not have been possi- Gilden RV: C-type virus associated with gibbon ble. My special thanks (in no particular order) goes to Bernie Poiesz, Frank lymphosarcoma. Nat New Biol 1972, 235:170-171. 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Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuki Y, Shiraishi Y, polymerase distinct from viral RNA-directed DNA et al.: Type C virus particles in a cord T-cell line derived by co- polymerase. Proc Natl Acad Sci U S A 1972, 69:3228-3232. cultivating normal human cord leukocytes and human leu- 15. Sarngadharan MG, Sarin PS, Reitz MS, Gallo RC: Reverse tran- kaemic T cells. Nature 1981, 294:770-771. scriptase activity of human acute leukaemic cells: purifica- 37. Yoshida M, Miyoshi I, Hinuma Y: Isolation and characterization tion of the enzyme, response to AMV 70S RNA, and of retrovirus from cell lines of human adult T-cell leukemia characterization of the DNA product. Nat New Biol 1972, 240:67-72. Page 6 of 7 (page number not for citation purposes) Retrovirology 2005, 2:17 http://www.retrovirology.com/content/2/1/17 and its implication in the disease. Proc Natl Acad Sci U S A 1982, 79:2031-2035. 38. Popovic M, Reitz MS Jr, Sarngadharan MG, Robert-Guroff M, Kaly- anaraman VS, Nakao Y, et al.: The virus of Japanese adult T-cell leukaemia is a member of the human T-cell leukaemia virus group. Nature 1982, 300:63-66. 39. Gallo RC, Blattner WA, Reitz MS Jr, Ito Y: HTLV: the virus of adult T-cell leukaemia in Japan and elsewhere. Lancet 1982, 1:683. 40. Kalyanaraman VS, Sarngadharan MG, Robert-Guroff M, Miyoshi I, Golde D, Gallo RC: A new subtype of human T-cell leukemia virus (HTLV-II) associated with a T-cell variant of hairy cell leukemia. Science 1982, 218:571-573. 41. Takatsuki K: Discovery of adult T-cell leukemia. Retrovirology 2:16. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 7 of 7 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Retrovirology Springer Journals

The discovery of the first human retrovirus: HTLV-1 and HTLV-2

Retrovirology , Volume 2 (1) – Mar 2, 2005

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Springer Journals
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Copyright © 2005 by Gallo; licensee BioMed Central Ltd.
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Biomedicine; Virology; Infectious Diseases; Cancer Research
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1742-4690
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10.1186/1742-4690-2-17
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15743526
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Abstract

I describe here the history leading up to and including my laboratory's discovery of the first human retrovirus, HTLV-I, and its close relative, HTLV-II. My efforts were inspired by early work showing a retroviral etiology for leukemias in various animals, including non-human primates. My two main approaches were to develop criteria for and methods for detection of viral reverse transcriptase and to identify growth factors that could support the growth of hematopoietic cells. These efforts finally yielded success following the discovery of IL-2 and its use to culture adult T cell lymphoma/ leukemia cells. Discovery that human T cells made cytokines Background After arriving at NIH in 1965 I spent my first year as a ("lymphokines") and early hints of human retroviruses young physician caring and treating (mostly unsuccess- The main leukemia I worked on was acute lymphocytic fully) acute leukemias in children: a vivid experience and leukemias (ALL). After all, these were the most common one which made me absolute in a decision to be fully of the acute leukemias and gram quantities of these cells involved in laboratory research and not return to clinical were available from my clinical colleagues at NCI. Impor- medicine. My research interest almost from the very start tantly, these were the only leukemias for which reasona- was in the biology of blood cells, and I focused on com- bly similar normal control cells were available, namely, parisons of human leukemic cells with normal leukocytes. normal human lymphoblasts. Scientists in Philadelphia This was mainly limited to comparative biochemistry. had just discovered that a plant lectin, phytohemaggluti- Specifically, I studied enzymes of pyrimidine nucleoside nin (PHA), could induce human lymphocytes to become and nucleotide metabolism, tRNA species and their corre- activated and go through a mitotic cycle. These normal sponding amino acyl-tRNA synthetases, and finally DNA lymphoblasts looked like ALL cells, but these were days polymerases [see refs.[1-5] as examples] – though now before most of us would or could know of the great com- this approach would seem to be empirical in the extreme, plexity of subtypes of lumphocytes. Functional discrimi- because we have so many obvious rational things in can- natory assays were barely available and monoclonal cer research today. However, at that time "fishing expedi- antibodies with their capacity to provide surface markers tions" were "where things were at". I hoped to uncover were yet to come. Thus, we did not then sub-classify lym- clues that might help us better understand the nature of phocytic leukemias. Herb Cooper of NIH had learned leukemic cells and also their origin. how to purify lymphocytes from columns packed with nylon; myeloid cells would adhere, but lymphocytes passed through. Cooper generously provided this Page 1 of 7 (page number not for citation purposes) Retrovirology 2005, 2:17 http://www.retrovirology.com/content/2/1/17 technique to me. During this period (1968–1970) I the DNA of the target cell. He referred to this integrated became very impressed by the studies of Leo Sachs in form as provirus, the name given to his theory. In 1970 Israel and later also of Don Metcalf in Australia who Temin and his colleague Mizutani, and separately, David where showing that, like some lymphocytes, myeloid cells Baltimore, gave credence to the theory with their discov- could also be grown in the laboratory but not in liquid ery of the DNA polymerase carried by all retroviruses, culture. Instead, they used the technique previously reverse transcriptase (RT)[9,10]. For me it also meant a applied to virus transformed cells of cell growth on a convenient, inexpensive, and extremely sensitive assay for methylcellulose solid surface in the form of cell colonies. a retrovirus. (This would be one of two technologies that However, growth was transient and the amount of cells would be key for later discoveries of all human quite limited, precluding many types of biochemical, retroviruses). molecular biological, and virological experiments. None- theless, from this system, Sachs and his colleagues and RT forms in virions only upon budding from the cell. Metcalf and co-workers made seminal discoveries, includ- Consequently, finding this enzyme in media of cultured ing a growth/differentiation factor, granulocyte macro- cells implied release of retrovirus particles, and finding RT phage colony-stimulating factor (GM-CSF), which was from extracts of cells implied the presence of a cell associ- specific for the myeloid lineage. Sachs logically believed ated virus particles, as for example, virions associated with the main production of GM-CSF would be from myeloid the cell surface membrane. We found rare cases of leuke- cells, i.e., a feedback regulation – granulopoietic progeni- mia that scored positive in RT assays. The problem, how- tors proliferated and formed "dead end" granulocytes, ever, was that RT might be a product of a normal cellular which should produce their own granulopoietic factor gene. We needed to develop the assay not only as a very [see refs. [6,7] for reviews]. sensitive one but also one that would distinguish RT from all of the then known cellular DNA polymerases (alpha, Meanwhile, while comparing ALL cells to normal lym- beta, and gamma). This became a major objective [see phocytes, I decided to test the conditioned medium of the refs. [5] and [11-14] for examples]. PHA-stimulated normal cells for growth factor. The late Alan Wu had just joined me from the laboratories of Till Armed with these RT assays we did find a few cases of and McCulloch in Toronto shortly after the publication of adult lymphocytic leukemias with RT showing all the their famous paper describing hematopoietic stem cell characteristics of RT from a retrovirus (we had by then assays (in mice) for the first time. Alan and Joan Prival, a purified and characterized RT from many different animal post-doctoral fellow, joined me in reporting the then sur- retroviruses). We published on the one best characterized prising finding that lymphocytes (T cells) made GM-CSF in Nature New Biology in 1972 [15]. We believed this was [8]. This would be the start of my long involvement with a "footprint" of a human retrovirus, but we failed to iso- "conditioned medium" from PHA-stimulated lym- late virus from this patient. (Though we will never know, phocytes. Dane Boggs, F. Ruscetti, and co-workers in Pitts- it is interesting to speculate whether this young adult had burgh had described the same phenomena at almost ATL because of some clinical similarities to ATL). We also exactly the same time. These papers were likely among the thought it would attract wide interest and excitement in first to describe lymphokines (lymphocyte-derived the field. It did not. It was clear that we had to isolate a cytokines). replicating virus, one we could study, perpetuate, and give to others. In this period (early 1970s) I began to study animal retro- viruses because in several animals these kinds of viruses The obvious and easiest approach to virus isolation was caused leukemias. Thus, no matter whether human retro- by using cell lines. Cell culture technology had become viruses (leukemia-causing or otherwise) existed or not, a widely available by the 1960s, and many cell lines from study of animal retroviruses, especially focused on learn- different species were available. The approach is generally ing their mechanisms of leukemia causation, might pro- to co-culture the primary cells (in our case the leukemic vide insights into the mechanisms involved in human cells) with a wide variety of such lines and hope virus will leukemias. However, my co-workers and I also decided to take in one or more. This, of course, would be after scoring search for human retroviruses, an unpopular goal at this positive in the RT assay. However, by this period, there time, considering the decades of attempts and failures. I was increasing antagonism to research directed toward the was, nonetheless, encouraged by discussions with Wil- finding of human tumor viruses and especially of retrovi- liam Jarrett, the Scottish veterinarian who discovered ruses. The NCI had created the heavily funded Virus Can- feline leukemia virus, and by the work of the late Howard cer Program which was under attack for failing to find Temin. Temin, of course, had predicted that retroviruses clear evidence of tumor viruses. Moreover, by the mid- of animals replicated by having their RNA genome tran- 1970s there had been not only decades of failure to find scribed into a DNA form, which would then integrate into human retroviruses, there had been many false starts by Page 2 of 7 (page number not for citation purposes) Retrovirology 2005, 2:17 http://www.retrovirology.com/content/2/1/17 Table 1: Factors that led to consensus that human retroviruses did not exist 1. Failure to discover them after an extensive survey by many investigators in the 1950s, 1960s, and 1970s. 2. Ease of detection in animal models because of extensive virema. 3. Difficulties in growing primary human cells. 4. Results showing human sera with complement lysed animal retroviruses. many investigators utilizing the co-culture system that intensely nursing them daily for months. But they were involved cell lines, including one by me. The usual prob- lymphocytes, not myeloid cells. It was neither unique nor lem was a cross contamination with an animal retrovirus. interesting to grow human B cells. Even at this time For this reason I became convinced that we had to find Epstein-Barr virus (EBV) immortalized cell lines were well ways to grow primary blood cells, but not with the sys- known to grow often from normal blood or a bone mar- tems of Sachs and Metcalf. These methylcellulose colonies row mixed cell population. Indeed, they were the only of leukocytes provided too few cells and growth of these kind of blood cells that could be routinely grown in long- cells were limited in number and in time. When we had term culture, but analyses of the cells revealed that they our next hint of an RT positive leukemic sample it turned were T cells, which at that time had only recently been out to be from a patient with a myeloid leukemia, so we clearly delineated from B cells by certain functional assays searched for a growth factor that would maintain and pro- (the E rosette assay, for example). The factor we had found mote growth of human myeloid leukemic cells in liquid in the PHA-CM was a new growth factor. Francis Ruscetti suspension culture. This had not been achieved before. had then joined our group and carried out a set of experi- From an early-term (first few weeks) abortion, we ments that demonstrated this more fully, and we reported obtained some human embryonic cells that produced a these results in 1976–1977[18,19] and they were to be the factor that led to the first successful routine growth of first reports of what we termed a T cell mitogenic factor, these human leukemic cells in liquid suspension(16). We later called TCGF, and finally interleukin-2 (IL-2). The called these HL (human leukemia) cells, with a given purification was later [20]. IL-2 was among the first well- sequential number of the samples we had studied. One of defined cytokines. The combination of IL-2 growth of T these cultured cell populations became an immortalized cells with sensitive RT assays would be (and still is) the cell line, HL-60. It was the first human leukemic myeloid key to the discoveries of human retroviruses in T cell cell line [17] and like almost all the others, the HL-60 cells leukemias and AIDS. showed no evidence of virus. However, one growing human leukemic cell culture (not immortalized) did yield The debate about the possible existence of human virus, and it was anxiously propagated. Unfortunately, the retroviruses embryonic factor needed to keep these cells alive and In this same period the pressure against attempts to find growing was lost when the freezer in which they were human retroviruses intensified. It was not only the pre- stored broke down over a long holiday weekend, which vailing atmosphere of failure but also reasonable scientific was not recognized for some time. (My first lesson in arguments. For examples: (1) there was little evidence for never storing a divisible valuable all in one place!). This leukemia viruses in primates. (2) When retroviruses were led us to a frantic search to find another source. We found in animals they were not difficult to find. Extensive screened conditioned medium from a wide variety of cell viremia preceded disease, therefore, if they infected lines and cell strains, including many more fetal cells – all humans, they would be easy to find and would have been to no avail. discovered much earlier. (3) Human sera in the presence of complement lysed animal retroviruses, thereby provid- One approach was to culture many different types of cells ing a rational mechanism for the conclusion that humans from many different tissue sources (including human were protected. embryos) for several days, collect the media (conditioned media or CM), and add it to leukocytes from normal Finally, there were technical difficulties such as the ability human cord blood, samples of human bone marrow, and to culture primary human cells (see Table 1). myeloid leukemic cells. In this period (early mid-1970s), a post-doctoral fellow, Doris Morgan, joined our group We reasoned otherwise. Kawakami and colleagues had and took part in the search. As would be expected, CM just discovered gibbon ape leukemia virus, and linked it to from PHA-stimulated lymphocytes was one of the cell chronic myeloid leukemia in that species [21]. Later, we sources I asked to be screened. Doris was succeeding in discovered a variant of that virus which caused T cell growing cells from human bone marrow, and was leukemia [22]. Bovine leukemia virus (BLV) was discov- Page 3 of 7 (page number not for citation purposes) Retrovirology 2005, 2:17 http://www.retrovirology.com/content/2/1/17 Table 2: Factors encouraging us to continue searching for human retroviruses 1. The discovery of bovine leukemia virus (minimally replicates, difficult to find) 2. Technological advances – A. A sensitive specific assay for a footprint of a retrovirus, namely, reverse transcriptase. B. Capacity to grow significant numbers of primary human T cells in liquid suspension culture giving us access to virus detection and isolation, namely by using IL-2. 3. Discovery of a retrovirus causing leukemias in a species close to man, namely GaLV. 4. A documented example of a retrovirus transmission from one species of primates to another, namely GaLV from a gibbon ape to a wooly monkey [26]. 5. Purification and characterization of reverse transcriptase from a patient with an adult lymphocytic leukemia (type unknown) 1972 [15]. Discoveries of HTLV-1 and HTLV-2 ered [23,24], and it was noted that BLV replicated at very low levels thus putting to rest the notion of "extensive The first detection and isolation of HTLV-1 was in 1979, viremia always precedes animal retrovirus induced leuke- and the first detection came from the analysis of a T cell mias". The biased view came from the fact that the earlier line established by J. Minna and A. Gazdar from a patient small animal models were naturally selected for their util- these clinicians called a cutaneous T cell lymphoma. Alter- ity. Consequently, models in which virus is difficult to natively, such patients were also called mycosis fungoides detect would be selected against. As for human sera lysing or Sezary T cell leukemia depending upon clinical retroviruses, unfortunately those studies were limited to nuances. Though IL-2 was supplied by us for them to use tests of retroviruses from non-primates. Later, we would in their initial culturing of these cells, the cells rapidly learn that many primate retroviruses, including the retro- immortalized. An outstanding post-doctoral fellow, Ber- viruses of many, are not susceptible. nard Poiesz, carried out RT assays on these cells with pos- itive results, and we soon arranged for electron Our ultimate focus on T cell leukemias was dictated by microscopic analysis of concentrated RT plus cultures and several factors. First, most animal leukemias caused by ret- found retrovirus particles. Because putative human retro- roviruses are lymphocytic leukemias and of these T cell viruses viruses had been found many times before by sev- leukemias predominate. Second, the first and to this date eral investigators in established cell lines, only to be only leukemia of non-human primates is caused by a ret- subsequently shown to be accidental laboratory contami- rovirus [21], and a particular strain of this virus which we nants, by the late 1970s I was well aware that much more isolated caused T cell leukemia [22]. Third, fortune dic- had to be done before this work was presentable. For tated that we would end up focusing on human T cell instance, we had to (1) show that the same virus could be malignancies because of our discovery of IL-2 which isolated from primary tissue samples of the same patient allowed us to grow significant numbers of such cells in by culturing primary T cells with IL-2; (2) demonstrate many but not all instances (not all T cell leukemias or that the virus was novel, i.e., not any of the known animal lymphomas respond to IL-2). retroviruses; (3) show it could infect human T cells in vitro; (4) demonstrate specific antibodies to the virus in One other development also influenced our continuation the serum of the patient; (5) demonstrate that proviral of the pursuit of human retroviruses. This was a docu- DNA could be found integrated in the DNA of the cells mented interspecies transmission of a gibbon ape leuke- from which the virus was isolated; (6) provide evidence mia virus (GaLV) from a pet old world Gibbon ape to a that this was not a one-time affair by showing serological new world Wooly monkey. It was well known that retro- evidence of specific antibodies not only in the patient but viruses could move from one species to another, but in all in others as well. These results were successfully obtained cases these were very ancient events only discovered by in 1979–1980 and available by the time we submitted analyses of cellular DNA of many animals. But in this case and published our first report in 1980 [27], enabling us to the event occurred "right before our eyes", giving rise to follow quickly with several other essential reports [28-33], the virus from the Wooly monkey known as simian sar- also including independent isolates from other patients coma virus [25]. We felt humans could not be excluded, [29,34]. One of these patients was a black woman from and indeed later we would learn that the first human ret- the Caribbean, and the second was a white merchant rovirus discovered (HTLV-1) has close relatives among marine who acknowledged sexual contacts in southern many old world primates and may have arisen from an Japan and the Caribbean. These and all subsequent iso- ancient transmission from monkey to man. A more rele- lates of HTLV-1 in our laboratory were from primary cells vant example, of course, is HIV. There is much evidence cultured with IL-2. After an initial struggle to publish in that it came into humans as a much more recent infection the J. of Virology, fortunately, we were soon able to from African primates (see Table 2). publish the original report in PNAS, and this opened the door. It soon became clear that HTLV-1 was specifically associated with adult T cell malignancy (usually CD4+ Page 4 of 7 (page number not for citation purposes) Retrovirology 2005, 2:17 http://www.retrovirology.com/content/2/1/17 cells) in which the patients frequently had cutaneous forming capability of HTLV-l because the cell line that was abnormalities and hypercalcemia. Clinicians in the immortalized was from the normal donor [37]. Later, my United States had not at that time made any distinction of colleague M. Popovic was able to make this a routine, that HTLV-1-associated T cell malignancies from other neo- is, we would show that HTLV-1 could routinely immortal- plasms, and as noted above collectively referred to these ize normal human T cells [34]. It was obvious to all that patients with others (non-HTLV associated) as cutaneous the virus pictures shown by Hinuma were HTLV-1. By the T cell leukemia-lyumphomas. However, a few years earlier time of this meeting we had already published a few Kiyoshi Takatsuki and his co-workers Junji Yodoi and papers on HTLV-1. Hinuma called his isolate ATLV (adult Takashi Uchiyama defined clusters of leukemia in south- T cell leukemia virus), but argued against collaboration west Japan with special clinical features and cellular mor- claiming it was not possible to provide human sera from phology, which when coupled with the geographic Japan for "cultural reasons". In June 1982 Hinuma and clustering, led him to propose in 1977 that this was a dis- colleagues published on their isolate of ATLV [38]. After tinct form of leukemia. He named it adult T cell leukemia comparative analyses of isolates of ATLV and HTLV were (ATL) [35]. performed we published with Japanese colleagues M. Yoshida, T. Miyoshi and Y. Ito that HTLV-1 and ATLV were Two events significantly catalyzed the further develop- the same virus [39]. Consequently, we agreed that the ment of our work and of our understanding of HTLV-1 virus name should be HTLV to recognize the priority of and its role in T cell malignancies. The first of these (in the our virus work, and the disease would be referred to as summer of 1980) was information from Drs. Tom Wald- ATL in recognition of the Japanese priority in distinguish- mann and H. Uchiyama, who had come to NIH as a visit ing this malignancy as a specific identity which had been scientist. They brought to our attention the ATL cluster in "lumped" with other T cell leukemias/lymphomas in Japan so in the fall of 1980 I contacted two Japanese western countries and elsewhere as cutaneous T cell lym- friends, the late Yohei Ito, then Chair of Microbiology at phomas [40]. Yoshida was soon to make many of the Kyoto University and Tad Aoki for more information and major advances in the molecular biology of HTLV-1 but for sera from such patients to test for antibodies to HTLV. this is another story. This specific clinical entity had been described as early as 1977 by Takatsuki and his co-workers Yodoi and Uchi- The second meeting of considerable importance was in yama, and was called adult T cell leukemia by him. Aoki London chaired by the late hematologist Sir John Dacie and Ito sent sera from such patients to me in 1980, and and attended by Dacie, Drs. Daniel Catovsky, Robin these sera scored positive for antibodies to HTLV-1. Based Weiss, Mel Greaves, and William Jarrett among others on these results Ito organized a small meeting at Lake from Great Britain and by my collaborator in epidemio- Miwa outside of Kyoto attended by a few co-workers and logical studies, Dr. William Blattner, and myself. It was myself from the U.S. and Aoki, Ito, and several other Jap- Catvosky who called for this meeting because he noted anese scientists most notably Takatsuki, Y. Hinuma, and that we had found HTLV-1 mainly in African Americans T. Miyoshi. The meeting was held in March 1981. Several and black persons in the Caribbean and he had found an of my colleagues and I presented our results in detail. This unusual frequency of adult T cell malignancies in Carib- included description of several isolates of HTLV-1, charac- bean immigrants to England. He recognized the similari- teristics of purified HTLV-1 p24 as well as reverse tran- ties of their disease to Takatsuki's ATL. Thus, he postulated scriptase proteins, evidence of integrated HTLV-1 provirus they were one and the same disease and HTLV-1 would be T cell malignancies and healthy volunteers which pro- present in all. He was right. Promptly, Blattner accelerated vided clear evidence for the linkage of HTLV-1 to certain T his studies in the Caribbean and documented that HTLV- cell malignancies, and the positive serological results in 1 was endemic in some islands. He and Guy de Thé of Japanese ATL patients. In organizing this meeting the France would then show that this result depended upon intention of Ito was to foster wide collaboration in Japan the particular tribes in Africa from which the individuals with me and my co-workers on this disease. The meeting descended. summary was published in Cancer Research in November 1981 [36]. Some of these experiences would be a precursor of a per- sistent pattern, i.e., HTLVs are not easy to transmit, remain It was only at the end of the meeting when we were sum- within families and regions over long periods of time, and marizing and planning for this collaboration with the Jap- have old-world linkage. Ultimately, related viruses would anese investigators, that Dr. Yorio Hinuma "announced" be found in old-world primates and more distantly he too had a retrovirus. He presented EM pictures of virus related viruses in some ungulates. The modes of particles from a cell line established by Dr. Miyoshi by co- transmission would soon be forthcoming as sexual con- cultivation of ATL cells and normal human T cells. These tact, blood, and mother to child via breast feeding. Later results of Miyoshi were the first indication of the trans- in 1981 we isolated HTLV-2 from a leukemia described as Page 5 of 7 (page number not for citation purposes) Retrovirology 2005, 2:17 http://www.retrovirology.com/content/2/1/17 16. Gallagher RE, Salahuddin SZ, Hall WT, McCredie KB, Gallo RC: "a hairy cell T cell leukemia" [41], but this strain is far less Growth and differentiation in culture of leukemic leukocytes pathogenic that HTLV-1. Many of the features of these from a patient with acute myelogenous leukemia and re- viruses coupled with CD4 T cell tropism would prove to identification of type-C virus. Proc Natl Acad Sci U S A 1975, 72:4137-4141. be remarkably similar to those of the virus about to enter 17. Collins SJ, Gallo RC, Gallagher RE: Continuous growth and differ- our work, HIV. entiation of human myeloid leukaemic cells in suspension culture. Nature 1977, 270:347-349. 18. Ruscetti FW, Morgan DA, Gallo RC: Functional and morphologic A companion article in Retrovirology by Kiyoshi Takat- characterization of human T cells continuously grown in suki recounts the events surrounding the discovery of the vitro. J Immunol 1977, 119:131-138. 19. Morgan DA, Ruscetti FW, Gallo R: Selective in vitro growth of T disease, adult T-cell leukemia [41]. lymphocytes from normal human bone marrows. Science 1976, 193:1007-1008. Acknowledgements 20. Mier JW, Gallo RC: The purification and properties of human T cell growth factor. J Immunol 1982, 128:1122-1127. I would like to thank the past and present members of my laboratory, with- 21. Kawakami TG, Huff SD, Buckley PM, Dungworth DL, Synder SP, out whom the studies described in this article would not have been possi- Gilden RV: C-type virus associated with gibbon ble. My special thanks (in no particular order) goes to Bernie Poiesz, Frank lymphosarcoma. Nat New Biol 1972, 235:170-171. 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