I briefly review the discovery and characterization of the first human retrovirus, human T-cell leukemia virus type 1, focusing on contributions from Japanese researchers. The unique regulatory mechanisms for the viral regulation with Tax and Rex, etiology of ATL and possible leukemogenic mechanism with Tax are also discussed briefly.
When virologists and oncologists are challenged to identify human tumor viruses, they dream of the contribution of such viruses towards understanding cancers and answering questions such as how and why cancers develop and how to prevent viral infection in order to prevent cancers. There was a time when retroviruses were a particular focus because retroviruses had been isolated as tumor viruses from many animal species in the 1970s. Such studies provided the conceptual revolution in understanding cancers, ultimately contributing to the proposition of a ‘viral oncogene’. Indeed, over the course of research, it became apparent that viral oncogenes originated from their host cell ‘proto-oncogenes’, which are widely conserved in many animal species. Thus, ‘the oncogene’ became the ‘key player’ in cancer research linking findings between animals and humans and also the different origins of cancers. However, the daunting worldwide challenge remained to identify and isolate a bona fide retrovirus in a human cancer.
The discovery of the first human retrovirus proceeded rather independently in Japan and the USA. Below, I first summarize the history of discovery from Japanese researchers and then discuss human T-cell leukemia virus type 1 (HTLV-1) regulatory and pathogenic mechanisms.
In Japan something was there, but it was not clear
Discovery of ATL
The first key event for human retrovirus was the discovery of adult T-cell leukemia (ATL) by Kiyoshi Takatsuki and his colleagues in Japan in 1976 (Uchiyama et al., 1977). The reports from his laboratory described unique characteristics of the leukemia such as unusual morphology of leukemic cells with lobulated nuclei and surface phenotypes. The most striking feature of the disease was the Kyushu (West part of Japan) origin of most of the patients who were identified in Kyoto where Takatsuki's group performed their research. Takatsuki's findings were later confirmed by extensive epidemiology and a clear clustering of ATL cases in the Kyushu area strongly suggested a unique etiology (Hinuma et al., 1981a). At the time, this novel discovery elicited enormous attention from physicians, virologists, epidemiologists and oncologists, but still there was no clue as to how to search for an etiology.
Establishment of cell lines
Isao Miyoshi who was interested in karyotype of tumor cells tried to establish cell lines from peripheral lymphoid cells of ATL patients (Miyoshi et al., 1980). Prior to the availability of purified lymphokines, a cell line MT-1 was established from the sample of an ATL patient using cocultivation with cord blood lymphocytes. Similar attempts then led to the establishing of MT-2 and MT-4 cells, and these established lines exhibited extremely high chromosomal abnormalities. Otherwise, at that time, not much additional information was captured from these cell lines. Nevertheless, these cell lines later became essential tools for diagnosis of ATL and identification of the retrovirus, providing reproducible and consistent biological materials that could be propagated in large quantity. Surprisingly, some of these cell lines turned out to be of cord-blood origin and not of ATL origin according to chromosome analysis (Miyoshi et al., 1981). These unexpected results were later explained by the transmission of retrovirus from ATL cells into cord-blood cells and subsequent immortalization of the de novo infected cell.
Identification of a unique antibody in ATL patients
Identification of unique serum antibodies in ATL patients was another key criterion for a new retrovirus. Postulating a viral infection in ATL patients, Yorio Hinuma et al. (1981b) applied an indirect immunofluorescence assay to some ATL sera employing a large panel of cells of human origin. After laborious screening, a single cell line was identified to give a robustly positive signal. That was the MT-1 cell, and the putative antigens in the cell line were preliminary termed ATL antigens (ATLA). Later, MT-2 and -4 were also identified to give positive signals in this assay. Without knowing what ATLA were, screening for anti-ATLA was carried out with sera from various types of leukemias, and the results were importantly informative: anti-ATLA were detected in all ATL patients, also in a minor fraction of healthy individuals restricted to the Kyushu area in Japan (Hinuma et al., 1981a, 1981b). Therefore, it was evident that something was there, but it was not clear what was there.
The daybreak of HTLV-1 in Japan
Identification of retrovirus
During the corresponding period of these years, Robert C Gallo and his group at NIH, USA (Poeisz et al., 1980), identified and first reported a retrovirus in a T-cell line, HUT102, established from a patient with Mycosis fungoides, which was later diagnosed as a case of ATL lymphoma.
Independently, molecular approaches were being applied to identify a retrovirus in the MT-2 cell line in Japan by Yoshida et al. (1982). MT2 reacted positively to sera from ATL patients. Particle fraction with similar density to animal retrovirus was purified from MT-2 culture fluid, and cDNA was prepared with the reverse transcriptase activity endogenous to those particles. This cDNA preparation was demonstrated to hybridize with cellular DNA of MT-2 but not of other cells. Thus, the virus-like particles released from MT-2 cells were shown to contain both RNA templates and reverse transcriptase, and MT-2 cells had DNA sequence which hybridized with RNA from the particles, that is, the integrated provirus DNA. p24 and p19 were also identified as components of the particles, which are known today as viral core proteins. Thus, a retrovirus was convincingly demonstrated in MT-2 cells, and this retrovirus was reported as adult T-cell leukemia virus (ATLV). The US HTLV and Japanese ATLV were later demonstrated to be identical at the sequence level and named as HTLV-1 (Watanabe et al., 1984).
Evidences for new retrovirus
Generally, replication competent retroviruses have their proviral genome organized as LTR-gag-pol-env-LTR, which respectively encode the viral core proteins, reverse transcriptase and envelope proteins. The replication competent genomic structure is frequently deleted by acquisition of a cellular proto-oncogene resulting in the formation of a transforming, but replication defective, virus. The newly isolated HTLV-1 was unusual in that it has a complete undeleted replication competent genome, but it contained an additional pX sequence which could encode multiple proteins using alternative open reading frames. Thus, HTLV-1 has an ‘LTR-gag-pol-env-pX-LTR’ genomic structure (Figure 1; Seiki et al., 1982, 1983), with constitution similar to that of Rous sarcoma virus. This unique genome structure distinguished HTLV-1 from any other animal retroviruses, thus founding a new retroviral group. Other members of this viral group, HTLV-2, STLV or PTLV and BLV, are well characterized today.
Association with ATL and other diseases
As described above, unique antibodies that crossreact with MT-1 cells were detected in all ATL patients, but not in other leukemic or healthy individuals, except for a rare minor population (Shimoyama et al., 1986). The HTLV-1 proteins, p24 and p19, were identified at least as a part of the antigens for the unique antibodies in ATL patients; therefore, close association of HTLV-1 infection with ATL was immediately concluded (Yoshida et al., 1982). Furthermore, the epidemiology also revealed that the cumulative risk of ATL among virus carriers in Japan was about 3–5%, indicating that most of the HTLV-1 carriers are asymptomatic throughout their life (Tajima, 1990).
Subsequently, HTLV-1 infection was also discovered to associate with tropical spastic paraparesis (TSP) in Jamaica by Guy de The and his colleagues (Gessain et al., 1985) and with HTLV-1-associated myelopathy (HAM) in Japan by Osame M et al. (1986). TSP and HAM are now identified as identical myelopathies, which have lesions in the spinal cord. Interestingly, ATL and HAM/TSP are mutually exclusive. Onset of either disease may depend on HLA types which determine the magnitude of immune response to HTLV-1, or may depend on modes of infection, mucosal exposure to HTLV-1 or primary viral infection in the peripheral blood (Barmak et al., 2003). More studies are apparently required.
Other diseases including uveitis, arthropathy and Sjogren syndrome were also discussed in possible association with HTLV-1 infection, but further study is needed for the conclusion.
Unique regulation in the viral replication
HTLV-1 has an extra sequence pX, in addition to gag, pol and env, required for viral replication. However, pX shows no sequence homology to host cell DNA, and thus is not a typical oncogene. Molecular studies on pX ultimately revealed a unique regulatory system essential for HTLV-1 replication. In brief, pX encodes three proteins, Tax, Rex and p21, in overlapping reading frames. Tax is a transcriptional activator of the viral genome (Kiyokawa et al., 1984; Sodroski et al., 1984; Fujisawa et al., 1985) and Rex is a splicing suppressor of the viral transcripts (Kiyokawa et al., 1985; Seiki et al., 1985). The combination of these two proteins regulates sequential and transitory viral expression (Hidaka et al., 1988; Seiki et al., 1988).
As summarized in Figure 2, an initial HTLV-1 transcript is fully spliced into pX mRNA which encodes a protein termed p40-Tax and p27-Rex. Tax trans-activates the transcription of the viral genome; thus viral expression is potently enhanced. Next, p27-Rex, which is encoded by the same pX mRNA in an alternate reading frame, accumulates and suppresses splicing of the viral transcripts. As a consequence, unspliced gag-pol-env and env mRNAs are expressed, and the viral structural proteins are produced. The suppression of the viral RNA splicing reduces the level of fully spliced pX mRNA that codes for the trans-activator Tax, and thus results in downregulation of viral gene expression. Accordingly, the viral gene expression is efficient at the initial stage of infection/gene expression and then is moderated quickly before host immunologic surveillance could be effectively triggered against infected cells. Such feedback regulation through the viral products was uniquely described first for HTLV-1.
Additional new accessory proteins were identified including p12, p35 and some others, which were encoded by alternative splicing of the viral message (Koralnik et al., 1992). These genes seemed not essential for the viral replication in vitro, but might be significant in pathogenesis. Physiologies of these proteins are still under investigation.
Etiology for ATL
The most important aspect of the new retroviral isolation was not just novelty but an etiology for a human leukemia. However, etiological proof for a human disease is generally not easy unless an animal model is available. Here, molecular biology provided effective tools. Association of HTLV-1 with ATL was quickly demonstrated after viral identification by preceding findings on unique antibodies in ATL as introduced above.
The most critical question thereafter was whether ‘close association of HTLV-1 with ATL’ reflects its causative role or whether the virus was just a passenger. The nature of provirus integration of the retroviruses provided a critical tool for the discrimination. The retroviral genomes are generally reverse transcribed into provirus DNA, and the proviral genomes are integrated into host cell DNA at random sites. Since a tumor originates from unlimited expansion of a single malignant cell, the site for the proviral integration into tumor cells would be uniform in individuals if the retroviral infection plays a causative role; but if the virus fortuitously infects leukemic cells, then the integration sites would be random. Southern blot analysis of patients' leukemic cell DNA clearly indicated clonal integration in each patient revealing two distinct bands with cellular franking sequences. This finding clearly supported the virus playing a causative role in ATL (Yoshida et al., 1984). Virtually all ATL cases were clonally infected leukemic cells; therefore, the conclusion for a ‘causative role’ became generally accepted. As controls, the sites for the integration in viral carriers are random except only in a few cases which show clonal integration with higher vial burden.
Molecular pathogenesis of ATL
Animal retroviruses induce tumors basically through one of two mechanisms: either activation through a ‘viral oncogene’ or ‘insertional activation’ of a cellular gene such as a proto-oncogene (Teich et al., 1985). Neither mechanism was supported by early studies on HTLV-1 and ATL cells (Seiki et al., 1984). Naturally most attention has been focused on the Tax protein because it was implicated as a potent transcriptional activator for the viral gene expression. In fact, Tax was reported to transform Rat-1 cells, a rat fibroblastic cell line (Pozzatti et al., 1990; Tanaka et al., 1990), and to induce tumors in transgenic mice (Nerenberg et al., 1987).
Pleiotropic functions of Tax
Transcriptional activation and repression
Tax was a potent transcriptional activator for the viral gene expression. So, the natural expectation was that it would activate cellular gene(s). The first indication for the activation of cellular gene was very intriguing (Inoue et al., 1986). It was already known that ATL cells express a high level of IL-2 receptor (Uchiyama et al., 1985; α and β subunits for the receptor was not yet known), which is essential for T-cell growth; this suggested that the constitutive expression of IL-2R might support abnormal cell growth of leukemic cells. Tax expression vector was found to robustly activate a reporter construct consisting of IL-2R promoter and reporter gene, and also induce expression of cellular IL-2R gene in a certain T-cell line through activation of NF-κB. This was exciting evidence which demonstrated how viral infection induces and supports abnormal growth of ATL cells, although it is realized today that the activation of IL-2Rα may not be sufficient to explain the tumor states. This finding consequently triggered a huge numbers of investigators to uncover cellular gene targets, which might explain abnormal cellular phenotypes. Today, many target genes have been identified including those involved in cell cycle, lymphokine/receptor systems, apoptosis and differentiation. Thus, a mechanistic summary of Tax action is that it binds enhancer-binding proteins such as NF-κB and CREB and transcriptional cofactors such as CBP, and that it bridges several of these factors to accelerate complex formation on enhancer–promoter DNA, and thereby enhances transcriptional initiation. When Tax cannot bind either partner, Tax will repress the transcription. The latter case seems to be operating in Tax-induced repression of transcription of DNA polymerase β (Jeang et al., 1990). This unexpected latter effect raises the hypothesis that Tax-expressing cells would suffer from insufficient DNA repair, and thus accumulate mutations easily.
Inhibition of tumor-suppressor proteins
Study on protein–protein interaction focusing on a specific domain for understanding transcriptional activation (Hirai et al., 1994) opened up a new category of Tax function that targets tumor-suppressor proteins. Tax binds to and inactivates p16INK4 and p15INK4, which were known as tumor-suppressor proteins that keep the Rb signaling pathway active (Suzuki et al., 1996). As a result, Cdk4 is activated and phosphorylates and inactivates the Rb protein. Inactivated Rb releases E2F, and released E2F activates transcription of various genes required for DNA replication, thus compelling cells into a G1–S transition. The observation holds significant implication because it indicates that Tax abrogates cell cycle arrest in G1 phase by inactivating p16INK4 and p15INK4, which are otherwise frequently deleted or hypermethylated in many human tumors.
Tax was also found to bind to another tumor-suppressor protein, hDlg, and interfere with its interaction with another tumor-suppressor protein APC (Suzuki et al., 1999). Through this binding, Tax affects the Wnt signaling pathway which controls cell proliferation.
Suppression of apoptosis, DNA repair and cell cycle checkpoint
T-cell lines infected with HTLV-1 are resistant to apoptotic signals (Copeland et al., 1994). The mechanisms for this resistance were proposed to be activation of NF-κB, transcriptional activation of Bcl-X gene and repression of Bax gene. Most likely all or some of these pathways cooperate together in infected T cells.
Accumulation of mutations was enhanced by Tax expression in an indicator gene exogenously inserted in rat cell line (Miyake et al., 1999). This phenotype may involve reduction of DNA repair activity and/or suppression of checkpoint activity in infected cells. In this respect, Tax represses transcription of DNA polymerase β (Jeang et al., 1990) and inhibits DNA topoisomerase I (Suzuki et al., 2000), reducing the DNA repair activity. Furthermore, Tax binds to hsMad1 and suppresses G2/M cell cycle checkpoint (Jin et al., 1998). The latter function of Tax accelerates cells into and through the mitotic phase without checking for chromosomal abnormalities. This property of Tax prevents the cell from either repairing chromosomal abnormalities or sending unrepaired cells into apoptosis.
Pleiotropic functions of Tax cooperate as tumor promoter
After uncovering so many divergent functions of Tax, one can see a common directional theme on cellular proliferation. That is, each of the different categories of affected function in infected cells is directed towards promotion of accelerated cell cycle and proliferation. Therefore, it can be proposed that these multiple functions of Tax cooperate together for malignant transformation of HTLV-1-infected cells. Taken together, Tax induces abnormal cell growth by activating growth-promoting genes, by repressing growth-suppressing genes and also by inhibiting tumor suppressor proteins. During induced abnormal cell proliferation, Tax also suppresses DNA repair capacity and bypasses cell cycle checkpoint by inactivation of checkpoint function and thus enhances accumulation of mutations. Furthermore, Tax inhibits apoptotic cell death even in cells with abnormally damaged DNAs. Over repeated cell cycles, some cells will fortuitously accumulate the right combination of mutated DNA, which would trigger transformation and progress into malignant conversion. It can also be said that pleiotropism of Tax might correspond to the multiple steps required for tumorigenesis in general cancers, which take place sequentially and at a low rate of incidence.
Is the laboratory bench far from the clinical bed?
Molecular biology of Tax is mainly based on studies in vitro, which indicate that Tax targets various cellular genes frequently altered in spontaneous tumor cells. This, therefore, strongly suggests that Tax would play a central role in induction of ATL. However, it is generally known that expression of many genes is differentially controlled in cultured cells and in vivo, although the significance of this finding for transformation is not well understood. As a result of the limitations of extrapolating from ex vivo studies, the pathogenic roles of Tax protein for in vivo leukemia remain to be carefully established.
Leukemic cells without Tax expression
It is reported that a vast majority (over 95%) of ATL cells in vivo are absolutely negative for HTLV-1 expression (Kinoshita et al., 1989). However, most of them express some cellular genes targeted by Tax such as IL-2Rα. This indicates that in vivo expression of IL-2Rα does not continuously require Tax. The molecular mechanisms to explain such phenomena are not well understood. Chromosomal translocation at the T-cell receptor region was identified in some cases of ATL (Isobe et al., 1990), but such isolated data are not sufficient to establish a general mechanism. We need to identify one or more abnormalities fixed in leukemic cells that functionally mimic the role of Tax in activating the expression of a multitude of genes.
Tax is not sufficient to dictate gene expression in vivo
The viral expression in vivo is also mysterious in another aspect. T-cell lines infected with HTLV-1 were established, and many of them express the viral genome continuously at high levels. On the other hand, regardless of whether the cell is leukemic or nonleukemic, HTLV-1 is expressed in vivo at extremely low levels and in many cases can be detected only by PCR. However, once the cells are harvested ex vivo, viral expression is frequently and quickly switched on. The mechanism as to how viral expression is maintained in vivo in a mostly latent form is not understood. The elucidation of such mechanism may provide us with a possible clue as to how to limit viral expression, ultimately enabling prevention of pathogenesis.
Targeted genes are oriented for proliferation
Most of the genes so far identified as the targets of Tax are those for promoting cell cycle and proliferation. Abnormality of these genes might explain abnormal growth of infected cells. However, clonal expansion of leukemic cells is not explained by these genes. No one knows what kind of mechanism is involved additionally. Also, we can ask whether unlimited proliferation is equal to ‘malignancy’. This is a very basic question on the nature of tumors and can also be asked about general tumorigenesis.
Expected in 1981 and answered in 2005
Understanding the leukemogenesis
HTLV-1 was characterized as a causative agent of ATL, but the virus has no oncogene and does not function through promoter insertion to enhance cellular proto-oncogene. Consequently, we postulated a new mechanism for tumorigenesis in ATL. However, what subsequent HTLV-1 study discovered after extensive molecular investigation were mechanisms similar to those used by DNA tumor viruses. Molecules and signaling pathways targeted by Tax were and include tumor-suppressor proteins such as p16ink4/Rb, p53, APC, and were similar to those targeted by transforming proteins of DNA tumor viruses, T antigen of SV40, E6/E7 of HPV and E1a of adenovirus. Furthermore, as generally accepted, the actions of viral transforming genes are also shared with the activation mechanisms identified in spontaneously arising or genetically induced tumors in humans. These similarities between cellular alterations induced HTLV-1, DNA tumor viruses and in spontaneous tumors strongly suggest that general transforming mechanisms are operative in ATL induction.
Prevention of HTLV-1 infection
Upon the identification of a new retrovirus, virologists and oncologists excitedly harbored the expectation that we would be able to prevent virus infection and replication and thus ultimately prevent the development of specific leukemia. This goal has been partly achieved by elucidating and preventing the infection pathways. HTLV-1 is mainly transmitted from (1) a mother to her child (Sugiyama et al., 1986), (2) husband to wife (Tajima et al., 1986) and (3) donor to recipient of blood transfusion (Okochi et al., 1984). Viral transmission is thought to require the transfer of live infected T lymphocytes, that is, T cells in breast milk, T cells in semen and fresh T cells in blood carriers of HTLV-1 proviruses. Therefore, cessation of breast milk is now applied to prevent HTLV-1 infection from an infected mother to her child (Hino et al., 1994). In Japanese endemic areas, pregnant mothers are mostly examined for HTLV-1 antibodies, and once the mother is identified to be infected, bottlefeeding is strongly suggested. The trial is now working quite well and is rather successful in preventing HTLV-1 infection in the next generation. However, there is a concern whether this strategy can be the best choice in developing regions, where breast milk is valuable in transferring a mother's immunity to her children to protect against various infectious diseases.
To combat the transmission through blood transfusion, sero-positive bloods are excluded from the blood bank and infection through this pathway is now almost completely gone in Japan. Vaccine development for preventing new infection has been challenging at the basic study level, and has achieved some success in animal studies, but no vaccine has yet come to fruition for protecting humans.
Prevention of pathogenesis
A total of 12 million individuals are estimated to be HTLV-1 carriers throughout the world. Therefore, development of any strategy to halt viral replication and/or viral gene expression would save a half million carriers from death by ATL. Although molecular virology has progressed rather extensively, no possible strategy for this has been effective. HTLV-1 provirus replicates very poorly through free virions in vivo, but the provirus is efficiently amplified through abnormal replication of infected T cells whose DNA carries the proviral genomes. Therefore, the task of preventing pathogenesis remains extremely difficult.
Treatment of ATL
Acute ATL is aggressive T-cell leukemia and no effective treatment is available. Unfortunately, even after extensive advances in the molecular biology of HTLV-1 and ATL, no effective treatment for ATL is available. Although some therapies such as monoclonal antibody and combination therapies with available anticancer drugs were reported to be transiently effective, no convincing therapeutic benefits have been established. Long-term strategic challenges, such as genome-wide molecular profiling for understanding the landscape of ATL cells in vivo and molecular targeting strategy for identifying new drugs, are significant future objectives.
HTLV-1 offers valuable clues for in vivo study of human cancers
During the quarter century since the discovery of ATL, the HTLV/ATL biological model has provided great opportunities for scientists to study tumorigenesis directly in humans. Points of particular note are (1) it is efficient and reliable to identify high-risk individuals for ATL, (2) it is possible to follow malignant steps of smoldering, chronic and acute phase of ATL, (3) it is possible to obtain samples repeatedly from specific individuals, and (4) it is easy to isolate a single malignant cell intact. In view of these advantages, it should be possible for scientists to answer key questions in human tumorigenesis. Even though the ATL patient population is small, from this small group valuable understanding of information applicable to general tumorigenesis of other cancers can be accrued. The recent years have seen the total genome sequencing for humans and the advent of molecular and genomic technologies such as genome-wide molecular profiling. Unanswered questions and prematurely derived conclusions may merit revisiting with new modern technologies.