Of mice, men, and HTLV-1.

In this issue of Blood, Tezuka et al report the establishment of humanized mice infected by human T-cell leukemia virus type 1 (HTLV-1) that recapitulate adult T-cell leukemia (ATL)-like leukemic symptoms and display HTLV-1­specific adaptive immune responses.

HTLV-1 bZIP factor impedes the menin tumor suppressor and upregulates JunD-mediated transcription of the hTERT gene.

Telomerase activity in cancer cells is dependent on the transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic subunit of human telomerase. We have shown previously that HTLV-1 basic leucine zipper (HBZ), a viral regulatory protein encoded by the human retrovirus, human T-cell leukemia virus, type 1 (HTLV-1) cooperates with JunD to enhance hTERT transcription in adult T-cell leukemia (ATL) cells. Menin, the product of the tumor-suppressor MEN-1 gene, also interacts with JunD, represses its transcriptional activity and downregulates telomerase expression. The main objective of this study was to examine how menin and HBZ get involved in the regulation of hTERT transcription. In this study, we report that JunD and menin form a repressor complex of hTERT transcription in HBZ-negative cells. Conversely, in HBZ-positive cells, the formation of a JunD/HBZ/menin ternary complex and the recruitment of p300 histone acetyl transferase activity by HBZ lead to a decreased activity of the JunD-menin suppressor unit that correlates with the activation of hTERT transcription. Silencing HBZ or menin expression in ATL cells confirms that these proteins are differentially involved in telomerase regulation. These results propose that HBZ, by impeding the tumor-suppressor activity of menin, functions as a leukemogenic cofactor to upregulate gene transcription and promote JunD-mediated leukemogenesis.

What we are learning on HTLV-1 pathogenesis from animal models.

Isolated and identified more than 30 years ago, human T cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia/lymphoma, an aggressive lymphoproliferative disease of activated CD4(+) T cells, and other inflammatory disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis. A variety of animal models have contributed to the fundamental knowledge of HTLV-1 transmission, pathogenesis, and to the design of novel therapies to treat HTLV-1-associated diseases. Small animal models (rabbits, rats, and mice) as well as large animal models (monkeys) have been utilized to significantly advance characterization of the viral proteins and of virus-infected cells in the early steps of infection, as well as in the development of leukemogenic and immunopathogenic processes. Over the past two decades, the creation of new immunocompromised mouse strains that are robustly reconstituted with a functional human immune system (HIS) after being transplanted with human tissues or progenitor cells has revolutionized the in vivo investigation of viral infection and pathogenesis. Recent observations obtained in HTLV-1-infected humanized HIS mice that develop lymphomas provide the opportunity to study the evolution of the proviral clonality in human T cells present in different lymphoid organs. Current progress in the improvement of those humanized models will favor the testing of drugs and the development of targeted therapies against HTLV-1-associated diseases.

HTLV-1 propels thymic human T cell development in "human immune system" Rag2⁻/⁻ gamma c⁻/⁻ mice.

Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously demonstrated that Tax(HTLV-1) interferes with ß-selection, an important checkpoint of early thymopoiesis, indicating that human T-cell leukemia virus type 1 (HTLV-1) infection has the potential to perturb thymic human αß T-cell development. To verify that inference and to clarify the impact of HTLV-1 infection on human T-cell development, we investigated the in vivo effects of HTLV-1 infection in a "Human Immune System" (HIS) Rag2⁻/⁻γ(c)⁻/⁻ mouse model. These mice were infected with HTLV-1, at a time when the three main subpopulations of human thymocytes have been detected. In all but two inoculated mice, the HTLV-1 provirus was found integrated in thymocytes; the proviral load increased with the length of the infection period. In the HTLV-1-infected mice we observed alterations in human T-cell development, the extent of which correlated with the proviral load. Thus, in the thymus of HTLV-1-infected HIS Rag2⁻/⁻γc⁻/⁻ mice, mature single-positive (SP) CD4⁺ and CD8⁺ cells were most numerous, at the expense of immature and double-positive (DP) thymocytes. These SP cells also accumulated in the spleen. Human lymphocytes from thymus and spleen were activated, as shown by the expression of CD25: this activation was correlated with the presence of tax mRNA and with increased expression of NF-kB dependent genes such as bfl-1, an anti-apoptotic gene, in thymocytes. Finally, hepato-splenomegaly, lymphadenopathy and lymphoma/thymoma, in which Tax was detected, were observed in HTLV-1-infected mice, several months after HTLV-1 infection. These results demonstrate the potential of the HIS Rag2⁻/⁻γ(c)⁻/⁻ animal model to elucidate the initial steps of the leukemogenic process induced by HTLV-1.

Human T-cell lymphotrophic virus type I rex and p30 interactions govern the switch between virus latency and replication.

Human T-cell lymphotrophic virus type I Rex and p30 are both RNA binding regulatory proteins. Rex is a protein that interacts with a responsive element and stimulates nuclear export of incompletely spliced viral RNAs thereby increasing production of virus particles. In contrast, p30 is involved in the nuclear retention of the tax/rex mRNA leading to inhibition of virus expression and possible establishment of viral latency. How these two proteins, with apparent opposite functions, integrate in the viral replication cycle is unknown. Here, we demonstrate that Rex and p30 form ribonucleoprotein ternary complexes onto specific viral mRNA. Our results explain the selective nuclear retention of tax/rex but not other viral mRNAs by p30. Whereas p30 suppresses Rex expression, it did not affect Rex-mediated nuclear export of RNA containing the Rex response element. In contrast, Rex was able to counteract p30-mediated suppression of viral expression and restore cytoplasmic tax/rex mRNA and Tax protein expression. Together, our data demonstrate a complex regulatory mechanism of antagonizing post-transcriptional regulators evolved by human T-cell lymphotrophic virus type I to allow a vigilant control of viral gene expression.

Tax protein of human T-cell leukaemia virus type 1 induces interleukin 17 gene expression in T cells.

Tax protein of human T-cell leukaemia virus type 1 (HTLV-1) induces the expression of several cellular genes that are involved in T cell activation and proliferation. In this study, it was observed that Tax upregulated the expression of human interleukin 17 (IL17), a cytokine mainly produced by activated CD4(+) memory T cells. Indeed, IL17 mRNA was highly expressed in HTLV-1-infected T cells as well as in Tax-expressing Jurkat T cells, whereas it was not detectable in HTLV-1-negative T cell lines. The clinical relevance of these observations was further demonstrated by quantitative assessment of IL17 expression in lymphocytes isolated from one HTLV-1-infected patient. To define the transcriptional activation of the IL17 gene by Tax, the 5'-flanking region of this gene was cloned and a reporter gene analysis performed. The presence of a Tax-responsive region spanning 614 bp upstream of the initiation start site was identified, in HeLa as well as in Jurkat cells, stimulated with phorbol myristate acetate and Ca(2+) ionophore. Finally, Tax mutants were used to show that the transcriptional activation of the IL17 promoter by Tax was dependent on the CREB/ATF pathway. As IL17 upregulates the expression of several pro-inflammatory cytokines, these observations provide new insights into the involvement of the Tax protein in the pathophysiology of HTLV-1-associated inflammatory disorders.

Heterogeneous nuclear ribonucleoprotein A1 interferes with the binding of the human T cell leukemia virus type 1 rex regulatory protein to its response element.

The human T cell leukemia virus, type 1 (HTLV-1), Rex protein mediates the nuclear export of unspliced and incompletely spliced viral mRNAs. This post-transcriptional activity is dependent in part on the binding of this protein to cis-regulatory sequences termed the Rex-response element (XRE). We have proposed previously that the decreased functionality exhibited by Rex in human lymphoblastoid Jurkat T cells may be linked to alterations in the Rex/XRE interactions. The analysis of the ribonucleoprotein complexes formed between Jurkat nuclear proteins and XRE-RNA led to the identification of a 36-kDa protein as heterogeneous nuclear ribonucleoprotein (hnRNP) A1. In vitro binding assays revealed that hnRNP A1 proteins were found to interfere with the binding of Rex to XRE, whereas nuclear extracts depleted of these proteins were unable to disrupt Rex-XRE complexes. Furthermore, A1 proteins from Jurkat cells were acting in a concentration-dependent manner, suggesting that the amount of these RNA-binding proteins is a critical parameter in controlling Rex activity. We indeed observed a lower level of hnRNP A1 in in vitro HTLV-1-transformed virus-producing T cells than that detected in Jurkat cells. Likewise, overexpression of hnRNP A1 proteins in 293T cells and in Jurkat cells led to a decrease in the expression of a reporter gene dependent on Rex/XRE interactions. Such a decrease was not observed when the expression of the same reporter gene by cells overexpressing hnRNP A1 was dependent on the interactions of human immunodeficiency virus Rev protein with the Rev-response element. These findings indicate that hnRNP A1 by competing with Rex for the formation of REX-XRE complexes is specifically involved in the modulation of the post-transcriptional activity of Rex.