Madeleine Duc Dodon

Critical role of hnRNP A1 in HTLV-1 replication in human transformed T lymphocytes

BackgroundIn this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in viral gene expression in T lymphocytes transformed by HTLV-1.ResultsWe have previously observed that hnRNP A1 (A1) down-modulates the post transcriptional activity of Rex protein of HTLV-1. Here, we tested whether the ectopic expression of a dominant negative mutant (NLS-A1-HA) defective in shuttling activity or knockdown of the hnRNPA1 gene using RNA interference could inhibit Rex-mediated export of viral mRNAs in HTLV-1 producing C91PL T-cells. We show that the expression of NLS-A1-HA does not modify the export of Rex-dependent viral mRNAs. Conversely, inhibiting A1 expression in C91PL cells by RNA interference provoked an increase in the Rex-dependent export of unspliced and singly spliced mRNAs. Surprisingly, we also observed a significant increase in proviral transcription and an accumulation of unspliced mRNAs, suggesting that the splicing process was affected. Finally, A1 knockdown in C91PL cells increased viral production by these cells. Thus, hnRNP A1 is implicated in the modulation of the level of HTLV-1 gene expression in T cells transformed by this human retrovirus.ConclusionsThese observations provide an insight into a new cellular control of HTLV-1 replication and suggest that hnRNP A1 is likely part of the regulatory mechanisms of the life cycle of this human retrovirus in T cells.

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.

A balanced transcription between telomerase and the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 in resting, activated, HTLV-1-transformed and Tax-expressing human T lymphocytes

BackgroundThe functional state of human telomeres is controlled by telomerase and by a protein complex named shelterin, including the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 involved in telomere capping functions. The expression of hTERT, encoding the catalytic subunit of telomerase, plays a crucial role in the control of lymphocyte proliferation by maintaining telomere homeostasis. It has been previously found that hTERT activity is down-regulated by the human T cell leukaemia virus type 1 (HTLV-1) Tax protein in HTLV-1 transformed T lymphocytes. In this study, we have examined the effects of Tax expression on the transcriptional profile of telomerase and of shelterin in human T lymphocytes.ResultsWe first provide evidence that the up-regulation of hTERT transcription in activated CD4+ T lymphocytes is associated with a down-regulation of that of TERF1, TERF2 and POT1 genes. Next, the down-regulation of hTERT transcription by Tax in HTLV-1 transformed or in Tax-expressing T lymphocytes is found to correlate with a significant increase of TRF2 and/or Pot1 mRNAs. Finally, ectopic expression of hTERT in one HTLV-1 T cell line induces a marked decrease in the transcription of the POT1 gene. Collectively, these observations predict that the increased transcriptional expression of shelterin genes is minimizing the impact on telomere instability induced by the down-regulation of hTERT by Tax.ConclusionThese findings support the notion that Tax, telomerase and shelterin play a critical role in the proliferation of HTLV-1 transformed T lymphocytes.

Overexpression of caveolin-1 in adult T-cell leukemia

Caveolin-1 is implicated in the regulation of signal pathways. Adult T-cell leukemia (ATL) is a T-cell malignancy causatively associated with human T-cell leukemia virus type 1 (HTLV-1). To determine the role of caveolin-1 in leukemogenesis, we examined caveolin-1 expression levels in HTLV-1-infected T-cell lines and ATL cells. These cells expressed high levels of caveolin-1 compared with uninfected T-cell lines and normal peripheral blood mononuclear cells (PBMCs). Caveolin-1-positive ATL cells were detected in ATL lymph nodes and skin lesions, and caveolin-1 was also detected in the plasma of patients with ATL. Infection of a human T-cell line, an epithelial cell line, and normal PBMCs with HTLV-1 induced caveolin-1 expression. The viral protein Tax transcriptionally activated caveolin-1 gene through nuclear factor-kappaB and cAMP response element binding protein signal pathways. HTLV-1-infected T-cell lines, and ATL cells are known to be resistant to transforming growth factor beta (TGF-beta)-induced growth inhibition. Caveolin-1 was colocalized with TGF-beta type I receptor in HTLV-1-infected T-cell lines and suppressed TGF-beta signaling. Caveolin-1 knockdown in an HTLV-1-infected T-cell line exhibited susceptibility to TGF-beta. Thus, we describe a new function for Tax, repression of TGF-beta signaling through caveolin-1 expression, which may play a critical role in ATL leukemogenesis.

From Immunodeficiency to Humanization: The Contribution of Mouse Models to Explore HTLV-1 Leukemogenesis

The first discovered human retrovirus, Human T-Lymphotropic Virus type 1 (HTLV-1), is responsible for an aggressive form of T cell leukemia/lymphoma. Mouse models recapitulating the leukemogenesis process have been helpful for understanding the mechanisms underlying the pathogenesis of this retroviral-induced disease. This review will focus on the recent advances in the generation of immunodeficient and human hemato-lymphoid system mice with a particular emphasis on the development of mouse models for HTLV-1-mediated pathogenesis, their present limitations and the challenges yet to be addressed.

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-1–specific adaptive immune responses.

HBZ impedes the Menin function and up-regulates the transcription of the hTERT gene in leukemic cells

Leukemic cells from Adult T-cell leukemia (ATL) patients display elevated telomerase activity, resulting mainly from transcriptional up-regulation of the human telomerase catalytic subunit (hTERT). We have previously shown that HBZ (HTLV-1 bZip) protein cooperates with JunD transcription factor to enhance hTERT expression after JunD anchoring to Sp1 bound to Sp1 sites within the hTERT proximal promoter. In normal somatic cells, telomerase expression is negatively regulated by tumor suppressor gene products, such as Menin, encoded by the multiple endocrine neoplasia type 1 (MEN-1) gene. Interestingly, the interaction of Menin with JunD has been shown to repress its transcriptional activity. We report here that, in HBZ-expressing cells, Menin and HBZ exert opposite effects on JunD-mediated regulation of hTERT transcription. Chromatin immuno-precipitation as well as functional assays demonstrate that this antagonism is linked to the recruitment of p300 by HBZ and HDACs by Menin. Furthermore, knock-down of Menin in the ATL Tl-Om1 (only expressing HBZ) cells results in an increase of hTERT expression and of telomerase activity, whereas a knock-down of HBZ exerts opposite effects. Interestingly, primary leukemic cells isolated from ATL patients that express high amounts of hTERT transcripts, are also characterized by an elevated expression of HBZ and MEN-1 genes. Thus, in leukemic cells, HBZ behaves as a key factor impeding the Menin function and sustaining hTERT transcription. These findings underline the critical role of HBZ as a tumor-promoting protein during the development of the HTLV-1-induced leukemogenic process.

HTLV-1 propels thymic human T cell development in “human immune system” Rag2-/- IL-2R γc-/- Mice

Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously shown that HTLV-1 (Human T cell Leukemia Virus type 1) is able not only to infect immature thymocytes in vitro but also, through Tax expression, to alter the β-selection checkpoint critical for early T cell development. To further clarify the role of the natural HTLV-1 infection on human T-cell development, we developed an in vivo model by transplanting immunocompromised Rag2-/-γc-/- newborn mice with human cord blood CD34+ cells to obtain Human Immune System (HIS) mice. In these mice the development of human T cells in the thymus is fully developed within two months after human cell transplantation. Lethally irradiated HTLV-1 producing cells were then injected into these HIS mice. Herein we observed in the thymus of the infected animals an enlarged population of mature T cells when compared with the mock-infected mice. Furthermore, we noted an increased number of CD4+ cells expressing CD25. Infected animals also developed, several weeks after the infection, pathological features such as splenomegaly, adenopathy, thymomas, lymphomas and leukemias in which predominate human T cells, with a large proportion of CD25+ activated cells. Tax expression especially in the lymphomas and thymomas correlated with an up-regulation of NF-κB regulated genes. Altogether, these results underline that this HIS Rag2-/-γc-/- model might be of great interest to study the leukemogenic process induced by HTLV-1 as well as to validate new therapeutic approaches of ATL.

Aberrant expression of the transcription factor Twist in adult T-cell leukemia

Adult T-cell leukemia (ATL) is a T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). Twist, a highly conserved basic helix-loop-helix transcription factor, is a newly identified oncogene. However, there are no reports on Twist expression in ATL. To define the role of Twist in leukemogenesis of ATL, we examined its expression in T-cell lines and PBMC. HTLV-1-infected T-cell lines and ATL cells expressed high levels of Twist compared with uninfected T-cell lines and normal PBMC. Immunohistochemistry showed immunostaining for Twist in ATL cells in ATL lymph nodes and skin lesions. Infection of normal PBMC with HTLV-1 induced Twist expression. Induction of the viral protein Tax in a human T-cell line led to upregulation of Twist. Tax-induced Twist expression involved the NF-kappaB and CREB signaling pathways. Twist augmented Tax-mediated HTLV-1 LTR and NF-kappaB activation. Short interfering RNA against Twist inhibited cell growth of HTLV-1-infected T-cell lines and downregulation of Twist expression in an HTLV-1-infected T-cell line inhibited the expression of Akt1, interleukin-2 receptor alpha chain, and Tax as well as the known target genes of Twist, YB-1 and Akt2. In conclusion, the results suggest that Tax-induced induction of Twist contributes to leukemogenesis of ATL.

HTLV-1-infected HIS Rag2-/-γc-/- mice, a suitable model for in vivo investigating the effects of drugs in ATL treatment?

Adult T cell Leukemia, an aggressive T-cell malignancy linked to HTLV-1 infection, is resistant to chemotherapy. Recently, promising results were obtained with the combination of arsenic trioxide, interferon alpha and zidovudine. However, the cellular and molecular mechanisms of their anti-leukemia activity remain to be investigated. To that aim, we have relied on HIS (Human Immune System) Rag2-/-γc-/- mice. We have indeed observed that when infected with HTLV-1, these mice displayed, five months later, an elevated number of human Tax-expressing T cells in the thymus, the spleen and the mesenteric lymph nodes. Some of them also developed T lymphoproliferative diseases. To determine the effects of these three drugs on the apparition of these pathological features, Rag2-/-γc-/- mice were infected with HTLV-1 and, 16 weeks after infection, daily treated with the drugs for one week and sacrificed. Untreated HIS mice infected with HTLV-1 were used as control. Treatment resulted in a significant decrease in the spleen weight as compared to untreated controls. Interestingly, we also noted a decrease of the proviral load in thymocytes as well as a drop in the number of mature activated T cells in the spleen and in lymph nodes. These data clearly indicate that the HIS mouse model provides us with the opportunity not only for the pre-clinical evaluation of therapeutic approaches against the leukemogenic process associated with HTLV-1 infection, but also for unraveling the corresponding mechanisms.