Search

Background: Reprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects, HTLV-1 qualitatively modifies the pattern of cellular gene expression., Results: Exon expression analysis shows that patients' untransformed and malignant HTLV-1(+) CD4(+) T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involved in cancer and cell cycle. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patterns of 3977 exons that discriminate uninfected, infected, and transformed CD4(+) T-cells. Furthermore, untransformed infected CD4+ clones and ATLL samples shared 486 exon modifications distributed in 320 genes, thereby indicating a role of AEUs in HTLV-1 leukemogenesis. Exposing cells to splicing modulators revealed that Sudemycin E reduces cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provide news targets for treating ATLL., Conclusions: Taken together, these data reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets.

Viral expression varies widely between untransformed HTLV-1 positive clones derived from infected individuals without malignancy. Here we show that, in the absence of malignancy, 68% of HTLV-1 positive clones carry deleted (10%) or methylated (58%) 5' LTR. These changes were found to contribute to the fluctuation of viral expression between clones. 5' LTR deletions strongly impaired tax expression and thereby clonal expansion, telomere homeostasis, and genetic instability. The effects of CpG methylation on viral transcription were qualitatively and quantitatively different from those of LTR deletions and preserved the preleukemic features of HTLV-1(+)CD4(+) clones. 5' LTR methylation varied not only between clones but also between cells belonging to the same clones, between distinct integrated sequences within the same cells, and between CpG dyads along the 5' LTR. Such distributions suggest that a dynamic methylation spectrum might help sustain persistent infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The Human T-cell Leukemia Virus type 1 (HTLV-1) is the causative agent of several inflammatory diseases, including HTLV-1-associated inflammatory myopathies (HAIM). Little is known about the virological and immunological characteristics of this viral disease., Objectives: To characterize the histological and virological features of HAIM patients, in order to better understand the pathogenetic mechanisms and unravel new biological markers of this disease., Study Design: We conducted a retrospective study on 13 patients with HAIM, based on blood and muscle samples. We included blood samples from HTLV-1-infected individuals without myopathy as controls. Muscle biopsies were used for a broad immunohistological evaluation of tissue damage and inflammation, as well as identification of infected cells through in situ hybridization. DNA extracted from patients' PBMC was used to identify the virus genotype by sequencing and to assess the proviral load by quantitative PCR. Anti-viral antibodies in plasma samples were titrated by indirect immunofluorescence., Results: Patients originate from HTLV-1 endemic areas, the West Indies and West Africa. Histological alterations and inflammation in patients muscles were mostly moderate, with classical features of idiopathic myositis and rare HTLV-1-infected infiltrating cells. In all patients, HTLV-1 belonged to the A subtype, transcontinental subgroup. Anti-HTLV-1 antibodies titers were high, but the proviral load was not elevated compared to asymptomatic HTLV-1 carriers., Conclusion: We show here that muscle inflammation is moderate in HAIM, and accompanied by a low HTLV-1 proviral load, suggesting that the pathogenetic events do not exactly mirror those of other HTLV-1-associated inflammatory diseases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Upon antiretroviral therapy (ART) human immunodeficiency virus (HIV)/human T-cell lymphotropic virus type 1 (HTLV-1) co-infected individuals frequently develop neurological disorders through hitherto unknown mechanisms. Here, we show that effective anti-HIV ART increases HTLV-1 proviral load through a polyclonal integration pattern of HTLV-1 in both CD4(+) and CD8(+) T-cell subsets that is reminiscent of that typically associated with HTLV-1-related inflammatory conditions. These data indicate that preventing ART-triggered clonal expansion of HTLV-1-infected cells in co-infected individuals deserves investigation.

Untransformed HTLV-1 positive CD4(+) cells from infected individuals are selected for expressing tax and displaying morphological features consistent with telomere dysfunctions. We show that in resting HTLV-1 positive CD4(+) cells cloned from patients, hTERT expression parallels tax expression and cell cycling. Upon activation, these cells dramatically augment tax expression, whereas their increase in telomerase activity is about 20 times lower than that of their uninfected counterpart. Activated HTLV-1 positive CD4(+) but not uninfected CD4(+) or CD8(+) clones also repress the transcription of TRF1, TPP1, TANK1, POT1, DNA-PKc and Ku80. Both infected and uninfected lymphocytes from infected individuals shared common telomere gene deregulations toward a pattern consistent with premature senescence. ATLL cells displayed the highest telomerase activity (TA) whereas recovered a telomere gene transcriptome close to that of normal CD4(+) cells. In conclusion HTLV-1-dependent telomere modulations seem involved in clonal expansion, immunosuppression, tumor initiation and progression., (Copyright © 2012 UICC.)

Recruitment of virus-infected T lymphocytes into the CNS is an essential step in the development of virus-associated neuroinflammatory diseases, notably myelopathy induced by retrovirus human T leukemia virus-1 (HTLV-1). We have recently shown the key role of collapsin response mediator protein 2 (CRMP2), a phosphoprotein involved in cytoskeleton rearrangement, in the control of human lymphocyte migration and in brain targeting in animal models of virus-induced neuroinflammation. Using lymphocytes cloned from infected patients and chronically infected T cells, we found that HTLV-1 affects CRMP2 activity, resulting in an increased migratory potential. Elevated CRMP2 expression accompanies a higher phosphorylation level of CRMP2 and its more pronounced adhesion to tubulin and actin. CRMP2 forms, a full length and a shorter, cleaved one, are also affected. Tax transfection and extinction strategies show the involvement of this viral protein in enhanced full-length and active CRMP2, resulting in prominent migratory rate. A role for other viral proteins in CRMP2 phosphorylation is suspected. Full-length CRMP2 confers a migratory advantage possibly by preempting the negative effect of short CRMP2 we observe on T lymphocyte migration. In addition, HTLV-1-induced migration seems, in part, supported by the ability of infected cell to increase the proteosomal degradation of short CRMP2. Finally, gene expression in CD69(+) cells selected from patients suggests that HTLV-1 has the capacity to influence the CRMP2/PI3K/Akt axis thus to positively control cytoskeleton organization and lymphocyte migration. Our data provide an additional clue to understanding the infiltration of HTLV-1-infected lymphocytes into various tissues and suggest that the regulation of CRMP2 activity by virus infection is a novel aspect of neuroinflammation.

Here we investigate the mechanisms by which HTLV-1 infection prevents the cell death of CD8(+) T cells in vivo. We show that upon natural infection, cloned CD8(+) but not CD4(+) cells from patients without malignancy become resistant to Fas-mediated cell death and acquire an antiapoptotic transcriptome that includes the overexpression of cIAP-2 and c-FLIP(L). CD8(+) lymphocyte-restricted cIAP-2 overexpression correlates with resistance to Fas-mediated apoptosis and depends on tax expression via NF-KappaB. In contrast, in the same CD8(+) cells, the HTLV-1-dependent overexpression of c-FLIP(L) does not correlate with resistance to Fas-mediated cell death nor with tax expression. In the present model, infected CD8(+) clones are the only cell subtype in which cIAP-2 expression correlates with resistance to cell death. These results support a role for Tax-dependent cIAP-2 expression in preventing the death of naturally infected CD8(+) cells and thereby in their clonal expansion in vivo., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Adult T cell leukemia results from the malignant transformation of a CD4+ lymphoid clone carrying an integrated HTLV-1 provirus that has undergone several oncogenic events over a 30-60 year period of persistent clonal expansion. Both CD4+ and CD8+ lymphocytes are infected in vivo; their expansion relies on CD4+ cell cycling and on the prevention of CD8+ cell death. Cloned infected CD4+ but not CD8+ T cells from patients without malignancy also add up nuclear and mitotic defects typical of genetic instability related to the expression of the virus-encoded oncogene tax. HTLV-1 expression is cancer-prone in vitro, but in vivo numerous selection forces act to maintain T cell homeostasis and are possibly involved in clonal selection., Results: Here we demonstrate that the HTLV-1 associated CD4+ preleukemic phenotype and the specific patterns of CD4+ and CD8+ clonal expansion are in vivo selected processes. By comparing the effects of recent (1 month) experimental infections performed in vitro and those observed in cloned T cells from patients infected for >6-26 years, we found that in chronically HTLV-1 infected individuals, HTLV-1 positive clones are selected for tax expression. In vivo, infected CD4+ cells are positively selected for cell cycling whereas infected CD8+ cells and uninfected CD4+ cells are negatively selected for the same processes. In contrast, the known HTLV-1-dependent prevention of CD8+ T cell death pertains to both in vivo and in vitro infected cells., Conclusions: Therefore, virus-cell interactions alone are not sufficient to initiate early leukemogenesis in vivo.

As other deltaretroviruses HTLV-1 replication in vivo includes a first short step of reverse transcription that is followed by the persistent clonal expansion of infected cells. In vivo these cells include the CD4+ and CD8+ lymphocytes yet the virus induces adult T cell leukemia/lymphoma (ATLL) that is regularly of the CD4+ phenotype. Cloned infected cells from individuals without malignancy possess a dramatic increase in spontaneous proliferation, which predominated with CD8+ lymphocytes and depends on the amount of tax mRNA. In fact, the clonal expansion of HTLV-1 positive CD8+ and CD4+ lymphocytes relies on two distinct mechanisms: infection prevented cell death in the former whereas recruiting the latter into the cell cycle. Furthermore infected tax-expressing CD4+ lymphocytes cumulate cellular defects characteristic of genetic instability. Therefore, HTLV-1 infection establishes a preleukemic phenotype that is restricted to CD4+ infected clones. Investigating the mechanisms underlying apoptosis, cell cycling and DNA repair in cloned cells from naturally infected individuals will permit to deciphering the molecular pathogenesis of HTLV-1 infection.

The replication of the retrovirus human T-cell leukemia virus type 1 (HTLV-1) is linked to the development of lymphoid malignancies and inflammatory diseases. Data from in vitro, ex vivo, and in vivo studies have revealed that no specific treatment can prevent or block HTLV-1 replication and therefore that there is no therapy for the prevention and/or treatment of HTLV-1-associated diseases in infected individuals. HTLV-1 and human immunodeficiency virus type 1 (HIV-1) integrases, the enzymes that specifically catalyze the integration of these retroviruses in host cell DNA, share important structural properties, suggesting that compounds that inhibit HIV-1 integration could also inhibit HTLV-1 integration. We developed quantitative assays to test, in vitro and ex vivo, the efficiencies of styrylquinolines and diketo acids, the two main classes of HIV-1 integrase inhibitors. The compounds were tested in vitro in an HTLV-1 strand-transfer reaction and ex vivo by infection of fresh peripheral blood lymphocytes with lethally irradiated HTLV-1-positive cells. In vitro, four styrylquinoline compounds and two diketo acid compounds significantly inhibited HTLV-1 integration in a dose-dependent manner. All compounds active in vitro decreased cell proliferation ex vivo, although at low concentrations; they also dramatically decreased both normalized proviral loads and the number of integration events during experimental ex vivo primary infection. Accordingly, diketo acids and styrylquinolines are the first drugs that produce a specific negative effect on HTLV-1 replication in vitro and ex vivo, suggesting their potential efficiency for the prevention and treatment of HTLV-1-associated diseases.

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated adult T-cell leukemia/lymphoma (ATLL) has a poor prognosis owing to its intrinsic resistance to chemotherapy. Although zidovudine (AZT) and alpha interferon (IFN-alpha) give rise to some response and improve the prognosis of ATLL, alternative therapies are needed. Arsenic trioxide (As(2)O(3)) has been shown to synergize with IFN-alpha in arresting cell growth and inducing apoptosis of ATLL cells in vitro. In this study, we evaluated the toxicity and the efficacy of this combined treatment in HTLV-1-infected squirrel monkeys (Saimiri sciureus) and HTLV-1 infected cell lines derived therefrom. We first show that treatment with As(2)O(3) and IFN-alpha can induce growth arrest in HTLV-1-transformed monkey T-cell lines in vitro. We then show that treatment of squirrel monkeys with As(2)O(3) in vivo is highly toxic at 0.9 or 0.3mg/day but not at 0.14mg/day for up to 2 weeks. Although the combination of As(2)O(3) and IFN-alpha did not affect significantly the HTLV-1 proviral load in infected monkeys, it reduced the absolute numbers of CD3(+), CD4(+) and CD8(+) cells during treatment, with a significant reduction in the total number of circulating HTLV-1 flower cells in the infected monkeys with chronic ATLL-like disease.

Human T cell leukemia virus type 1 (HTLV-1) infects both CD4+ and CD8+ lymphocytes, yet it induces adult T cell leukemia/lymphoma (ATLL) that is regularly of the CD4+ phenotype. Here we show that in vivo infected CD4+ and CD8+ T cells displayed similar patterns of clonal expansion in carriers without malignancy. Cloned infected cells from individuals without malignancy had a dramatic increase in spontaneous proliferation, which predominated in CD8+ lymphocytes and depended on the amount of tax mRNA. In fact, the clonal expansion of HTLV-1-positive CD8+ and CD4+ lymphocytes relied on 2 distinct mechanisms--infection prevented cell death in the former while recruiting the latter into the cell cycle. Cell cycling, but not apoptosis, depended on the level of viral-encoded tax expression. Infected tax-expressing CD4+ lymphocytes accumulated cellular defects characteristic of genetic instability. Therefore, HTLV-1 infection establishes a preleukemic phenotype that is restricted to CD4+ infected clones.

Background: Antisense transcription in retroviruses has been suggested for both HIV-1 and HTLV-I, although the existence and coding potential of these transcripts remain controversial. Thorough characterization is required to demonstrate the existence of these transcripts and gain insight into their role in retrovirus biology., Results: This report provides the first complete characterization of an antisense retroviral transcript that encodes the previously described HTLV-I HBZ protein. In this study, we show that HBZ-encoding transcripts initiate in the 3' long terminal repeat (LTR) at several positions and consist of two alternatively spliced variants (SP1 and SP2). Expression of the most abundant HBZ spliced variant (SP1) could be detected in different HTLV-I-infected cell lines and importantly in cellular clones isolated from HTLV-I-infected patients. Polyadenylation of HBZ RNA occurred at a distance of 1450 nucleotides downstream of the HBZ stop codon in close proximity of a typical polyA signal. We have also determined that translation mostly initiates from the first exon located in the 3' LTR and that the HBZ isoform produced from the SP1 spliced variant demonstrated inhibition of Tax and c-Jun-dependent transcriptional activation., Conclusion: These results conclusively demonstrate the existence of antisense transcription in retroviruses, which likely plays a role in HTLV-I-associated pathogenesis through HBZ protein synthesis.

Understanding cell dynamics in animal models have implications for therapeutic strategies elaborated against leukemia in human. Quantification of the cell turnover in closely related primate systems is particularly important for rare and aggressive forms of human cancers, such as adult T-cell leukemia. For this purpose, we have measured the death and proliferation rates of the CD4+ T lymphocyte population in squirrel monkeys (Saimiri sciureus) infected by human T-lymphotropic virus type 1 (HTLV-1). The kinetics of in vivo bromodeoxyuridine labeling revealed no modulation of the cell turnover in HTLV-1-infected monkeys with normal CD4 cell counts. In contrast, a substantial decrease in the proliferation rate of the CD4+ T population was observed in lymphocytic monkeys (e.g. characterized by excessive proportions of CD4+ T lymphocytes and by the presence of abnormal flower-like cells). Unexpectedly, onset of HTLV-associated leukemia thus occurs in the absence of increased CD4+ T-cell proliferation. This dynamics significantly differs from the generalized activation of the T-cell turnover induced by other primate lymphotropic viruses like HIV and SIV.

HPRT mutations in vivo in human T-lymphocytes are useful probes for mechanistic investigations. Molecular analyses of isolated mutants reveal their underlying mutational changes as well as the T-cell receptor (TCR) gene rearrangements present in the cells in question. The latter provide temporal reference points for other perturbations in the in vivo clones as well as evidence of clonal relationships among mutant isolates. Immunological studies and investigations of genomic instability have benefited from such analyses. A method is presented describing a T-cell lineage analysis in a patient with HTLV-1 infection. Lineage reconstruction of an in vivo proliferating HPRT mutant clone allows timing of the integration event to a postthymic differentiated cell prior to the occurrence of HPRT mutations.

Human T cell lymphotropic virus (HTLV) type I is the agent of adult T cell leukemia and HTLV-I-associated myelopathy. Because its pathogenesis is not well understood, a mouse model of HTLV-I infection would be valuable. We report the infection of adult BALB/c, C3H/He, 129Sv, and 129Sv IFNAR(-/-) mice with an infectious chimeric HTLV-I provirus bearing the Moloney-murine leukemia virus (Mo-MuLV) envelope glycoprotein truncated for the C-terminal R peptide. Mice were persistently infected (500-800 proviral DNA copies/10(5) splenocytes) for at least 20 weeks after inoculation. The chimeric virus disseminated to several organs, such as spleen, thymus, lung, brain, and spinal cord. The amplification of proviral integration sites showed an oligoclonal integration resembling that reported in HTLV-I-infected humans. Infected mice developed lasting humoral and cellular immune responses. This DeltaR HTLV-I/Mo-MuLV chimeric virus, with the Mo-MuLV Env tropism and HTLV-I replication characteristics, could provide a mouse model of HTLV-I infection.

Adult T-cell leukaemia/lymphoma (ATLL) is a malignant T-cell proliferation that occurs in 3-5% of individuals infected with human T-cell leukaemia virus-1 (HTLV-1). HTLV-1 infection is also linked to the development of infective dermatitis (ID), an exudative dermatitis of children that has been proposed as a cofactor of ATLL. Here, HTLV-1 replication was investigated over time in a girl with ID and multiparasitic infestation including strongyloidiasis, a disease also known to predispose HTLV-1 carriers to ATLL. Quantitative polymerase chain reaction (PCR) revealed extremely high proviral loads. During the 2-year period of the present study, the proportion of circulating infected cells ranged between 12% and 36%. Quadruplicate linker-mediated PCR amplification of HTLV-1 flanking sequences identified a pattern of extensive and persistent oligoclonal expansion of infected lymphocytes. As viral loads, both the number and the degree of infected T-cell expansion were independent of treatment or clinical signs. However, the temporal fluctuation of proviral loads correlated significantly with the degree of infected T-cell expansion, but not with the overall number of detected clones. This pattern of HTLV-1 replication over time is very different from that observed in asymptomatic carriers and reminiscent of that observed in ATLL, a result consistent with the proposal of ID as an ATLL cofactor.

Having isolated somatically mutated HTLV-1 3' LTR sequences from six infected individuals, the effect of these mutations on the integration process in vitro was investigated. Double-strand pre-processed HTLV-1 3' LTR ends (53-54 bp) were used in an in vitro strand-transfer reaction, together with HTLV-1 purified integrase and using a synthetic double-strand naked DNA oligonucleotide as target. Integration efficiency was measured by a fluorescent PCR assay. No significant difference in the pattern of strand transfer was observed between the distinct patients consensus sequences. For each patient, the effect of acquired somatic mutations was then assessed by comparing the strand-transfer efficiency of the mutated sequences (n=8, each harboring one to two substitutions) with that of the corresponding patient consensus sequence. Five somatic mutations or deletions at positions 7, 10, 21, 30, and 53 from the proviral 3' end did not alter the reaction efficiency. By contrast, a single G-->A transition at position 52 was found to result in 33% gain of function. Furthermore, a C-->T transition at 41 bp from the provirus 3' end decreased the reaction efficiency by 80%. This is the first study investigating the effect of naturally acquired substitutions on the strand-transfer capacity of long LTR sequences in vitro. Disproving the hitherto assumed opinion that integration specificity is restricted to the extreme boundary of the LTR end, i.e. the last 12-20 bp of the unintegrated provirus, the present results demonstrate that naturally occurred substitutions of the HTLV-1 LTR can alter significantly its strand-transfer capacity.