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Human T cell leukemia virus type-1 (HTLV-1) was the first retrovirus found to cause cancer in humans, but the mechanisms that drive the development of leukemia and other diseases associated with HTLV-1 infection remain to be fully understood. This review describes the functional properties of p13, an 87-amino acid protein coded by HTLV-1 open reading frame II (orf-II). p13 is mainly localized in the inner membrane of the mitochondria, where it induces potassium (K + ) influx and reactive oxygen species (ROS) production, which can trigger either proliferation or apoptosis, depending on the ROS setpoint of the cell. Recent evidence indicates that p13 may influence the cell's innate immune response to viral infection and the infected cell phenotype. Association of the HTLV-1 transcriptional activator, Tax, with p13 increases p13's stability, leads to its partial co-localization with Tax in nuclear speckles, and reduces the ability of Tax to interact with the transcription cofactor CBP/p300. Comparison of p13 sequences isolated from HTLV-1-infected individuals revealed a small number of amino acid variations in the domains controlling the subcellular localization of the protein. Disruptive mutations of p13 were found in samples obtained from asymptomatic patients with low proviral load. p13 sequences of HTLV-1 subtype C isolates from indigenous Australian patients showed a high degree of identity among each other, with all samples containing a pattern of 5 amino acids that distinguished them from other subtypes. Further characterization of p13's functional properties and sequence variants may lead to a deeper understanding of the impact of p13 as a contributor to the clinical manifestations of HTLV-1 infection.

Many investigations of the replication and pathogenesis of human T-cell leukemia virus type 1 (HTLV-1) employ chronically infected cell lines, cell lines stabilized from primary adult T-cell leukemia cells, and noninfected T-cell lines. The validity of data obtained from such studies depends on the unambiguous identification of each cell line, which can be performed by short-tandem-repeat (STR) profiling (DNA fingerprinting). While kit-based profiling represents the standard method for cell line authentication, not all labs have ready access to the required capillary electrophoresis equipment, and the costs of such tests can become substantial, especially if the cell lines are to be tested frequently. We analyzed DNA from a panel of HTLV-1-infected cell lines and noninfected T-cell lines using a commercial STR kit and then analyzed the same DNA for individual STR markers followed by nondenaturing polyacrylamide gel electrophoresis. This simplified method should facilitate routine confirmation of cell line identity in diverse laboratory settings.

Unlabelled: Human T-cell leukemia virus type 1 (HTLV-1) expression depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of viral expression. In the present study, we investigated the Rex dependence of the complete set of alternatively spliced HTLV-1 mRNAs. Analyses of cells transfected with Rex-wild-type and Rex-knockout HTLV-1 molecular clones using splice site-specific quantitative reverse transcription (qRT)-PCR revealed that mRNAs encoding the p30Tof, p13, and p12/8 proteins were Rex dependent, while the p21rex mRNA was Rex independent. These findings provide a rational explanation for the intermediate-late temporal pattern of expression of the p30tof, p13, and p12/8 mRNAs described in previous studies. All the Rex-dependent mRNAs contained a 75-nucleotide intronic region that increased the nuclear retention and degradation of a reporter mRNA in the absence of other viral sequences. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis revealed that this sequence formed a stable hairpin structure. Cell cycle synchronization experiments indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. These findings indicate a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system., Importance: HTLV-1 is a complex retrovirus that causes two distinct pathologies termed adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy in about 5% of infected individuals. Expression of the virus depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of virus expression. The findings reported in this study revealed a novel cis-acting regulatory element and indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. Our results add a layer of complexity to the mechanisms controlling the expression of alternatively spliced HTLV-1 mRNAs and suggest a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Human T cell leukemia virus type 1 (HTLV-1) gene expression is controlled by the key regulatory proteins Tax and Rex. The concerted action of these proteins results in a two-phase kinetics of viral expression that depends on a time delay between their action. However, it is difficult to explain this delay, as Tax and Rex are produced from the same mRNA. In the present study we investigated whether HTLV-1 may produce novel mRNA species capable of expressing Rex and Tax independently., Findings: Results revealed the expression of three alternatively spliced transcripts coding for novel Rex isoforms in infected cell lines and in primary samples from infected patients. One mRNA coded for a Tax isoform and a Rex isoform, and two mRNAs coded for Rex isoforms but not Tax. Functional assays showed that these Rex isoforms exhibit activity comparable to canonic Rex. An analysis of the temporal expression of these transcripts upon ex vivo culture of cells from infected patients and cell lines transfected with a molecular clone of HTLV-1 revealed early expression of the dicistronic tax/rex mRNAs followed by the monocistronic mRNAs coding for Rex isoforms., Conclusion: The production of monocistronic HTLV-1 mRNAs encoding Rex isoforms with comparable activity to canonical Rex, but with distinct timing, would support a prolonged duration of Rex function with gradual loss of Tax, and is consistent with the two-phase expression kinetics. A thorough understanding of these regulatory circuits will shed light on the basis of viral latency and provide groundwork to develop strategies for eradicating persistent infections.

Unlabelled: The present study employed mass sequencing of small RNA libraries to identify the repertoire of small noncoding RNAs expressed in normal CD4(+) T cells compared to cells transformed with human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma (ATLL). The results revealed distinct patterns of microRNA expression in HTLV-1-infected CD4(+) T-cell lines with respect to their normal counterparts. In addition, a search for virus-encoded microRNAs yielded 2 sequences that originated from the plus strand of the HTLV-1 genome. Several sequences derived from tRNAs were expressed at substantial levels in both uninfected and infected cells. One of the most abundant tRNA fragments (tRF-3019) was derived from the 3' end of tRNA-proline. tRF-3019 exhibited perfect sequence complementarity to the primer binding site of HTLV-1. The results of an in vitro reverse transcriptase assay verified that tRF-3019 was capable of priming HTLV-1 reverse transcriptase. Both tRNA-proline and tRF-3019 were detected in virus particles isolated from HTLV-1-infected cells. These findings suggest that tRF-3019 may play an important role in priming HTLV-1 reverse transcription and could thus represent a novel target to control HTLV-1 infection., Importance: Small noncoding RNAs, a growing family of regulatory RNAs that includes microRNAs and tRNA fragments, have recently emerged as key players in many biological processes, including viral infection and cancer. In the present study, we employed mass sequencing to identify the repertoire of small noncoding RNAs in normal T cells compared to T cells transformed with human T-cell leukemia virus type 1 (HTLV-1), a retrovirus that causes adult T-cell leukemia/lymphoma. The results revealed a distinct pattern of microRNA expression in HTLV-1-infected cells and a tRNA fragment (tRF-3019) that was packaged into virions and capable of priming HTLV-1 reverse transcription, a key event in the retroviral life cycle. These findings indicate tRF-3019 could represent a novel target for therapies aimed at controlling HTLV-1 infection.

Recent efforts to understand the molecular networks governing normal T cell development and driving the neoplastic transformation of T cells have brought to light the involvement of microRNAs (miRNAs), a class of noncoding RNAs of approximately 22 nucleotides that regulate gene expression at the posttranscriptional level. In the present review, we compare the expression profiles of miRNAs in normal T cell development to that of transformed T cells using as a model adult T cell leukemia/lymphoma, an aggressive malignancy of mature CD4+ T cells that is caused by infection with human T cell leukemia virus type 1., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Human T-cell leukemia virus type 1 (HTLV-1) codes for 9 alternatively spliced transcripts and 2 major regulatory proteins named Tax and Rex that function at the transcriptional and posttranscriptional levels, respectively. We investigated the temporal sequence of HTLV-1 gene expression in primary cells from infected patients using splice site-specific quantitative RT-PCR. The results indicated a two-phase kinetics with the tax/rex mRNA preceding expression of other viral transcripts. Analysis of mRNA compartmentalization in cells transfected with HTLV-1 molecular clones demonstrated the strict Rex-dependency of the two-phase kinetics and revealed strong nuclear retention of HBZ mRNAs, supporting their function as noncoding transcripts. Mathematical modeling underscored the importance of a delay between the functions of Tax and Rex, which was supported by experimental evidence of the longer half-life of Rex. These data provide evidence for a temporal pattern of HTLV-1 expression and reveal major differences in the intracellular compartmentalization of HTLV-1 transcripts.

Human T-cell leukemia virus type 1 (HTLV-1) infection is characterized by life-long persistence of the virus in the host. While most infected individuals remain asymptomatic, 3-5% will eventually develop adult T-cell leukemia/lymphoma (ATLL) or tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM) after a clinical latency that can span years (TSP/HAM) to decades (ATLL). The major oncogenic determinant among HTLV-1 proteins is the Tax transactivator, which influences the expression and function of a great number of cellular proteins, drives cell proliferation, reduces cell death, and induces genetic instability. The present review is focused on the current knowledge of p13, an HTLV-1 accessory protein targeted to the inner mitochondrial membrane and, under certain conditions, to the nucleus. In mitochondria, p13 produces an inward K+current that results in an increased production of ROS by mitochondria. These effects are linked to the protein's effects on cell turnover which include activation of primary T-cells and reduced proliferation/sensitization to death of tumor cells. Recent findings suggest that in the presence of Tax, p13 is subjected to ubiquitylation and partly targeted to the nucleus. Nuclear p13 binds Tax and inhibits its transcriptional activity. These findings suggest that the protein might exert distinct functions depending on its intracellular localization and influence both the turnover of infected cells and the balance between viral latency and productive infection., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Human T-cell leukemia virus type 1 (HTLV-1), a retrovirus that infects more than 20 million people worldwide, is the etiological agent of ATLL (adult T-cell leukemia/lymphoma), an aggressive leukemia of CD4+ T lymphocytes which arises in a small percentage of infected individuals after a long clinical latency. Tumor emergence is attributed primarily to the oncogenic activity of the viral protein Tax, which drives the expression of viral transcripts and controls the expression and function of a broad variety of host-cell genes involved in proliferation, genetic stability and apoptosis. Nevertheless, many aspects of HTLV-1 replication, persistence and pathogenesis remain to be understood. The emerging role of microRNAs in tumor development and viral infection has prompted investigations on the interactions between HTLV-1 and the microRNA regulatory network. In the present review we discuss recent data demonstrating changes in cellular microRNA expression in HTLV-1-infected cell lines and ATLL cells, and the functional impact of a subset microRNAs deregulated by HTLV-1 on cellular gene expression and signal transduction pathways. Mechanisms through which the viral proteins may influence microRNA expression are discussed. Results of searches for potential cellular microRNAs that target viral transcripts and for microRNAs produced by HTLV-1 are described. Observations along with regarding the expression of tRNA-derived small regulatory RNAs in HTLV-1-infected cells are presented., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The present study investigated the function of p13, a mitochondrial protein of human T-cell leukemia virus type 1 (HTLV-1). Although necessary for viral propagation in vivo, the mechanism of function of p13 is incompletely understood. Drawing from studies in isolated mitochondria, we analyzed the effects of p13 on mitochondrial reactive oxygen species (ROS) in transformed and primary T cells. In transformed cells (Jurkat, HeLa), p13 did not affect ROS unless the cells were subjected to glucose deprivation, which led to a p13-dependent increase in ROS and cell death. Using RNA interference we confirmed that expression of p13 also influences glucose starvation-induced cell death in the context of HTLV-1-infected cells. ROS measurements showed an increasing gradient from resting to mitogen-activated primary T cells to transformed T cells (Jurkat). Expression of p13 in primary T cells resulted in their activation, an effect that was abrogated by ROS scavengers. These findings suggest that p13 may have a distinct impact on cell turnover depending on the inherent ROS levels; in the context of the HTLV-1 propagation strategy, p13 could increase the pool of "normal" infected cells while culling cells acquiring a transformed phenotype, thus favoring lifelong persistence of the virus in the host.

Human T cell leukemia virus type 1 (HTLV-1) encodes p13, an 87-amino-acid protein that accumulates in the inner mitochondrial membrane. Recent studies performed using synthetic p13 and isolated mitochondria demonstrated that the protein triggers an inward potassium (K+) current and inner membrane depolarization. The present study investigated the effects of p13 on mitochondrial inner membrane potential (Deltapsi) in living cells. Using the potential-dependent probe tetramethyl rhodamine methyl ester (TMRM), we observed that p13 induced dose-dependent mitochondrial depolarization in HeLa cells. This effect was abolished upon mutation of 4 arginines in p13's alpha-helical domain that were previously shown to be essential for its activity in in vitro assays. As Deltapsi is known to control mitochondrial calcium (Ca2+) uptake, we next analyzed the effect of p13 on Ca2+ homeostasis. Experiments carried out in HeLa cells expressing p13 and organelle-targeted aequorins revealed that the protein specifically reduced mitochondrial Ca2+ uptake. These observations suggest that p13 might control key processes regulated through Ca2+ signaling such as activation and death of T cells, the major targets of HTLV-1 infection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Human T-cell leukemia virus type-1 (HTLV-1) encodes a mitochondrial protein named p13. p13 mediates an inward K(+) current in isolated mitochondria that leads to mitochondrial swelling, depolarization, increased respiratory chain activity and reactive oxygen species (ROS) production. These effects trigger the opening of the permeability transition pore and are dependent on the presence of K(+) and on the amphipathic alpha helical domain of p13. In the context of cells, p13 acts as a sensitizer to selected apoptotic stimuli. Although it is not known whether p13 influences the activity of endogenous K(+) channels or forms a channel itself, it shares some structural and functional analogies with viroporins, a class of small integral membrane proteins that form pores and alter membrane permeability., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K(+) current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K(+) in the assay medium. The effects of p13 on ROS are mimicked by the K(+) ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K(+) vs. H(+) currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K(+) permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells.

Individuals infected with HTLV-1 harbor the virus mainly in CD4+ memory T-cells as a lifelong infection that remains subclinical in the majority of cases. However, about 3-5% of HTLV-1-infected individuals develop an aggressive T-cell neoplasia (ATLL) or a neurodegenerative disease (TSP/HAM) after a latency period ranging from years to decades. This review summarizes the current knowledge of the effects of the HTLV-1 proteins Tax, p13 and p12 on cell death and survival pathways. Tax, the major oncogenic determinant of HTLV-1, enhances cell survival through its effects on the NF-kappaB, CREB and AKT pathways and on the tumor suppressors p53 and Rb. p13 is targeted to the inner mitochondrial membrane and sensitizes cells to the Fas/ceramide apoptotic pathway and reactive oxygen species-mediated cell death. p12 enhances release of calcium from the endoplasmic reticulum and therefore may influence calcium-dependent apoptotic signals, including opening of the mitochondrial permeability transition pore. The long-term fate of HTLV-1-infected cells (apoptosis, survival, transformation) may therefore depend on the balance of the effects of Tax, p13 and p12 on cell death pathways.

Human T cell leukemia virus type 1 encodes an "accessory" protein named p13(II) that is targeted to mitochondria and triggers a rapid flux of K(+) and Ca(2+) across the inner membrane. In this study, we investigated the effects of p13(II) on tumorigenicity in vivo and on cell growth in vitro. Results showed that p13(II) significantly reduced the incidence and growth rate of tumors arising from c-myc and Ha-ras-cotransfected rat embryo fibroblasts. Consistent with these findings, HeLa-derived cell lines stably expressing p13(II) exhibited markedly reduced tumorigenicity, as well as reduced proliferation at high density in vitro. Mixed culture assays revealed that the phenotype of the p13(II) cell lines was dominant over that of control lines and was mediated by a heat-labile soluble factor. The p13(II) cell lines exhibited an enhanced response to Ca(2+)-mediated stimuli, as measured by increased sensitivity to C2-ceramide-induced apoptosis and by cAMP-responsive element-binding protein (CREB) phosphorylation in response to histamine. p13(II)-expressing Jurkat T cells also exhibited reduced proliferation, suggesting that the protein might exert similar effects in T cells, the primary target of HTLV-1 infection. These findings provide clues into the function of p13(II) as a negative regulator of cell growth and underscore a link between mitochondria, Ca(2+) signaling, and tumorigenicity.

Human T-cell leukemia virus type 1 encodes a number of "accessory" proteins of unclear function; one of these proteins, p13(II), is targeted to mitochondria and disrupts mitochondrial morphology. The present study was undertaken to unravel the function of p13(II) through (i) determination of its submitochondrial localization and sequences required to alter mitochondrial morphology and (ii) an assessment of the biophysical and biological properties of synthetic peptides spanning residues 9-41 (p13(9-41)), which include the amphipathic mitochondrial-targeting sequence of the protein. p13(9-41) folded into an alpha helix in micellar environments. Fractionation and immunogold labeling indicated that full-length p13(II) accumulates in the inner mitochondrial membrane. p13(9-41) induced energy-dependent swelling of isolated mitochondria by increasing inner membrane permeability to small cations (Na(+), K(+)) and released Ca(2+) from Ca(2+)-preloaded mitochondria. These effects as well as the ability of full-length p13(II) to alter mitochondrial morphology in cells required the presence of four arginines, forming the charged face of the targeting signal. The mitochondrial effects of p13(9-41) were insensitive to cyclosporin A, suggesting that full-length p13(II) might alter mitochondrial permeability through a permeability transition pore-independent mechanism, thus distinguishing it from the mitochondrial proteins Vpr and X of human immunodeficiency virus type 1 and hepatitis B virus, respectively.