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Ontogenesis of T cells in the thymus is a complex process whose molecular control is poorly understood. The present study investigated microRNAs involved in human thymocyte differentiation by comparing the microRNA expression profiles of thymocytes at the double-positive, single-positive CD4+ and single-positive CD8+ maturation stages. Microarray analysis showed that each thymocyte population displays a distinct microRNA expression profile that reflects their developmental relationships. Moreover, analysis of small-RNA libraries generated from human unsorted and double-positive thymocytes and from mature peripheral CD4+ and CD8+ T lymphocytes, together with the microarray data, indicated a trend toward up-regulation of microRNA expression during T-cell maturation after the double-positive stage and revealed a group of microRNAs regulated during normal T-cell development, including miR-150, which is strongly up-regulated as maturation progresses. We showed that miR-150 targets NOTCH3, a member of the Notch receptor family that plays important roles both in T-cell differentiation and leukemogenesis. Forced expression of miR-150 reduces NOTCH3 levels in T-cell lines and has adverse effects on their proliferation and survival. Overall, these findings suggest that control of the Notch pathway through miR-150 may have an important impact on T-cell development and physiology.

Ontogenesis of T cells in the thymus is a complex process whose molecular control is poorly understood. The present study investigated microRNAs involved in human thymocyte differentiation by comparing the microRNA expression profiles of thymocytes at the double-positive, single-positive CD4+and single-positive CD8+maturation stages. Microarray analysis showed that each thymocyte population displays a distinct microRNA expression profile that reflects their developmental relationships. Moreover, analysis of small-RNA libraries generated from human unsorted and double-positive thymocytes and from mature peripheral CD4+and CD8+T lymphocytes, together with the microarray data, indicated a trend toward up-regulation of microRNA expression during T-cell maturation after the double-positive stage and revealed a group of microRNAs regulated during normal T-cell development, including miR-150, which is strongly up-regulated as maturation progresses. We showed that miR-150 targets NOTCH3, a member of the Notch receptor family that plays important roles both in T-cell differentiation and leukemogenesis. Forced expression of miR-150 reduces NOTCH3 levels in T-cell lines and has adverse effects on their proliferation and survival. Overall, these findings suggest that control of the Notch pathway through miR-150 may have an important impact on T-cell development and physiology.

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) 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/rexmRNA 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 HBZmRNAs, 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.

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.

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 encodes a number of “accessory” proteins of unclear function; one of these proteins, p13II, is targeted to mitochondria and disrupts mitochondrial morphology. The present study was undertaken to unravel the function of p13IIthrough (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 (p139–41), which include the amphipathic mitochondrial-targeting sequence of the protein. p139–41folded into an α helix in micellar environments. Fractionation and immunogold labeling indicated that full-length p13IIaccumulates in the inner mitochondrial membrane. p139–41induced energy-dependent swelling of isolated mitochondria by increasing inner membrane permeability to small cations (Na+, K+) and released Ca2+from Ca2+-preloaded mitochondria. These effects as well as the ability of full-length p13IIto alter mitochondrial morphology in cells required the presence of four arginines, forming the charged face of the targeting signal. The mitochondrial effects of p139–41were insensitive to cyclosporin A, suggesting that full-length p13IImight 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.

ABSTRACTThe activity of human immunodeficiency virus Rev as a regulator of viral mRNA expression is tightly linked to its ability to shuttle between the nucleus and cytoplasm; these properties are conferred by a leucine-rich nuclear export signal (NES) and by an arginine-rich nuclear localization signal/RNA binding domain (NLS/RBD) required for binding to the Rev-responsive element (RRE) located on viral unspliced and singly spliced mRNAs. Structure predictions and biophysical measurements indicate that Rev consists of an unstructured region followed by a helix-loop-helix motif containing the NLS/RBD and sequences directing multimerization and by a carboxy-terminal tail containing the NES. We present evidence that the loop portion of the helix-loop-helix region is an essential functional determinant that is required for binding to the RRE and for correct intracellular routing. Data obtained using a protein kinase CK2 phosphorylation assay indicated that the loop region is essential for juxtaposition of helices 1 and 2 and phosphorylation by protein kinase CK2. Deletion of the loop resulted in partial accumulation of Rev in SC35-positive nuclear bodies that resembled nuclear bodies that form in response to inhibition of transcription. Accumulation of the ?Loop mutant in nuclear bodies depended on the presence of an intact NES, suggesting that both the loop and the NES play a role in controlling intranuclear compartmentalization of Rev and its association with splicing factors.

In addition to the essential regulatory proteins Rex and Tax, the HTLV-1 genome encodes several accessory proteins of yet undefined function. One of these "orphan" proteins, named p13II, was recently shown to be selectively targeted to mitochondria and to induce specific changes in mitochondrial morphology suggestive of altered inner membrane permeability and swelling. This represented the first report of a retroviral gene product targeted to mitochondria, and suggested that p13II-induced alterations in the function of this organelle may play a role in HTLV-1 replication and/or pathogenesis. The more recent findings that both Vpr and Tat of HIV-1 are targeted to mitochondria reinforces the proposed relevance of mitochondrial metabolism to the life cycle of retroviruses. Thus, p13II, Vpr, and Tat can be added to the growing list of mitochondrial proteins produced by clinically important human viruses, including Epstein-Barr virus, human cytomegalovirus, and hepatitis B virus. Mitochondria are known to play a critical role by providing an amplification loop required for the execution of signaling pathways leading to programmed cell death. The functional consequences of the interactions between viral proteins and mitochondria described so far have been attributed to either the positive or negative control of apoptotic responses mediated by this organelle. Further analysis of the effects of p13II on mitochondrial function is likely to add to our understanding of the mechanisms underlying the development of HTLV-1-associated diseases.

The HIV‐1 Rev transactivator is phosphorylated in vitro by protein kinase CK2 at two residues, Ser‐5 and Ser‐8; these sites are also phosphorylated in vivo. Here we show that the mechanism by which CK2 phosphorylates Rev is unique in several respects, notably: (i) it is fully dependent on the regulatory, β‐subunit of CK2; (ii) it relies on the integrity of an acidic stretch of CK2β which down‐regulates the phosphorylation of other substrates; (iii) it is inhibited in a dose‐dependent manner by polyamines and other polycationic effectors that normally stimulate CK2 activity. In contrast, a peptide corresponding to the amino‐terminal 26 amino acids of Rev, including the phosphoacceptor site, is readily phosphorylated by the catalytic subunit of CK2 even in the absence of the β‐subunit. These data, in conjunction with the observation that two functionally inactive derivatives of Rev with mutations in its helix‐loop‐helix motif are refractory to phosphorylation, indicate the phosphorylation of Rev by CK2 relies on conformational features of distinct regions that are also required for the transactivator's biological activity.

Human T-cell leukemia/lymphotropic virus type I (HTLV-I) infection causes a variety of human diseases, including adult T-cell leukemia/lymphoma. The viral transactivator Tax has been implicated as a key factor in the HTLV-I-induced transformation pathway. To investigate the components of this pathway, we derived fibroblast-like cell lines, designated T6 and T9, from tail biopsies oftax-transgenic C57BL/6 mice that do not develop tumors. Phenotypic characterization of T6 and T9 cells and T6-derived subclones revealed that they differ in their abilities to form fociin vitroand tumorsin vivo.The observed differences in the levels of Tax expression did not correlate with their degree of neoplastic potential. However, a control cell line derived from a nontransgenic C57BL/6 mouse did not form fociin vitroor tumorsin vivo,indicating that Tax was required for the transformation process. Results of Northern analyses showed that the T9 cells and the highly malignant derivatives of T6 cells expressed elevated levels of c-mycmRNA. These findings suggest that progression of thetax-transgenic cells toward a more malignant phenotype might involve c-mycderegulation.

Expression of the incompletely spliced HTLV-I mRNAs relies on the viral posttranscriptional activator Rex, whose interaction with the Rex-responsive element (RXRE) overcomes effects of cis-acting repressive sequences (CRSs). Studies based on heterologous reporter plasmids identified an intronic CRS in the 5' LTR and a CRS that overlaps with the RXRE. The present study investigated the effects of these elements in the context of spliced viral mRNAs encoding p21Rex (mRNA 1-3), Tax/Rex (mRNA 1-2-3), and Tof (mRNA 1-2-B). All three mRNAs were inefficiently expressed when transcribed in their mature intronless form, with the p21Rex mRNA showing the weakest expression. In contrast, efficient expression of p21Rex was obtained from a plasmid containing the 5' LTR and 3' portion of the genome that encoded a spliceable RNA. The defective expression of the intronless mRNAs reflected the inhibitory activity of the RXRE and the lack of 5' intronic sequences. Insertion of an intronic 5' LTR segment located upstream of the 5' CRS overcame Rex dependence conferred by the RXRE. The activity of this segment was mapped to the major splice donor and sequences overlapping with, but functionally distinct from, a previously described transcriptional enhancer. The three mRNAs responded differently to Rex and to insertion of the constitutive transport element of simian retrovirus type 1. Taken together, these results suggest that expression of the spliced mRNAs is controlled by the relative influence of positive and negative sequences present on the primary transcript as well as the Rex-RXRE interaction.

HIV-1 Rev transactivator is readily phosphorylated at separate regions by protein kinase CK2 and MAP kinase. Protein kinase CK1 cannot replace CK2 as phosphorylating agent and cdc2 only slowly phosphorylates Rev at one of the two sites affected by MAP kinase. Mutational analysis shows that Ser-8 and, to a lesser extent, Ser-5 are phosphorylated by CK2. In contrast, a mutation (R14TV → EED) which suppresses Rev activity dramatically enhances Rev phosphorylation eitherin vitroby CK2 orin vivo,suggesting that phosphorylation by CK2 could play a role in Rev down-regulation.

In previous studies we showed that human T-cell leukemia/lymphotropic virus type I (HTLV-1) may produce novel proteins encoded in the X region. To investigate a possible correlation between expression of viral genes and different biologic properties of HTLV-I and HTLV-II, we analyzed expression of HTLV-II in the chronically infected cell line MoT. Reverse transcription-polymerase chain reaction analyses revealed that the virus produces several mRNAs singly or doubly spliced into the X region. Corresponding cDNAs were cloned and transfected into a HeLa cell line; resulting proteins were designated according to their sizes and coding open reading frames (ORFs). p10xIand p11xVwere produced by a dicistronic doubly spliced mRNA. p10xIwas generated by translation of the first rex of rex linked to the x-I ORF; p11xVwas translated from the tax initiation codon linked to the x-V ORF. Two singly spliced polycistronic mRNAs produced p28xII, coded by the x-II ORF, and several isoforms generated by initiation within the x-III ORF. Studies of the proteins' subcellular localization revealed that they exhibited distinct targeting patterns. Comparison of these proteins with their HTLV-I counterparts indicated intriguing differences between these two viruses, suggesting that further study of the X region products may aid in defining genetic determinants of pathogenicity.

ABSTRACTA DNA immunization approach was used to induce an immune response against the tumor-specific antigen P815A in DBA/2 mice. The P1Agene, which encodes the P815A antigen, was modified by the addition of a short sequence coding for a tag epitope recognized by the monoclonal antibody AU1, and cloned into the eukaryotic expression vector pBKCMV, resulting in plasmid pBKCMV-P1A. L1210 cells stably transfected with pBKCMV-P1A expressed P1A mRNA and were lysed by the syngeneic P815A-specific cytotoxic clone CTL-P1:5, thus confirming that the tag-modified P1A protein underwent correct processing and presentation. A single intramuscular injection of 100 g of pBKCMV-P1A induced the expression of P1A mRNA for at least 4 months. Eighty percent of DBA/2 mice injected three times with 100 g of pBKCMV-P1A generated cytotoxic T lymphocytes (CTL) that lysed P815 tumor cells, whereas mock-inoculated animals failed to show any cytotoxicity. Moreover, experiments designed to evaluate the protection of pBKCMV-P1A-immunized mice against a lethal challenge with P815 tumor cells showed that 6 of 10 immunized mice rejected the tumor, and 2 mice showed prolonged survival compared to control animals.