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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 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.)

Retrovirus HTLV-1 gene circuit is characterized by positive and negative feedback phenomena, thus candidating it as a potential relaxation oscillator deliverable into eukaryotes. Here we describe a model of HTLV-1 which, by providing predictions of genes and proteins kinetics, can be helpful for designing gene circuits for eukaryotes, or for optimizing gene therapy approaches which are currently carried out by means of lentiviral vectors or re-engineered adenoviruses. Oscillatory patterns of HTLV-1 gene circuit are predicted when positive feedback is faster than negative feedback. Techniques to mutate the retroviral genome in order to implement practically the above conditions are discussed. Finally, the effect of stochasticity on the system behavior is tested by means of Gillespie algorithm. Simulations show the difficulties to preserve synchronization in viral expression for a multiplicity of cells, while the long tail of the density probability function of the master regulator gene tax/rex, due to its steady state fluctuations, suggests an activation mechanism of HTLV-1 similar to that recently proposed for HIV(1): the virus tends to latency but under certain circumstances, the master regulator gene reaches high values of expression, whose persistence induces the viral replication.

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.

Amiodarone interferes with the endocytic pathway, inhibits proteolysis, and causes the formation of vacuoles, but uptake and intracellular distribution of the drug, origin of vacuoles, and functional consequences of amiodarone accumulation remain unclear. Our objective was to study amiodarone uptake, clarify the origin of vacuoles, and investigate the effect of amiodarone on the life cycle of the coronavirus responsible for the Severe Acute Respiratory Syndrome (SARS), which, to enter cells, relies on the proteolytic cleavage of a viral spike protein by the endosomal proteinase cathepsin L. Using alveolar macrophages, we studied uptake of (125)I-amiodarone and (125)I-B2, an analog lacking the lateral group diethylamino-beta-ethoxy, and analyzed the effects of amiodarone on the distribution of endosomal markers and on the uptake of an acidotropic dye. Furthermore, using Vero cells, we tested the impact of amiodarone on the in vitro spreading of the SARS coronavirus. We found that (1) amiodarone associates with different cell membranes and accumulates in acidic organelles; (2) the diethylamino-beta-ethoxy group is an important determinant of uptake; (3) vacuoles forming upon exposure to amiodarone are enlarged late endosomes; (4) amiodarone inhibits the spreading in vitro of SARS coronavirus; and (5) trypsin cleavage of the viral spike protein before infection, which permits virus entry through the plasma membrane, does not impair amiodarone antiviral activity. We conclude that amiodarone alters late compartments of the endocytic pathway and inhibits SARS coronavirus infection by acting after the transit of the virus through endosomes.

Human T-lymphotropic virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia, encodes unique regulatory and accessory proteins in the pX region of the provirus, including the open reading frame II product p13(II). p13(II) localizes to mitochondria, binds farnesyl pyrophosphate synthetase, an enzyme involved in posttranslational farnesylation of Ras, and alters Ras-dependent cell signaling and control of apoptosis. The role of p13(II) in virus infection in vivo remains undetermined. Herein, we analyzed the functional significance of p13(II) in HTLV-1 infection. We compared the infectivity of a human B-cell line that harbors an infectious molecular clone of HTLV-1 with a selective mutation that prevents the translation of p13(II) (729.ACH.p13) to the infectivity of a wild-type HTLV-1-expressing cell line (729.ACH). 729.ACH and 729.ACH.p13 producer lines had comparable infectivities for cultured rabbit peripheral blood mononuclear cells (PBMC), and the fidelity of the start codon mutation in ACH.p13 was maintained after PBMC passage. In contrast, zero of six rabbits inoculated with 729.ACH.p13 cells failed to establish viral infection, whereas six of six rabbits inoculated with wild-type HTLV-1-expressing cells (729.ACH) were infected as measured by antibody responses, proviral load, and HTLV-1 p19 matrix antigen production from ex vivo-cultured PBMC. Our data are the first to indicate that the HTLV-1 mitochondrion-localizing protein p13(II) has an essential biological role during the early phase of virus infection in vivo.

The human T-cell leukemia/lymphoma virus (HTLV) genome, in addition to the structural Gag and Env proteins and retroviral enzymes, carries a region at its 3' end originally designated pX. To date, we know that this region encodes two essential transcriptional and post-transcriptional positive regulators of viral expression, the Tax and Rex proteins, respectively (reviewed elsewhere in this issue). Here, we will review current knowledge of the functions of three additional proteins encoded in the pX region, p12I, p13II, and p30II.

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia. In addition to typical retroviral structural and enzymatic gene products, HTLV-1 encodes unique regulatory and accessory proteins, including a singly spliced pX open reading frame II (ORF II) product, p13(II). We have demonstrated that proviral clones of HTLV-1 which are mutated in pX ORF II fail to obtain typical proviral loads and antibody responses in a rabbit animal model. p13(II) localizes to mitochondria and reduces cell growth and tumorigenicity in mice, but its function in human lymphocytes remains undetermined. For this study, we analyzed the functional properties of Jurkat T cells expressing p13(II), using both transient and stable expression vectors. Our data indicate that p13(II)-expressing Jurkat T cells are sensitive to caspase-dependent, ceramide- and FasL-induced apoptosis. p13(II)-expressing Jurkat T cells also exhibited reduced proliferation when cultured at a high density. Furthermore, preincubation of the p13(II)-expressing cells with a farnesyl transferase inhibitor, which blocks the posttranslational modification of Ras, markedly reduced FasL-induced apoptosis, indicating the participation of the Ras pathway in p13(II)'s influence on lymphocyte survival. Our data are the first to demonstrate that p13(II) alters Ras-mediated apoptosis in T lymphocytes, and they reveal a potential mechanism by which HTLV-1 alters lymphocyte proliferation.

Molecular analyses of tumor virus-host cell interactions have provided key insights into the genes and pathways involved in neoplastic transformation. Recent studies have revealed that the human tumor viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), and human T-cell leukemia virus type 1 (HTLV-1) express proteins that are targeted to mitochondria. The list of these viral proteins includes BCL-2 homologues (BHRF1 of EBV; KSBCL-2 of KSHV), an inhibitor of apoptosis (IAP) resembling Survivin (KSHV K7), proteins that alter mitochondrial ion permeability and/or membrane potential (HBV HBx, HPV E[wedge]14, HCV p7, and HTLV-1 p13(II)), and K15 of KSHV, a protein with undefined function. Consistent with the central role of mitochondria in energy production, cell death, calcium homeostasis, and redox balance, experimental evidence indicates that these proteins have profound effects on host cell physiology. In particular, the viral BCL-2 homologues BHRF1 and KSBCL-2 inhibit apoptosis triggered by a variety of stimuli. HBx, p7, E1[wedge]4, and p13(II) exert powerful effects on mitochondria either directly due to their channel-forming activity or indirectly through interactions with endogenous channels. Further investigation of these proteins and their interactions with mitochondria will provide important insights into the mechanisms of viral replication and tumorigenesis and could aid in the discovery of new targets for anti-tumor therapy.

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.

G4 and p13(II) are accessory proteins encoded by the X region of bovine leukemia virus and human T-cell leukemia virus type 1 (HTLV-1), respectively. Disruption of the G4 and p13(II) open reading frames interferes with viral spread in animal model systems, indicating that the corresponding proteins play a key role in viral replication. In addition, G4 is oncogenic in primary cell cultures and is absolutely required for efficient onset of leukemogenesis in sheep. To gain insight into the function of these proteins, we utilized the yeast two-hybrid system to identify protein partners of G4. Results revealed that G4 interacts with farnesyl pyrophosphate synthetase (FPPS), a protein involved in the mevalonate/squalene pathway and in synthesis of FPP, a substrate required for prenylation of Ras. The specificity of the interaction was verified by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation experiments. Furthermore, confocal microscopy showed that the subcellular localization of G4 was profoundly affected by FPPS. The G4 protein itself was not prenylated, at least in rabbit reticulocyte lysate-based assays. The domain of G4 required for binding to FPPS was restricted to an amphipathic alpha-helix rich in arginine residues. Subtle mutation of this alpha-helix abrogated G4 oncogenic potential in vitro, providing a biological relevance for FPPS-G4 complex formation in cells. Finally, HTLV-1 p13(II) was also found to specifically interact with FPPS (in yeast as well as in GST pull-down assays) and to colocalize with G4 in mitochondria, suggesting a functional analogy between these oncoviral accessory proteins. Identification of FPPS as a molecular partner for p13(II) and G4 accessory proteins opens new prospects for treatment of retrovirus-induced leukemia.