The human T-cell leukemia virus type-1 Tax protein regulates the activity of the IkappaB kinase complex

Two cytokine-inducible kinases, IKKalpha and IKKbeta, are components of a 700-kDa kinase complex that specifically phosphorylates IkappaB. Phosphorylation of IkappaB by IKK leads to its ubiquitination and subsequent degradation, resulting in the nuclear translocation of NF-kappaB. The oncogenic protein Tax, encoded by human T-cell leukemia virus type-1 (HTLV-1), stimulates IKK activity to result in constitutive nuclear levels of NF-kappaB. In an attempt to gain insights into the mechanism by which Tax mediates constitutive activation of the NF-kappaB pathway, we analyzed the chromatographic distribution of IKK proteins using cellular extracts prepared from three T lymphocytes either lacking or containing Tax. IKK kinase activity and the distribution of proteins in the IKK complex were characterized. In extracts prepared from cells containing Tax, the activity of both IKKalpha and IKKbeta present in the 700-kDa IKK complex were increased. Surprisingly, cell lines expressing Tax also contained an additional peak of IKKbeta, but not IKKalpha activity, that migrated at 300 kDa rather than at 700 kDa. We noted that extracts containing Tax had extremely low levels of IkappaBbeta, but not IkappaBalpha, and contained predominantly a truncated form of the MAP3K MEKK1. These results suggest that Tax may target several components of the NF-kappaB pathway leading to constitutive activation of this important regulator of cellular gene expression.     

Cell-type-specific trans-activation of herpes simplex virus thymidine kinase promoter by the human T-cell leukemia virus type I Tax protein.

The human T-cell leukemia virus type I Tax protein (HTLV-I Tax) is known as a trans-activating factor for a variety of genes, including those of cytokines. Here, we show that Tax is capable of activating the herpes simplex virus thymidine kinase (HSV-TK) promoter in certain mammalian cell lines. In murine NIH 3T3 fibroblasts and human HeLa cells, trans-activation by Tax was remarkably strong, whereas in human chondrocytic HCS-2/8 and monkey kidney Cos-7 cells, the responsiveness of the TK promoter to Tax was poor. Deletion analysis revealed that one of the two previously described Sp1 sites is required for the Tax responsiveness, whereas the CTF binding site is not. The results suggest possible interactions between the oncogenic Tax protein and the viral TK in coinfected cells in vivo. Care should be taken in the context of HTLV-I research, as the HSV-TK promoter has been widely used in molecular biology and gene therapeutics.     

Enzyme activities associated with an invertebrate iridovirus: protein kinase activity associated with iridescent virus type 6 (chilo iridescent virus)

Iridescent virus type 6 was found to contain an endogenous protein kinase activity which can phosphorylate some viral proteins and exogenous basic proteins. The enzyme required a divalent metal ion but was not stimulated by cyclic nucleotides. Procedures which are known to solubilize the viral envelope indicated that the protein kinase was an internal component of the virion. Conditions for protein kinase activity are described.     

Activation of endogenous c-fos proto-oncogene expression by human T-cell leukemia virus type I-encoded p40tax protein in the human T-cell line, Jurkat.

We examined the ability of the trans-acting factor p40tax of human T-cell leukemia virus type I (HTLV-I), which is thought to be a crucial molecule in T-cell transformation by HTLV-I, to activate expression of a set of endogenous cellular genes related to T-cell proliferation. For this purpose we established a subclone (JPX-9) of Jurkat cells that was stably transfected with an expression plasmid containing the p40tax gene, whose expression is definitely dependent on heavy-metal ions. Expression of the interleukin-2 receptor alpha chain in JPX-9 cells was induced in response to the induction of p40tax expression, as has been demonstrated by others in transient transfection experiments with Jurkat cells. In addition, we found that significant enhancement of expression of the nuclear proto-oncogene c-fos was closely associated with expression of p40tax. Continous enhancement in the level of c-fos mRNA was observed in the presence of p40tax. In contrast, mRNA levels of other nuclear proto-oncogenes (c-myc, c-myb, and c-jun) were not appreciably effected by the expression of p40tax. These results suggest that (i) in addition to the interleukin-2-interleukin-2 receptor system, cellular genes such as c-fos, which regulate normal T-cell growth, are also activated directly or indirectly by p40tax and (ii) p40tax-induced modulation of gene expression plays a crucial role in T-cell transformation by HTLV-I.     

Inhibition of T-cell receptor signal transduction and viral expression by the linker for activation of T cells-interacting p12(I) protein of human T-cell leukemia/lymphoma virus type 1

The p12(I) protein of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is a small oncoprotein that increases calcium release following protein kinase C activation by phorbol myristate acetate, and importantly, this effect is linker for activation of T cells (LAT) independent. Here, we demonstrate that p12(I) inhibits the phosphorylation of LAT, Vav, and phospholipase C-gamma 1 and decreases NFAT (nuclear factor of activated T cells) activation upon engagement of the T-cell receptor (TCR) with anti-CD3 antibody. Furthermore, we demonstrate that p12(I) localizes to membrane lipid rafts and, upon engagement of the TCR, relocalizes to the interface between T cells and antigen-presenting cells, defined as the immunological synapse. A p12(I) knockout molecular clone of HTLV-1 expresses more virus upon antigen stimulation than the isogenic wild type, suggesting that, by decreasing T-cell responsiveness, p12(I) curtails viral expression. Thus, p12(I) has contrasting effects on TCR signaling: it down-regulates TCR in a LAT-dependent manner on one hand, and on the other, it increases calcium release in a LAT-independent manner. The negative regulation of T-cell activation by p12(I) may have evolved to minimize immune recognition of infected CD4(+) T cells, to impair the function of infected cytotoxic CD8(+) T cells, and to favor viral persistence in the infected host.     

Free major histocompatibility complex class I heavy chain is preferentially targeted for degradation by human T-cell leukemia/lymphotropic virus type 1 p12(I) protein

Human T-cell leukemia virus type 1 (HTLV-1) establishes a persistent infection in the host despite a vigorous virus-specific immune response. Here we demonstrate that an HTLV-1-encoded protein, p12(I), resides in the endoplasmic reticulum (ER) and Golgi and physically binds to the free human major histocompatibility complex class I heavy chains (MHC-I-Hc) encoded by the HLA-A2, -B7, and -Cw4 alleles. As a result of this interaction, the newly synthesized MHC-I-Hc fails to associate with beta(2)-microglobulin and is retrotranslocated to the cytosol, where it is degraded by the proteasome complex. Targeting of the free MHC-I-Hc, and not the MHC-I-Hc-beta(2)-microglobulin complex, by p12(I) represents a novel mechanism of viral interference and disrupts the intracellular trafficking of MHC-I, which results in a significant decrease in surface levels of MHC-I on human T-cells. These findings suggest that the interaction of p12(I) with MHC-1-Hc may interfere with antigen presentation in vivo and facilitate escape of HTLV-1-infected cells from immune recognition.     

Modulation of mitochondrial K permeability and reactive oxygen species production by the p13 protein of human T-cell leukemia virus type 1

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.     

Mitochondrial alterations induced by the p13II protein of human T-cell leukemia virus type 1. Critical role of arginine residues

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.     

Characterization and properties of a protein kinase associated with murine sarcoma-leukemia virus (MSV-MuLV). Endogenous phosphorylation of a unique p10 polypeptide.

MSV-MuLV virions produced by the rat embryo fibroblast 78 A₁ cell line and extensively purified by treatment with trypsin (M. Rucheton, D. Blaas and Ph. Jeanteur, Biochimie, in press) were shown to contain an endogenous cyclic AMP independent protein kinase activity which is located within the viral core. Enzymatic properties were investigated and an apparent molecular weight of 40,000 daltons determined by gel filtration. It shows a marked preference for acidic exogenous substrates and especially α-casein. During endogenous reaction, only the viral protein p10 was phosphorylated.     

Vesicular stomatitis virus produced from infected LSTRA lymphoma cells bear tyrosine protein kinase activity (p56)

Murine LSTRA lymphoma cells contain a very active tyrosine protein kinase of 56 kDa (p56) which is not related to any of the other known tyrosine kinases. In the past the purification and characterization of the p56 have been hampered because of the low amount of this protein in LSTRA membranes. In this study, we have utilized a different approach for purification which consisted of trapping the protein in the membrane of vesicular stomatitis virus. Incubation of the virions with [gamma-32P]ATP resulted in the phosphorylation of p56 on tyrosine residues. Moreover, the phosphopeptide digest profile of vesicular stomatitis virus-p56 was identical to that observed with authentic LSTRA-p56. The p56 from such virions could be resolved from other proteins by two-dimensional gels, and furthermore, such virions have been used to prepare several antisera directed against the p56.