Autocrine role of macrophage inflammatory protein-1 beta in human T-cell lymphotropic virus type-I tax-transfected Jurkat T-cells

The interaction of human T-cell lymphotropic virus I (HTLV-I) tax gene with host cell factors plays an important role in the maintenance of the transformed state. There have been numerous reports that have demonstrated the role of tax in transactivating several cytokines. In this study, we show that upon mitogen stimulation, macrophage inflammatory protein-1 beta (MIP-1beta) is expressed and secreted in tax-transfected Jurkat cells. Furthermore, expression of CC-chemokine receptor-5 (CCR5) mRNA in these cells suggests an autocrine role for MIP-1beta in HTLV-I-infected T-cells. These results, coupled with our earlier observations, demonstrate the influence that extracellular Tax protein might have on modulating host chemokines for proliferation and transformation of uninfected cells.     

Target epitope in the Tax protein of human T-cell leukemia virus type I recognized by class I major histocompatibility complex-restricted cytotoxic T cells.

A trans-acting regulatory gene product p40tax (Tax) of human T-cell leukemia virus type I (HTLV-I) is one of the main target antigens recognized by cytotoxic T lymphocytes (CTL) specific for HTLV-I. A CTL epitope within the Tax protein was identified in this report. HTLV-I-specific CD8+ CTL lines established from two HTLV-I carriers with HTLV-I-associated myelopathy or Sj?gren syndrome were previously demonstrated to kill predominantly the target cells expressing HTLV-I Tax. The CTL from two patients showed significant levels of cytotoxicity to autologous target cells pulsed with a synthetic peptide of 24 amino acids corresponding to the amino-terminal sequences of the Tax protein. Allogeneic target cells were also sensitized for CTL by this peptide when the target cells have HLA-A2. Tax-specific cytotoxicity, detected as cytolysis of the target cells infected with vaccinia virus-HTLV-I recombinant expressing Tax protein, was almost completely inhibited by competitor cells pulsed with the synthetic peptide. This indicates that a major CTL epitope is present in this peptide. Further analysis using shorter peptides revealed that the core sequence of the CTL epitope was LLFGYPVYV at positions 11 through 19. This sequence can be aligned with the HLA-A2-specific motifs reported recently.     

Activation of HTLV-I long terminal repeat by stress-inducing agents and protection of HTLV-I-infected T-cells from apoptosis by the viral tax protein.

HTLV-I is etiologically implicated with tropical spastic paraparesis/HTLV-I associated myelopathy, adult T-cell leukemia and certain other diseases. However, after infection the virus enters into a dormant state, whereas the characteristics of the HTLV-I related diseases indicate that their genesis requires activation of the dormant virus by a Tax-independent mechanism. In the present study we demonstrate that a variety of stress-inducing agents (TPA, cisplatin, etoposide, taxol, and 3-methylcholanthrene) are capable of Tax-independent activation of HTLV-I LTR and that this activation is detected mainly in cells that are undergoing through the apoptotic process. Furthermore, it is demonstrated that both apoptosis induction and HTLV-I LTR activation are inhibited by Bcl-2 and by PKC, indicating that these two processes are mechanistically cross-linked. In addition, using an HTLV-I producing human T-cell line which permanently express the negatively transdominant tax mutant, Delta58tax, under the Tet-Off control system, we prove that the virally encoded Tax protein protects the host cells from apoptosis. Together, these data suggest that activation of the dormant virus in the carriers' infected T-cells by certain stress-inducing conditions and protecting these cells from the consequent apoptotic death by the viral Tax protein emerging after this activation, might be the basis for switching the virus from latency to a pathogenic phase.     

Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax.

To understand the mechanism of p56lck protein downregulation observed in human T cells infected by human T-cell leukemia virus type 1 (HTLV-1), we have investigated the ability of the 3' end of the HTLV-1 genome as well as that of the tax and rex genes to modulate p56lck protein expression and p56lck mRNA synthesis. By using Jurkat T cells stably transfected with constructs that expressed either the 3' end of the HTLV-1 genome (JK C11-pMTEX), the tax gene (JK52-Tax) or the rex gene (JK9-Rex), we found that the expression of p40tax (Tax) was sufficient to modulate p56lck protein expression. Similarly, we found that the expression of the mRNA which encoded p56lck was repressed in Jurkat T cells which expressed Tax. This downregulation was shown to be proportional to the amount of tax mRNA found in the transfected cells, as evidenced by experiments that used cells (JPX-9) stably transfected with a tax gene driven by a cadmium-inducible promoter. Furthermore, cadmium induction of Tax in JPX-9 cells transiently transfected with a construct containing the chloramphenicol acetyltransferase (CAT) gene under control of the lck distal promoter (lck DP-CAT) resulted in the downregulation of CAT gene expression. In contrast, cadmium induction of Tax in JPX-9 cells transiently transfected with a CAT construct driven by a lck DP with a deletion extending from position -259 to -253 (a sequence corresponding to a putative E-Box) did not modulate CAT gene expression, suggesting that the effect of Tax on p56lck is mediated through an E-Box binding protein.     

Genetic stability of human T lymphotropic virus type I despite antiviral pressures by CTLs

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurological disease. Patients with HAM/TSP show high proviral load despite increased HTLV-I Tax-specific CTL. It is still unknown whether the CTL efficiently eliminate the virus in vivo and/or whether a naturally occurring variant virus becomes predominant by escaping from the CTL. To address these issues, we sequenced a large number of HTLV-I tax genes from HLA-A*02 HAM/TSP patients and estimated synonymous and nonsynonymous changes of the genes to detect positive selection pressure on the virus. We found the pressures in three of six CTL epitopes in HTLV-I Tax, where amino acid substitutions preferentially occurred. Although some of variant viruses were not recognized by the CTL, no variant viruses accumulated within 3-8 years, indicating genetic stability of HTLV-I tax gene. These results suggest that CTL eliminate the infected cells in vivo and naturally occurring variant viruses do not predominate. As Tax is a regulatory protein which controls viral replication, the amino acid substitutions in Tax may reduce viral fitness for replication. Viral fitness and host immune response may contribute to the viral evolution within the infected individuals. Furthermore, the genetic stability in the epitopes despite the antiviral pressures suggests that the three epitopes can be the candidate targets for HTLV-I vaccine development.     

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.     

Overexpression of transforming growth factor-beta in transgenic mice carrying the human T-cell lymphotropic virus type I tax gene.

Human T-cell lymphotropic virus type I (HTLV-I) has been associated with an adult form of T-cell leukemia as well as tropical spastic paraparesis, a neurodegenerative disease. Adult T-cell leukemia patients express high levels of the type 1 isoform of transforming growth factor-beta (TGF-beta 1), which is mediated by the effects of the HTLV-I Tax transactivator protein on the TGF-beta 1 promoter. To understand further the regulation of TGF-beta 1 expression by Tax, we examined its expression in transgenic mice carrying the HTLV-I tax gene. We show that tumors from these mice and other tissues, such as submaxillary glands and skeletal muscle, which express high levels of tax mRNA selectively express high levels of TGF-beta 1 mRNA and protein. Moreover, TGF-beta 1 significantly stimulated the incorporation of tritiated thymidine into one of three cell lines derived from neurofibromas of tax-transgenic mice, which suggests that the excessive production of TGF-beta 1 may play a role in tumorigenesis and that these mice may serve as a useful model for studying the biological effects of TGF-beta in vivo.     

Naturally occurring variants of human T-cell leukemia virus type I Tax protein impair its recognition by cytotoxic T lymphocytes and the transactivation function of Tax.

There is a high degree of intraisolate sequence heterogeneity in the tax gene of human T-cell leukemia virus type I (HTLV-I), although the sequence variation between patients is small compared with that of human immunodeficiency virus type 1. In the present study, we investigated whether naturally occurring amino acid substitutions changed the properties of the Tax protein in two respects: first, recognition of the protein by cytotoxic T lymphocytes (CTL), and second, the ability of the Tax protein to transactivate various promoters. We found that (i) all of the observed amino acid substitutions that occur in known CTL epitopes abolished the recognition of the synthetic peptide representing the respective epitope; (ii) these substitutions occurred significantly more frequently in subjects carrying HLA-A2; and (iii) most of the amino acid substitutions severely reduced the ability of Tax protein to transactivate three promoters: the HTLV-I long terminal repeat, the c-fos promoter, and the interleukin-2 receptor alpha chain promoter.     

Transfection of macrophage inflammatory protein 1 alpha into B16 F10 melanoma cells inhibits growth of pulmonary metastases but not subcutaneous tumors

Macrophage inflammatory protein 1 alpha (MIP-1 alpha), a CC chemokine, is a chemoattractant for T cells and immature dendritic cells. Plasmacytoma cells expressing MIP-1 alpha generate a cytotoxic T cell response without affecting tumor growth. To understand this discrepancy, we compared a local tumor model with a metastatic one using MIP-1 alpha-transfected B16 F10 melanoma cells. Clonal idiosyncrasies were controlled by selecting three lipotransfected tumor clones and two pcDNA vector transfected control clones with equivalent in vitro proliferative capacities. No significant differences were seen between the MIP-1 alpha-producing and control melanoma cells after s.c. injection in the hind leg. All animals had a leg diameter of 10 cm in 18.5-21.5 days. However, after i.v. injection the number of pulmonary foci was significantly reduced in the MIP-1 alpha-producing clones. Injection of 10(6) control transfected cells resulted in a median of 98.5 tumor foci in 2 wk, whereas the injection of the MIP-1 alpha-producing clones resulted in 89.5, 26.5, and 0 foci. The number of metastatic foci was inversely proportional to the amount of MIP-1 alpha produced by the clone in vitro. Flow cytometry showed a significant increase in CD8(+) cells in lungs of mice with MIP-1 alpha-transfected tumors 3 days after injection. This increase was not maintained 10 days later despite continued production of MIP-1 alpha. The protection offered by transfection with MIP-1 alpha was significantly impaired in beta(2)-microglobulin(-/-) mice. Our findings suggest that MIP-1 alpha is effective in preventing the initiation of metastasis, but not at sustaining an effective antitumor response.     

The transactivator gene of human T-cell leukemia virus type I is more variable within and between healthy carriers than patients with tropical spastic paraparesis.

Human T-cell leukemia virus type I (HTLV-1) causes T-cell leukemia and tropical spastic paraparesis (TSP) in a minority of infected people, whereas the majority remain healthy. No association between a particular HTLV-I sequence and disease manifestation has been found in previous studies. We studied here the sequence variability of the gene for the HTLV-I Tax protein, which is the dominant target antigen of the very strong cytotoxic T-lymphocyte response to the virus. In HTLV-I infection, the intraisolate nucleotide variability is much greater than the variability between isolates. The predicted protein sequence of Tax was significantly more variable in the healthy seropositive individuals' provirus than in those of the patients with TSP. Thus, tax sequence heterogeneity, rather than the presence of particular sequences, distinguishes healthy HTLV-I-seropositive individuals from patients with TSP.     

Transcriptional suppression of the human T-cell leukemia virus type I long terminal repeat occurs by an unconventional interaction of a CREB factor with the R region.

To analyze regulation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR), cell lines were generated from LTR-tax x LTR-beta-galactosidase (beta-Gal) doubly transgenic mouse fibroblastic tumors. The HTLV-I LTR directs expression of both the tax and lacZ genes, and Tax up-modulates both promoters in primary cells. However, once cells were transformed by tax, beta-Gal but not tax expression was suppressed. Supertransformation of these cells with v-src suppressed both beta-Gal and tax expression. This suppression was reversed by treatment with the tyrosine kinase inhibitor herbimycin A or protein kinase A inhibitor H8. Electrophoretic mobility shift assays demonstrated augmented binding in the R but not U3 region. This binding was competitively inhibited by a high-affinity CREB oligodeoxynucleotide and super-shifted with a specific CREB antibody. Treatment of cells with the cyclic AMP analog dibutyryl cyclic AMP also transiently increased the R region binding dramatically. In vitro DNase I footprint analysis identified a protein-binding sequence in the R region which corresponded with suppression. However, this target sequence lacked a conventional CREB-binding site. A 70.5-kDa DNA-binding protein was partially purified by affinity chromatography, along with a 49-kDa protein which reacted with CREB-specific sera. These data demonstrate that HTLV-I LTR suppression is associated with CREB factor binding in the R region, probably by direct interaction with a 70.5-kDa protein, and provide a novel mechanism for maintenance of viral latency.     

Mutational analysis of the HTLV-I trans-activator, Tax

The gene coding for the trans-activating factor (Tax) of the human T-cell leukemia virus, type I (HTLV-I) was mutagenized in vitro using oligonucleotide-directed mutagenesis and recombinant DNA techniques. All except one of the mutagenized tax constructs failed to trans-activate the HTLV-I LTR in a eukaryotic test system. Moreover, negative Tax mutant Arg-39----Gly was found to be trans-dominant. This observation suggests that Tax contains distinct functional domains mediating different interactions of the protein in the process of trans-activation.     

Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

We have studied the role of proteoglycans in the function of Macrophage Inflammatory Protein-1 alpha (MIP-1alpha), a member of the proteoglycan binding chemokine family. Sequence and peptide analysis has identified a basic region within MIP-1alpha which appears to be the major determinant of proteoglycan binding and we have now produced a mutant of MIP-1alpha lacking the basic charges on two of the amino acids within this proteoglycan binding site. This mutant (Hep Mut) appears to have lost the ability to bind to proteoglycans. Bioassay of Hep Mut indicates that it has retained stem cell inhibitory properties but has a compromised activity as a monocyte chemoattractant, thus suggesting uncoupling of these two properties of MIP-1alpha. Receptor studies have indicated that the inactivity of Hep Mut on human monocytes correlates with its inability to bind to CCR1, a cloned human MIP-1alpha receptor. In addition, studies using proteoglycan deficient cells transfected with CCR1 have indicated that the proteoglycan binding site in MIP-1alpha is a site that is also involved in the docking of MIP-1alpha to the monocyte receptor. The site for interaction with the stem cell receptor must therefore be distinct, suggesting that MIP-1alpha utilizes different receptors for these two different biological processes.