Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human T-cell lymphotropic virus type III trans activator genes.

Recombinant retroviruses containing the trans activator genes of human T-cell leukemia virus (HTLV) type II and human T-cell lymphotropic virus type III were constructed. The trans activator genes tat II and tat III were inserted into the murine retroviral vector pZIPNEOSV(X)1. Recombinant plasmids were transfected into the psi 2 and psi AM packaging cell lines that produce murine leukemia virions containing no retroviral RNA. Functional tat II and tat III gene products were expressed as demonstrated by trans activation of HTLV type I and II and human T-cell lymphotropic virus type III long terminal repeat-directed gene expression in the respective infected cells. Use of these recombinant vectors permits high-efficiency gene transfer into a wide variety of cells, thereby providing the opportunity to study the biochemical effects associated with tat II and tat III gene expression.     

Identification of a protein encoded by the trans activator gene tatIII of human T-cell lymphotropic retrovirus type III.

The human T-cell lymphotropic virus type III (HTLV-III/LAV) is a retrovirus associated with acquired immune deficiency syndrome. The region on the viral genome that is necessary for trans-activation of the HTLV-III/LAV long terminal repeat called tatIII has previously been determined to lie between nucleotides 5365 and 5607. Here we report that a bacterial fusion protein containing amino acid sequences specified by the first coding exon of the tatIII gene is recognized by some patient antisera. We also demonstrate that lymphoid and epithelial cells that express the trans activator function express a 14-kilodalton (kDa) protein recognized by a patient antiserum that reacts with the bacterial tatIII fusion protein. Cells transiently transfected with a deletion mutant of the trans activator protein produce a 12-kDa protein rather than the 14-kDa protein. These observations indicate that the tatIII region contains a functional gene and is capable of expressing a protein that migrates with an apparent molecular size of 14 kDa in some lymphoid and epithelial cells transfected with plasmids containing the tatIII region. We propose that the product of the trans activator gene be designated p14tat-III.     

Induction of tumor necrosis factor alpha in human neuronal cells by extracellular human T-cell lymphotropic virus type 1 Tax.

To examine the role of human T-lymphotropic virus type 1 (HTLV-1) Tax1 in the development of neurological disease, we studied the effects of extracellular Tax1 on gene expression in NT2-N cells, postmitotic cells that share morphologic, phenotypic, and functional features with mature human primary neurons. Treatment with soluble HTLV-1 Tax1 resulted in the induction of tumor necrosis factor alpha (TNF-alpha) gene expression, as detected by reverse-transcribed PCR and by enzyme-linked immunosorbent assay. TNF-alpha induction was completely blocked by clearance with anti-Tax1 monoclonal antibodies. Furthermore, cells treated with either a mock bacterial extract or with lipopolysaccharide produced no detectable TNF-alpha. Synthesis of TNF-alpha in response to soluble Tax1 occurred in a dose-dependent fashion between 0.25 and 75 nM and peaked within 6 h of treatment. Interestingly, culturing NT2-N cells in the presence of soluble Tax1 for as little as 5 min was sufficient to result in TNF-alpha production, indicating that the induction of TNF-alpha in NT2-N does not require Tax1 to be continually present in the culture medium. Treatment of the undifferentiated parental embryonal carcinoma cell line NT2 with soluble Tax1 did not result in TNF-alpha synthesis, suggesting that differentiation-dependent, neuron-specific factors may be required. These results provide the first experimental evidence that neuronal cells are sensitive to HTLV-1 Tax1 as an extracellular cytokine, with a potential role in the pathology of HTLV-1-associated/tropical spastic paraparesis.     

Analysis of potential phosphorylation sites in human T cell leukemia virus type 1 Tax

The human T cell leukemia virus type 1 (HTLV-1) Tax is a phosphoprotein, however, the contribution of phosphorylation to Tax activity is unknown. Previous studies have shown that phosphorylation of Tax occurs on serine residue(s), within one tryptic fragment, in response to 4-phorbol-12-myristate-13-acetate, in both mouse and human cells. Studies were conducted in multiple cell lines to identify the specific phosphorylated serines as a prelude to functional analysis. The phosphorylation pattern of Tax was found to be different in 293T and COS-7 cells in comparison with MT-4 and Px-1 cells. However, one tryptic fragment remained consistent in comigration analyses among all cell lines. Using selected Tax serine mutants a tryptic fragment containing a serine at residue 113 believed to be the site of phosphorylation of Tax did not comigrate with the common phosphorylated tryptic fragment. Analysis of selected Tax mutants for ability totrans-activate the cytomegalovirus promoter demonstrated mutation of serine 77 to alanine reducedtrans-activation by 90% compared to wild-type Tax. However, examination of the phosphorylation pattern of the serine 77 mutant demonstrated that it is not the site of phosphorylation. These studies demonstrate the importance of using relevant cell lines to characterize the role of phosphorylation in protein function.     

Human T-cell leukemia virus type I trans activator induces class I major histocompatibility complex antigen expression in glial cells

Transfection of the tax gene encoding the trans activator of human T-cell leukemia virus type I into glial line cells induced class I major histocompatibility complex (MHC) antigens on these cells. This occurred through the interaction of tax protein with the gene encoding class I MHC antigens but not through any soluble factors, such as interferons, or factors from glial cells. Since neural cells do not usually express MHC antigens, this novel mechanism may be an intermediate event between viral infection and subsequent immune-mediated pathology in the central nervous system.     

Immunodominant sites of human T cell lymphotropic virus type 1 envelope protein for murine helper T cells

HTLV-I (human T cell lymphotropic virus type 1) is the retrovirus causally related to adult T cell leukemia/lymphoma and is also associated with a neurological disorder, tropical spastic paraparesis, or HTLV-I-associated myelopathy. The development of these two different diseases among HTLV-I-infected individuals may depend in part on differences in their T cell immunity associated with a difference of HLA phenotype. Peptides corresponding to 17 sites in the HTLV-I envelope protein were tested for their antigenicity for lymph node cells from B10.BR, B10.D2, B10.A(5R), and B10.HTT congenic mice, representing four independent MHC haplotypes, immunized with the native envelope protein. Ten of the 17 tested sites were predicted to be amphipathic alpha-helical sites and all of them were found to be antigenic for at least one of the four MHC congenic strains of mice. Three of the 17 sites were amphipathic 3(10)-helical sites and four sites were predicted to be non-helical sites: none of the 3(10)-helical sites were antigenic and only one of four non-predicted sites was found to be immunodominant. Furthermore, three potent immunodominant peptides, V1E1 (342-363), V1E8/SP4a (191-209), and V1E10 (141-156) were also shown to be immunogenic; i.e., these peptides could be used to immunize mice to elicit proliferative responses of lymph node cells to the native HTLV-I envelope protein. Furthermore, these three peptides were able to prime animals for an enhanced antibody response to the native protein. Because this priming followed the same Ir gene control as the proliferative response, it probably reflects the ability of these peptides to prime helper T cells. The localization of immunodominant sites in HTLV-I envelope protein in mice may be useful for finding antigenic and immunogenic sites in humans, for developing a peptide vaccine for the virus, and possibly for aiding in prognosis for the development of different disease manifestations of HTLV-I infection.     

trans activation of nerve growth factor in transgenic mice containing the human T-cell lymphotropic virus type I tax gene.

Three lines of transgenic mice containing the human T-cell lymphotropic virus type I (HTLV-I) tax gene develop neurofibromas composed of perineural fibroblasts (S. H. Hinrichs, M. Nerenberg, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1340-1343, 1987; M. Nerenberg, S. H. Hinrichs, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1324-1327, 1987). Tumors and tumor cell lines derived from these mice produce neurite outgrowth from PC-12 cells and nerve growth factor (NGF), as determined by RNA (Northern) blot analysis and enzyme-linked immunosorbent assays. In vitro cotransfection studies demonstrate that Tax is able to trans activate the NGF promoter in NIH 3T3 fibroblast cells. The major cis-acting tax-responsive element in the NGF promoter (AGGGTGTGACGA) has 92% homology with a tax-responsive element contained within the 21-bp repeats of the HTLV-I long terminal repeat. The receptor for NGF is also expressed in the transgenic tumor cells, suggesting that Tax may activate an autocrine mechanism through the upregulation of NGF.     

A source of glycosylated human T-cell lymphotropic virus type 1 envelope protein: expression of gp46 by the vaccinia virus/T7 polymerase system.

Heterologous expression of the human T-cell lymphotropic virus type 1 (HTLV-1) envelope surface glycoprotein (gp46) in a vaccinia virus/T7 polymerase system resulted in the production of authentic recombinant gp46. Five differentially glycosylated forms of the surface envelope protein were produced by this mammalian system, as demonstrated by tunicamycin inhibition of N-glycosylation and N-glycan removal with endoglycosidase H and glycopeptidase F. These studies revealed that all four potential N-glycosylation sites in gp46 were used for oligosaccharide modification and that the oligosaccharides were mannose-rich and/or hybrid in composition. Conformational integrity of the recombinant HTLV-1 envelope protein was determined by the ability to bind to various HTLV-1-infected human sera and a panel of conformational-dependent human monoclonal antibodies under nondenaturing conditions. Furthermore, this recombinant gp46 was recognized by a series of HTLV-2-infected human sera and sera from a Pan paniscus chimpanzee infected with the distantly related simian T-cell lymphotropic virus STLVpan-p. Maintenance of highly conserved conformational epitopes in the recombinant HTLV-1 envelope protein structure suggests that it may serve as a useful diagnostic reagent and an effective vaccine candidate.     

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.