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At least 343 records · Page 19Linked to original sources
PubMed1991

Isolation of cDNAs for DNA-binding proteins which specifically bind to a tax-responsive enhancer element in the long terminal repeat of human T-cell leukemia virus type I.

One of the gene products of human T-cell leukemia virus type I (HTLV-I), p40tax, activates its own viral transcription in trans through tax-responsive enhancers in viral long terminal repeats. Five species of cDNA clones for proteins that bind to the tax-responsive enhancer element in HTLV-I were isolated from the Jurkat cell library. The beta-galactosidase fusion protein prepared from the lysogen of a clone specifically recognized the cyclic AMP-responsive element in HTLV-I enhancer. The nucleotide sequence of a full-length cDNA clone (TAXREB67) had a coding capacity of 351 amino acids, which contained a basic motif followed by a leucine zipper structure near the carboxy terminus. Its mRNA was detected in human cell lines, including HTLV-I-infected or noninfected hematopoietic cell lines. The mRNA level in Jurkat cells was decreased temporarily by increasing cyclic AMP concentration but increased by increasing Ca2+ concentration. Polyclonal antibodies against the fusion protein specifically recognized a 52-kDa protein in Jurkat cells. Analyses of the function of this protein and its interactions with other cellular factors will be useful to help understand the regulatory mechanism through tax-responsive enhancers in HTLV-I.

Amino Acid Sequence↗
PubMed1991

In vivo transcription of the bovine leukemia virus tax/rex region in normal and neoplastic lymphocytes of cattle and sheep.

Expression of bovine leukemia virus (BLV) has been considered to be blocked at the transcriptional level in vivo, since viral RNA species are not readily detected in freshly isolated leukocytes from BLV-infected animals. However, the presence of a persistent antiviral antibody response in infected animals suggests that some degree of virus expression must occur in vivo. The purpose of this study was to determine whether BLV RNA species could be detected by using the polymerase chain reaction in normal or neoplastic lymphoid cells freshly isolated from naturally or experimentally BLV-infected cattle and sheep, respectively. Primers designed to detect a 2.1-kb doubly spliced BLV tax/rex-specific mRNA were used to amplify cDNA copies of RNA derived from infected animals. The amplified viral product was then detected with a radiolabeled BLV tax/rex-specific probe. BLV-specific RNA was detected readily in freshly isolated peripheral blood leukocytes derived from BLV-seropositive cattle or sheep with persistent lymphocytosis and less readily in peripheral blood leukocytes from BLV-seropositive but hematologically normal animals. BLV-specific RNA was also detected in fresh samples of BLV-induced lymphosarcomas. Normal and neoplastic lymphoid cells from BLV-seronegative animals were uniformly negative under similar conditions. These primers also amplified the same viral product from genomic DNA derived from BLV-seropositive animals, providing further evidence for in vivo transcription and suggesting that BLV RNA-dependent DNA polymerase is capable of reverse transcribing the 2.1-kb mRNA in vivo. The demonstration of transcriptional products of BLV in vivo proves that viral latency in BLV infection is incomplete.

Animals↗
PubMed1991

Transdominant human T-cell lymphotropic virus type I TAX1 mutant that fails to localize to the nucleus.

Human T-cell lymphotropic virus type I (HTLV-I) encodes a 40-kDa nuclear transactivating phosphoprotein, TAX1. The results presented in this study demonstrate that deletion of amino acids 2 through 59 of TAX1 (delta 58 TAX1) decreased transactivation of the HTLV-I long terminal repeat 10- to 20-fold. S1 nuclease analysis revealed that the decrease in transactivation of the HTLV-I long terminal repeat was associated with a lack of RNA synthesis. In contrast to the nuclear localization of the wild-type TAX1 protein, indirect immunofluorescence analysis demonstrated that delta 58 TAX1 failed to localize to the nucleus, indicating that the TAX1 nuclear localization sequence is present in amino acids 2 through 59. Cotransfection of wild-type and mutant TAX1 DNAs resulted in the cytoplasmic accumulation of TAX1 and a 25-fold decrease in transactivation. Although several possibilities which may account for this transdominant effect exist, we favor a model in which delta 58 TAX1 interferes with the nuclear localization of wild-type TAX1 protein, perhaps by forming heterodimer complexes.

Amino Acid Sequence↗
PubMed1992

Bovine leukemia virus gene expression in vivo.

The in vivo transcriptional status of bovine leukemia virus was assessed at three stages of infection during the progression of the disease: aleukemic stage, persistent lymphocytosis, and leukemia/lymphosarcoma. Bovine leukemia virus transcripts could be amplified from total or cytoplasmic enriched lymphocyte RNA by reverse transcription polymerase chain reaction in cells from all but a few aleukemic animals. With primer pairs diagnostic for differentially spliced transcripts (full length-genomic, envelope, tax/rex, and alternatively spliced), a trend toward exclusion of both full-length and envelope RNAs, with retention of the tax/rex message, appears as leukemia/lymphosarcoma develops.

Base Sequence↗
PubMed1992

Mutational analysis of human T-cell leukemia virus type I Tax: regions necessary for function determined with 47 mutant proteins.

We have made 47 mutations that span the length of the human T-cell leukemia virus type I (HTLV-I) Tax open reading frame. Of the 47 mutations, 38 were substitutions of single amino acids, 5 were missense changes in two or more amino acids, and 4 were deletions. A subset of these mutations includes individual changes of all 26 naturally occurring serines to alanines. By assaying each mutant protein separately on the HTLV-I long terminal repeat (LTR) and the human immunodeficiency virus type 1 (HIV-1) LTR in parallel, we were able to identify regions of Tax selectively necessary for each promoter. A small region in the carboxyl terminus, amino acids 315 to 325, was found to be selectively important for activation of the HTLV-I LTR. Three changes at serine 113, serine 160, and serine 258 were found to specifically affect function on the HIV-1 LTR. Surprisingly, we found that the great preponderance of missense changes (32 of 42) in Tax did not affect function.

Animals↗
PubMed1992

Strong trans activation of the human cytomegalovirus major immediate-early enhancer by p40tax of human T-cell leukemia virus type I via two repetitive tax-responsive sequence elements.

The immediate-early 1 and 2 gene locus of human cytomegalovirus (HCMV) that encodes trans-activator proteins with effects on both homologous and heterologous promoters is expressed under control of a complex enhancer/promoter regulatory region. This enhancer contains four types of repetitive sequence elements with 17, 18, 19, and 21 bp that bind cellular transcription factors. Although the HCMV enhancer acts as a powerful stimulator of transcription in most cell types examined, human T cells do not support strong activity. The present study demonstrates that the tax gene product of human T-cell leukemia virus type I trans activates the major enhancer of HCMV more than 60-fold in the T-cell line Jurkat. When a series of chloramphenicol acetyltransferase expression plasmids containing synthetic oligonucleotides with the 17-, 18-, 19-, or 21-bp motif upstream of a minimal immediate-early 1 and 2 gene promoter was tested, two of the four repeat motifs could be identified as Tax-responsive elements. Both the 18- and the 19-bp motifs were able to act as strong Tax-responsive elements even when they were present as single copies. Thus, in addition to interacting with human immunodeficiency virus, HCMV is able to interact with a second retrovirus of clinical importance.

Avian Sarcoma Viruses↗
PubMed1993

Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.

The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown.

Alternative Splicing↗
PubMed1993

Identification and characterization of an extended Tax protein in human T-cell lymphotropic virus type II subtype b isolates.

The tax gene sequence of human T-cell lymphotropic virus type II isolate G12 (HTLV-IIG12) was found to encode an extended Tax protein when compared with that of HTLV-IIMoT. In vitro transcription-translation of the HTLV-IIG12 tax gene produced a 40-kDa Tax protein that specifically reacted with serum specimens from HTLV-II-infected individuals. Limited sequence analysis demonstrated that isolates with an extended Tax protein were all HTLV-II subtype b (HTLV-IIb). Therefore, the extended Tax protein appears to be a unique characteristic of most HTLV-IIb isolates and may be useful in designing immunoassays to distinguish between HTLV-IIa and HTLV-IIb.

Amino Acid Sequence↗
PubMed1993

Influence of human T-cell leukemia virus type I tax and rex on interleukin-2 gene expression.

The X region of human T-cell leukemia virus type I (HTLV-I) encodes two proteins that regulate viral gene expression. The tax protein is the product of the transactivator gene and has been shown to up-regulate the expression of some cellular genes controlling T-cell replication, including that of the interleukin-2 (IL-2) T-cell growth hormone and the alpha chain of its receptor (IL-2R). Several studies have shown that tax transactivation of the IL-2R alpha-chain promoter is mediated by binding sites for the transcriptional activator NF-kappa B, and this mechanism has also been implicated in the tax activation of IL-2 promoter activity. The rex gene product of HTLV-I regulates viral protein production by influencing mRNA expression and has been implicated in the stabilization of IL-2R alpha-chain mRNA. In the present studies, the ability of the tax and rex proteins to transactivate IL-2 gene expression has been reinvestigated. The ability of the tax protein to transactivate IL-2 promoter activity appears, at least in part, to be mediated by the recognition sequence for a DNA-binding complex known as CD28RC. Consistent with this hypothesis is the observation that tax-mediated activation of IL-2 gene expression is resistant to the immunosuppressive affects of cyclosporin A, a property postulated for the CD28RC binding complex. Unexpectedly, this tax-mediated up-regulation of IL-2 expression is synergized by the presence of the rex protein. These findings demonstrate that transactivation of IL-2 gene expression by tax is augmented by mechanisms distinct from NF-kappa B and raise the possibility that rex, as well as tax, contributes to the oncogenic capability of HTLV-I by altering the expression of the IL-2 gene in T cells infected with this retrovirus.

Antigens, CD↗
PubMed1994

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.

Base Sequence↗
PubMed1995

Human T-cell leukemia virus type II nucleotide sequences between env and the last exon of tax/rex are not required for viral replication or cellular transformation.

Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) and bovine leukemia virus contain a region of approximately 600 nucleotides located 3' to the env gene and 5' to the last exon of the tax and rex regulatory genes. This region was originally termed nontranslated or untranslated (UT) since it did not appear to be expressed. Several studies have identified novel mRNAs in HTLV-I-, HTLV-II-, a bovine leukemia virus-infected cells that splice into open reading frames (ORFs) contained in the UT region and, thus, have the potential to produce proteins that might contribute to the biological properties of these viruses. The HTLV-II infectious molecular clone pH6neo has several ORFs in the UT region (nucleotides 6641 to 7213) and a large ORF which overlaps the third exon of tax/rex. To investigate the importance of these ORF-containing sequences on viral replication and transformation in cell culture, proviral clones containing deletions in UT (pH6neo delta UT) or a stop codon insertion mutation (pH6neoST) were constructed. Lymphoid cells were transfected with mutant proviral constructs, and stable cell clones, designated 729pH6neo delta UT and 729pH6neoST, were characterized. Viral protein production, reverse transcriptase activity, and the capacity to induce syncytia were indistinguishable from cells transfected with the wild-type clone. Finally, 729pH6neo delta UT- and 729pH6neoST-producer cells cocultured with primary blood T lymphocytes resulted in cellular transformation characteristic of HTLV. These results indicate that putative protein-coding sequences between env and the last exon of tax/rex are not required for viral replication or transformation in cell culture.

Amino Acid Sequence↗
PubMed1995

Genetically simpler bovine leukemia virus derivatives can replicate independently of Tax and Rex.

Retrovirus genomes have a conserved modular organization that consists of trans-acting gag, pol, and env genes that function through cis-acting sequences to replicate the RNA genome to the DNA provirus. Genetically more complex retroviruses also encode regulatory genes and cis-acting sequences that are essential for their replication. We sought to convert a more complex retrovirus into a simpler retrovirus derivative that can replicate. We constructed novel, hybrid retrovirus vectors to replicate the gag, pol, and env genes of the more complex bovine leukemia virus (BLV) in the absence of regulatory genes and cis-acting response sequences. Most of the cis-acting sequences involved in the replication and regulation of BLV were replaced by the cis-acting transcriptional control sequences of a simpler retrovirus, spleen necrosis virus. We found that the resulting hybrid BLV derivatives can replicate independently of BLV Tax and Rex and the Tax and Rex cis-acting response sequences, as measured by successive passages of virus on target cells, detection of provirus sequences, and analyses of provirus and encapsidated RNAs.

Genes, pX↗
PubMed1995

Tax mutation associated with tropical spastic paraparesis/human T-cell leukemia virus type I-associated myelopathy.

Tumaco, Colombia, is an area with elevated rates of tropical spastic paraparesis/human T-cell leukemia virus type I (HTLV-I)-associated myelopathy (TSP/HAM). We have identified a mutation in nucleotide 7959 of the tax gene of 14 Tumaco HTLV-I isolates (14 positive of 14 tested) that was present in 5 of 14 (35%) TSP/HAM patients from Japan and in 8 of 11 (72%) TSP/HAM patients from other geographic locations. In contrast, this mutation was found in only 2 of 21 (9.5%) HTLV-I-infected subjects outside of Tumaco who did not have TSP/HAM. tax clones with nucleotide mutations including one at nucleotide 7959 showed a greater ability to transactivate the HTLV-I U3 promoter. However, this effect was not observed when two clones that differed only in nucleotide 7959 were compared. These results suggest that HTLV-I-infected individuals carrying isolates with this tax mutation are at higher risk for developing TSP/HAM.

Colombia↗
PubMed1996

Repression of the NF1 gene by Tax may expain the development of neurofibromas in human T-lymphotropic virus type 1 transgenic mice.

In familial neurofibromatosis type 1 (NF1), individuals with a germ line-transmitted NF1 mutation develop multiple neurofibromas. To explain the observation that transgenic mice expressing the human T-lymphotropic virus type 1 (HTLV-1) tax gene under the control of the viral regulatory element also develop multiple neurofibromas, we demonstrate that the Tax trans-regulator can functionally repress NF1 gene expression through a cis-acting element located immediately upstream of its transcriptional start site, thereby allowing the development of benign neurofibromas without the need for direct mutations in NF1. We propose that such a mechanism would suffice to epigenetically alter NF1 gene expression. The fact that transgenic animals have localized rather than diffuse neurofibroma formation, however, suggests that additional genetic or epigenetic events may be required for neurofibroma formation.

3T3 Cells↗
PubMed1996

The tax gene of human T-cell leukemia virus type 2 is essential for transformation of human T lymphocytes.

The mechanism of human T-cell leukemia virus (HTLV)-mediated transformation and induction of malignancy is unknown; however, several studies have implicated the viral gene product, Tax. Conclusive evidence for the role of Tax in the HTLV malignant process has been impeded by the inability to mutate tax in the context of an infectious virus and dissociate viral replication from cellular transformation. To circumvent this problem we constructed a mutant of HTLV type 2 (HTLV-2) that replicates by a Tax-independent mechanism. For these studies, the Tax response element in the viral long terminal repeat was replaced with the cytomegalovirus immediate-early promoter enhancer (C-enh). Transcription of the chimeric HTLV-2 (HTLVC-enh) was efficiently directed by this heterologous promoter. Also, the chimeric virus transformed primary human T lymphocytes with an efficiency similar to that of wild-type HTLV-2. A tax-knockout virus, termed HTLVC-enhDeltaTax, was constructed to directly assess the importance of Tax in cellular transformation. Transfection and infection studies indicated that HTLVC-enhDeltaTax was replication competent; however, HTLVC-enhDeltaTax failed to transform primary human T lymphocytes. We conclude that Tax is essential for HTLV-mediated transformation of human T lymphocytes. Furthermore, this chimeric HTLV, that replicates in the absence of Tax, should facilitate studies to determine the precise mechanism of T-lymphocyte transformation by HTLV.

Cell Line↗
PubMed1996

A cooperative interaction of human T-cell leukemia virus type 1 Tax with the p21 cyclin-dependent kinase inhibitor activates the human immunodeficiency virus type 1 enhancer.

Interactions between the Tax transactivator of human T-cell leukemia virus type 1 (HTLV-1) and a cell cycle regulatory protein have been examined. We report cooperative stimulation of human immunodeficiency virus type 1 gene expression by Tax and a regulator of cell cycle progression, the p21 cyclin-dependent kinase inhibitor (CKI). This cooperativity results from the effect of p21 on transcriptional coactivation by Tax-induced NF-kappaB. This effect was abrogated by a mutation in Tax which specifically eliminated NF-kappaB induction, was inhibitable by IkappaB-alpha, and was markedly reduced in human immunodeficiency virus reporter plasmids with mutant kappaB sites. These studies demonstrate that transcriptional activation by Tax is influenced by cell cycle regulatory proteins.

Cyclin-Dependent Kinase Inhibitor p21↗