Human T-cell Leukemia Virus Type I Long Terminal Repeat Research Articles

A new sensitive and quantitative HTLV-I-mediated cell fusion assay in T cells

Similar to several other viruses, human T cell leukemia virus type I (HTLV-I) induces the formation of multinucleated giant cells (also known as syncytium) when amplified in tissue culture. These syncytia result from the fusion of infected cells with uninfected cells. Due to the intrinsic difficulty of infecting cells with cell-free HTLV-I virions, syncytium formation has become an important tool in the study of HTLV-I infection and transmission. Since most HTLV-I-based cell fusion assays rely on the use of non-T cells, the aim of this study was to optimize a new HTLV-I-induced cell fusion assay in which HTLV-I-infected T cell lines are co-cultured with T cells that have been transfected with an HTLV-I long terminal repeat (LTR) luciferase reporter construct. We demonstrate that co-culture of various HTLV-I-infected T cells with different transfected T cell lines resulted in induction of luciferase activity. Cell-to-cell contact and expression of the viral gp46 envelope protein was crucial for this induction while other cell surface proteins (including HSC70) did not have a significant effect. This quantitative assay was shown to be very sensitive. In this assay, the cell fusion-mediated activation of NF-κB and the HTLV-I LTR occurred through previously described Tax-dependent signaling pathways. This assay also showed that cell fusion could activate Tax-inducible cellular promoters. These results thus demonstrate that this new quantitative HTLV-I-dependent cell fusion assay is versatile, highly sensitive, and can provide an important tool to investigate cellular promoter activation and intrinsic signaling cascades that modulate cellular gene expression.

Enhanced Activation of Tax-dependent Transcription of Human T-cell Leukemia Virus Type I (HTLV-I) Long Terminal Repeat by TORC3

Tax, a protein encoded by the env-pX gene of human T-cell leukemia virus type I (HTLV-I), interacts with various host cell transcription factors. Tax activates transcription from the long terminal repeat (LTR) of HTLV-I through association with cyclic AMP-responsive element-binding protein (CREB). Here, we present evidence that transducer of regulated cyclic AMP-response element-binding protein 3 (TORC3), a co-activator of CREB, is involved in Tax-induced transcriptional activation from the HTLV-I LTR. By using a luciferase assay system, we show that TORC3 alone can enhance transcription from the HTLV-I LTR, as well as from a cellular cyclic AMP-response element (CRE). Interestingly, we find that co-expression of TORC3 and Tax dramatically increased transcriptional activation at the HTLV-I LTR. We also show by glutathione S-transferase pull-down and co-immunoprecipitation experiments that TORC3 interacts with Tax. Using deletion mutant analysis, we identify the Tax interaction domain of TORC3 as a region spanning from amino acid 1 to 103, which contains a coiled-coil domain. These results provide important clues toward understanding the molecular mechanism of Tax-dependent transcriptional activation of the HTLV-I LTR.

Activation of HTLV-I gene transcription by protein tyrosine phosphatase inhibitors

Human T-cell leukemia virus type I (HTLV-I) transcription generally depends on the ability of the viral Tax protein to bind the CREB transcription factor and form an active complex by recruiting CBP/p300 coactivators to the long terminal repeat (LTR). Studies have demonstrated that T-cell activating agents that stimulate CREB are potent inducers of HTLV-I transcription. Herein, we demonstrate that bpV[pic], a protein tyrosine phosphatase (PTP) inhibitor activates the HTLV-I LTR in the presence and absence of Tax expression. Optimal activation occurred at 8 h and was synergistic with forskolin or PGE 2. Infected cell lines and cells transfected with HTLV-I proviral DNA were equally responsive to the synergistic effect of bpV and forskolin on HTLV-I gene expression. Activation of the LTR by bpV[pic] was T-cell receptor-independent, but required ZAP70, calcineurin activity and functional calcium entry. Inhibition of the SHP-1 PTP was suggested to be important. Transfection experiments with a CREB dominant-negative mutant and with isolated TRE1- or CREB-responsive reporter constructs and treatment with the MDL-12,330A adenylate cyclase inhibitor all supported the involvement of a CREB/ATF family member in this bpV-dependent activation of the HTLV-I LTR, although CREB itself did not seem to be involved. Analysis of HTLV-I reporter constructs containing mutated CREB-binding sites also implied the involvement of another element in this activation. These results demonstrate for the first time a powerful effect of PTP inhibitors on HTLV-I LTR activity and suggest participation of both CREB-dependent and -independent pathways in this activation.

Evidence that protein kinase A activity is required for the basal and tax-stimulated transcriptional activity of human T-cell leukemia virus type-I long terminal repeat

The present study was undertaken to investigate the role of protein kinase A (PKA) in the control of human T-cell leukemia virus type-I (HTLV-I) long terminal repeat (LTR) expression, since this issue is still controversial. For this purpose we employed two human T-cell lines; the Jurkat cells in which long exposure to diBu-cAMP severely down-regulated the catalytic subunit of PKA (PKA-C), and H-9 cells in which such exposure markedly increased PKA-C level. Transient transfection assays revealed that addition of diBu-cAMP 1 h before or after transfection profoundly increased HTLV-I LTR directed CAT expression and synergistically enhanced its stimulation by the viral transactivator tax gene product in both cell lines. However longer exposure to diBu-cAMP before transfection reduced LTR-CAT expression to below its basal level and completely abolished its stimulation by tax in Jurkat cells, and this diBu-cAMP inhibitory effect could be abrogated by co-transfection of a PKA-C expressing vector. By contrast, in H-9 cells, this long exposure to diBu-cAMP continued enhancing LTR-CAT expression and its tax-mediated transactivation, and this stimulatory effect of diBu-cAMP could be diminished by the PKA-specific inhibitor N-[2-( p-bromocinnamylamine)ethyl]-5- isoquinolinsulfonamide (H-89). Notably, in the absence of diBu-cAMP treatment H-89 reduced LTR-CAT expression to below its basal level and prevented its stimulation by tax in both cell lines. Together these findings indicate not only that cAMP-activated PKA stimulates HTLV-I LTR expression and its transactivation by tax, but even in the absence of PKA activating signals the basal HTLV-I LTR expression as well as its stimulation by tax are both dependent on a basal PKA activity.

Repression of Transcription From the Human T-Cell Leukemia Virus Type I Long Terminal Repeat and Cellular Gene Promoters by Wild-Type p53

Human T-cell leukemia virus type-I (HTLV-I), the etiologic agent of adult T-cell leukemia (ATL) transforms human T cells both in vivo and in vitro. However, the long latency period between infection and development of ATL, as well as the small fraction of the infected population that actually develops this disease, suggest that factors in addition to the virus are involved in its pathogenesis. Mutation of tumor suppressor gene p53 has been found in both HTLV-I–transformed T-cell lines and ATL cases at relatively low frequency. However, increasing evidence supports p53 functional impairment in HTLV-I–transformed T cells. Tax, the major transactivator of HTLV-I, is critical for the initial events involved in transformation. We have considered the possibility that p53 may regulate transcription of viral and cellular genes important for viral replication and transformation. Inactivation of p53 function might then permit constitutive expression of these viral and cellular genes. We have investigated the effects of wild-type and mutant p53 on Tax-mediated activation of the HTLV-I long terminal repeat (LTR) and the promoters of several cellular genes including the interleukin (IL)-1α, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF ), and IL-2 receptor α chain gene. Jurkat, HuT78, and U937 cells were cotransfected with plasmids containing a chloramphenicol acetyltransferase (CAT ) reporter gene under viral or cellular promoter control and the Tax expression vector, in addition to vectors for a wild-type or mutant p53. Wild-type p53 is a potent repressor of viral and cellular activation by Tax. Mutations within p53 severely inhibit this downregulation. We also show that wild-type p53 suppresses transcription from the HTLV-I LTR in Jurkat-Tax, a T-cell line stably expressing Tax, and MT-2, a HTLV-I–transformed T-cell line. Wild-type, but not mutant, p53 interfered with the binding of TATA-binding protein (TBP) to the TATA motif of the HTLV-I LTR. These results suggest that p53 inactivation may lead to upregulation of viral and cellular genes and may also be important for establishment of productive viral infection and development of ATL.

Activation of the HTLV-I Long Terminal Repeat by the Hepatitis B Virus X Protein

The human T-cell leukemia virus type I (HTLV-I) Tax protein and the hepatitis B virus (HBV) X protein have each been shown to activate transcription of their respective viral promoters as well as a subset of cellular gene promoters. Here we show that the HTLV-I long terminal repeat (LTR) is responsive to HBV X transactivation. Maximum levels of X-mediated transactivation of the LTR were 8-fold. An X-responsive-region (XRR) of the LTR is located between nucleotides −355 and −276 and contains an AP-2 binding site, a previously recognized X-responsive element. We demonstrated that Tax and X synergize to activate transcription from the HTLV-I LTR, although the AP-2 binding site was not required for this synergy. These results raise the possibility that the HBV X protein may affect the level of HTLV-I gene expression in co-infected individuals.

CAMP-response Element-binding Protein Induces Directed DNA Bending of the HTLV-I 21-Base Pair Repeat

Gene expression from the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is mediated by three cis-acting regulatory elements known as 21-base pair (bp) repeats in addition to the transactivator protein Tax. Each of the 21-bp repeats contain nucleotide sequences which are homologous to a cAMP response element (CRE) which bind members of the ATF/CREB family of transcription factors. In this study, we investigated whether CREB alone or in the presence of Tax was able to induce DNA structural changes when bound to CRE sites in the HTLV-I 21 bp, the cellular somatostatin promoter, or a hybrid CRE construct comprised of both the somatostatin and 21-bp repeat sequences. Circular permutation analysis indicated that CREB was able to induce DNA flexure upon binding to each of these elements. However, phasing analysis, which is a more sensitive method to determine the degree and orientation of directed DNA bending, demonstrated that CREB induced DNA bending of the HTLV-I 21-bp repeat and the hybrid CRE but not the somatostatin CRE. The addition of Tax did not change CREB-mediated bending of the 21-bp repeat or the hybrid CRE although it markedly increased the amount of CREB bound to each of these DNA elements. These results indicate that sequence motifs flanking the CRE in the 21-bp repeat are critical for inducing DNA structural changes and that these changes are likely important in mediating Tax activation of the HTLV-I LTR.

Protein domains involved in both in vivo and in vitro interactions between human T-cell leukemia virus type I tax and CREB.

Gene expression from the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is mediated by three cis-acting regulatory elements known as 21-bp repeats and the transactivator protein Tax. The 21-bp repeats can be subdivided into three motifs known as A, B, and C, each of which is important for maximal gene expression in response to Tax. The B motif contains nucleotide sequences known as a cyclic AMP response element (CRE) or tax-response element which binds members of the ATF/CREB family of transcription factors. Though mutations of this element in the HTLV-I LTR eliminate tax activation, Tax will not activate most other promoters containing these CRE sites. In this study, we investigated the mechanism by which Tax activates gene expression in conjunction with members of the ATF/CREB family. We found that Tax enhanced the binding of one member of the ATF/CREB family, CREB 1, to each of the three HTLV-I LTR 21-bp repeats but not another member designated CRE-BP1 or CREB2. Tax enhanced the binding of CREB1 to nonpalindromic CRE binding sites such as those found in the HTLV-I LTR, but Tax did not enhance the binding of CREB1 to palindromic CRE binding sites such as found in the somatostatin promoter. This finding may help explain the failure of Tax to activate promoters containing consensus CRE sites. These studies were extended by use of the mammalian two-hybrid system. Tax was demonstrated to interact directly with CREB1 but not with other bZIP proteins, including CREB2 and Jun. Site-directed mutagenesis of both Tax and CREB1 demonstrated that the amino terminus of Tax and both the basic and the leucine zipper regions of CREB1 were required for direct interactions between these proteins both in vivo and in vitro. This interaction occurred in vivo and thus did not require the presence of the HTLV-I 21-bp repeats, as previously suggested. These results define the domains required for interaction between Tax and CREB that are likely critical for the activation of HTLV-I gene expression.

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.

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.

Phorbol esters modulate the phosphorylation of human T-cell leukemia virus type I Tax

The Tax protein from human T-cell leukemia virus type I (HTLV-I) is a 40-kDa phosphoprotein capable of activating transcription from its own long terminal repeat (LTR), as well as increasing the transcription of cellular genes. Transcriptional activation of the HTLV-I LTR has been demonstrated via a cyclic-AMP-responsive element within the 21-bp Tax-responsive elements of the LTR. Phorbol esters also upregulate expression via the LTR. Since phosphorylation of Tax may play a role in these processes, we investigated the relative effects of kinase-stimulating agents on 32P incorporation into Tax. Our studies demonstrated that the phorbol ester 4 beta-phorbol-12 beta-myristate-13 alpha-acetate greatly stimulated Tax phosphorylation in a time- and dose-dependent manner. In contrast, 8-bromoadenosine 3'-5'-cyclic monophosphate induced little stimulation of Tax phosphorylation. Tax phosphorylation occurred only on serine residues and was mapped to a single tryptic fragment in both Tax-producing human lymphocytes and mouse fibroblast cells.

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.

Functional analysis of two long terminal repeats from the HTLV-I retrovirus

Human T-cell leukemia virus type I (HTLV-I) is associated with a large spectrum of clinical manifestations in man. Viral and host factors are probably involved in determining the consequences of infection. Although most of the genome of HTLV-I appears remarkably stable, considerable variation is observed in the long terminal repeat (LTR) which harbors the promoter region. So far, no correlation between specific mutations and pathogenesis has been found, and the current opinion is that sequence variations reflect the geographical origin of the isolate more than the associated pathology. To assess whether the mutations observed between two HTLV-I LTRs were functionally significant, two LTRs, which differ by ten mutations, were coupled to the highly sensitive eukaryotic luciferase-encoding reporter gene, luc, and tested by transfection in a variety of cell lines. Marked differences in promoter activity were observed in some of the cells tested, whereas in others both LTRs were equally active. This result demonstrates that the minor differences observed between two HTLV-I LTRs can affect the activity level of the promoter in some cellular environments, a result which could point to the LTR as one determinant of HTLV-I cell tropism in vivo.

An RNA secondary structure juxtaposes two remote genetic signals for human T-cell leukemia virus type I RNA 3'-end processing

Sequence analysis of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) does not reveal a polyadenylation consensus sequence, AAUAAA, close to the polyadenylation site at the 3' end of the viral RNA. Using site-directed mutagenesis, we demonstrated that two cis-acting signals are required for efficient RNA processing in HTLV-I LTR: (i) a remote AAUAAA hexamer at a distance of 276 nucleotides upstream of the polyadenylation site, and (ii) the 20-nucleotide GU-rich sequence immediately downstream from the poly(A) site. It has been postulated that the folding of RNA into a secondary structure juxtaposes the AAUAAA sequence, in a noncontiguous manner, to within 14 nucleotides of the polyadenylation site. To test this hypothesis, we introduced deletions and point mutations within the U3 and R regions of the LTR. RNA 3'-end processing occurred efficiently at the authentic HTLV-I poly(A) site after deletion of the sequences predicted to form the secondary structure. Thus, the genetic analysis supports the hypothesis that folding of the HTLV-I RNA in the U3 and R regions juxtaposes the AAUAAA sequence and the poly(A) site to the correct functional distance. This unique arrangement of RNA-processing signals is also found in the related retroviruses HTLV-II and bovine leukemia virus.

Role of protein kinase A in tax transactivation of the human T-cell leukemia virus type I long terminal repeat

The human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is inducible both by the retroviral tax gene product and by cyclic AMP in the murine thymoma S49 cell line. The cis-acting sequences that control transcriptional induction by tax and by cyclic AMP are in close proximity within the HTLV-I promoter. By using a protein kinase A (PKA)-deficient S49 mutant cell line, the response of the viral promoter to cyclic AMP was shown to depend on PKA, whereas the response to tax did not require the activity of this enzyme. Transactivation of the HTLV-I LTR by tax, however, decreased in PKA-deficient and adenylate cyclase-deficient cells. The evidence presented supports largely independent mechanisms of promoter induction by cyclic AMP and tax but also suggests a role for PKA-mediated phosphorylation in the regulation of HTLV-I LTR-driven gene expression by tax.

Protein kinase A-dependent binding of a nuclear factor to the 21-base-pair repeat of the human T-cell leukemia virus type I long terminal repeat

The long terminal repeat (LTR) of the human T-cell leukemia virus type I (HTLV-I) contains an imperfect repeat of 21 nucleotides which governs the response to the virus trans-activator protein tax and to cyclic AMP. In a murine thymocyte cell line defective in the catalytic subunit of protein kinase A, the response of the HTLV-I LTR to cyclic AMP is abolished and the response to tax is substantially diminished. This report shows that a factor present in nuclear extracts of wild-type cells binds to the HTLV-I 21-nucleotide sequence and that this binding activity is missing from the extracts of protein kinase A-defective cells. Treatment of nuclear extracts of protein kinase A-defective cells with the bovine protein kinase A catalytic subunit restores the binding activity, whereas treatment of wild-type nuclear extracts with a protein phosphatase destroys the binding activity. The binding factor is referred to as protein kinase A-dependent factor (PKAF). These results indicate that in murine thymocytes the response of the HTLV-I LTR to cyclic AMP depends upon the binding of a phosphorylated protein to the 21-nucleotide repeat sequence and that the response to tax is partially dependent upon binding of the phosphorylated protein. The results suggest a model in which the phosphorylation of a transcription factor by protein kinase A regulates HTLV-I gene expression.

Activation of the human T-cell leukemia virus type I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate and by tax (p40x) occurs through similar but functionally distinct target sequences.

One potent transcriptional activator of human T-cell leukemia virus type I (HTLV-I) is virally encoded protein Tax (p40x). p40x trans-activates HTLV-I through the long terminal repeat (LTR) by using a triply repeated 21-base-pair sequence as the target. In this report we have characterized the induction of the HTLV-I LTR by 12-O-tetradecanoylphorbol-13-acetate (TPA). By assaying progressively deleted mutations in the HTLV-I LTR, we have delimited a 60-base-pair sequence in the LTR which is capable of conferring TPA responsiveness, but not p40x responsiveness, to heterologous promoters in a position-independent fashion. This HTLV-I TPA-responsive element is specifically recognized by preexisting factors from uninfected cells. We show that activation of this sequence by phorbol ester does not require de novo cellular protein synthesis. When the HTLV-I LTR was simultaneously activated by both Tax and TPA, an additive effect was seen. This suggests the use of distinct regulatory pathways by the two respective trans-activators.

Characterization of cellular factors that interact with the human T-cell leukemia virus type I p40x-responsive 21-base-pair sequence.

Transcriptional activation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) by viral protein p40x requires a 21-base-pair (bp) sequence which is repeated three times within the LTR. This sequence contains a core octanucleotide (TGACGTCT) which has been attributed to be a cyclic-AMP (cAMP)-responsive element. We demonstrate here that the HTLV-I LTR can be specifically stimulated by cAMP regulators and have identified four proteins in HeLa cells that bind to the HTLV-I 21-bp sequence. We correlated the in vitro binding and transcriptional activity of one of these cellular factors (Mr, 180,000) to the trans-activation of the HTLV-I LTR by p40x. Point mutations were generated within the cAMP octanucleotide of the HTLV-I 21-bp sequence that simultaneously abolished biological responsiveness to trans-activation by p40x and to stimulation by cAMP. We found that these mutations also eliminated the binding of the 180-kilodalton HeLa factor to the HTLV-I 21-bp element. In the absence of a demonstrable DNA-binding property for p40x, we hypothesize that cellular proteins are involved, possibly through signal transduction pathways, in its trans-activation of responsive promoters.

12- O-Tetradecanoylphorbol-13-acetate induces the enhancer function of human T-cell leukemia virus type I

Phorbol esters were employed in studies on the molecular mechanism of the induction of expression of human T-cell leukemia virus type I (HTLV-I) by a tumor promoter, 12- O-tetradecanoylphorbol-13-acetate (TPA). Experiments using the chloramphenicol acetyltransferase (CAT) system showed that CAT expression directed by the long terminal repeat (LTR) of HTLV-I was induced by TPA, but not by 4α-phorbol-12,13-didecanoate, which is not an activator of protein kinase C, and that like other known enhancers, irrespective of its position and orientation, a 230-bp fragment in the U3 region of the HTLV-I LTR confers susceptibility to induction by TPA.

Identification of p40x-responsive regulatory sequences within the human T-cell leukemia virus type I long terminal repeat.

Distinct transcriptional regulatory sequences located within the upstream sequences required for p40x trans-activation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) were chemically synthesized and cloned upstream of the basal HTLV-I LTR promoter. Plasmids containing a single 21-base-pair (bp) repeat were weakly inducible by p40x. The level of trans-activation by p40x was increased when two (30-fold) or three (40-fold) 21-bp repeats were present in the upstream control region. In the mutant containing two 21-bp repeats, the upstream 21-bp repeat could be positioned in either the sense (30-fold) or the antisense (16-fold) orientation. Plasmids containing a 51-bp repeat element, which included a single 21-bp repeat, were induced to levels similar to that obtained with the 21-bp repeat sequence alone. Template DNAs containing a single copy of the HTLV-I sequences between -117 and -160 were stimulated approximately 10-fold by p40x when one copy of the 21-bp element was located downstream.