Tax Expression Plasmid Research Articles

Human T-lymphotropic Virus Type 1 p30 Interacts with REGγ and Modulates ATM (Ataxia Telangiectasia Mutated) to Promote Cell Survival

Human T-lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T cell leukemia/lymphoma and a variety of inflammatory disorders. HTLV-1 encodes a nuclear localizing protein, p30, that selectively alters viral and cellular gene expression, activates G(2)-M cell cycle checkpoints, and is essential for viral spread. Here, we used immunoprecipitation and affinity pulldown of ectopically expressed p30 coupled with mass spectrometry to identify cellular binding partners of p30. Our data indicate that p30 specifically binds to cellular ATM (ataxia telangiectasia mutated) and REGγ (a nuclear 20 S proteasome activator). Under conditions of genotoxic stress, p30 expression was associated with reduced levels of ATM and increased cell survival. Knockdown or overexpression of REGγ paralleled p30 expression, suggesting an unexpected enhancement of p30 expression in the presence of REGγ. Finally, size exclusion chromatography revealed the presence of p30 in a high molecular mass complex along with ATM and REGγ. On the basis of our findings, we propose that HTLV-1 p30 interacts with ATM and REGγ to increase viral spread by facilitating cell survival.

Site-specific Phosphorylation Differentiates Active from Inactive Forms of the Human T-cell Leukemia Virus Type 1 Tax Oncoprotein

Site-specific Phosphorylation Differentiates Active from Inactive Forms of the Human T-cell Leukemia Virus Type 1 Tax Oncoprotein

Tax-dependent Displacement of Nucleosomes during Transcriptional Activation of Human T-Cell Leukemia Virus Type 1

The human T-cell leukemia virus type 1 (HTLV-1) is integrated into the host cell DNA and assembled into nucleosomes. Within the repressive chromatin environment, the virally encoded Tax protein mediates the recruitment of the coactivators CREB-binding protein/p300 to the HTLV-1 promoter, located within the long terminal repeats (LTRs) of the provirus. These proteins carry acetyltransferase activity that is essential for strong transcriptional activation of the virus in the context of chromatin. Consistent with this, the amino-terminal tails of nucleosomal histones at the viral promoter are acetylated in Tax-expressing cells. We have developed a system in which we transfect Tax into cells carrying integrated copies of the HTLV-1 LTR driving the luciferase gene to analyze changes in "activating" histone modifications at the LTR. Unexpectedly, Tax transactivation led to an apparent reduction of these modifications at the HTLV-1 promoter and downstream region that correlates with a similar reduction in histone H3 and linker histone H1. Micrococcal nuclease protection analysis showed that less LTR-luciferase DNA is nucleosomal in Tax-expressing cells. Furthermore, nucleosome depletion correlated with RNA polymerase II recruitment and loss of SWI/SNF. The M47 Tax mutant, deficient in HTLV-1 transcriptional activation, was also defective for nucleosome depletion. Although this mutant formed complexes with CREB and p300 at the HTLV-1 promoter in vivo, it was unable to mediate RNA polymerase II recruitment or SWI/SNF displacement. These results support a model in which nucleosomes are depleted from the LTR and transcribed region during Tax-mediated transcriptional activation and correlate RNA polymerase II recruitment with nucleosome depletion.

A Novel NF-κB Pathway Involving IKKβ and p65/RelA Ser-536 Phosphorylation Results in p53 Inhibition in the Absence of NF-κB Transcriptional Activity

Nuclear factor kappaB (NF-kappaB) plays an important role in regulating cellular transformation and apoptosis. The human T-cell lymphotropic virus type I protein, Tax, which is critical for viral transformation, modulates the transcription of several cellular genes through activation of NF-kappaB. We have demonstrated previously that Tax inhibits p53 activity through the p65/RelA subunit of NF-kappaB. We now present evidence that suggests that the upstream kinase IKKbeta plays an important role in Tax-induced p53 inhibition through phosphorylation of p65/RelA at Ser-536. First, mouse embryo fibroblast (MEF) IKKbeta-/-cells did not support Tax-mediated p53 inhibition, whereas MEFs lacking IKKalpha allowed Tax inhibition of p53. Second, transfection of IKKbeta wild type (WT), but not a kinase-dead mutant, into IKKbeta-/-cells rescued p53 inhibition by Tax. Third, the IKKbeta-specific inhibitor SC-514 decreased the ability of Tax to inhibit p53. Fourth, we show that phosphorylation of p65/RelA at Ser-536 is important for Tax inhibition of p53 using MEF p65/RelA-/-cells transfected with p65/RelA WT or mutant plasmids. Moreover, Tax induced p65/RelA Ser-536 phosphorylation in WT or IKKalpha-/- cells but failed to induce the phosphorylation of p65/RelA Ser-536 in IKKbeta-/-cells, suggesting a link between IKKbeta and p65/RelA phosphorylation. Consistent with this observation, blocking IKKbeta kinase activity by SC-514 decreases the phosphorylation of p65/RelA at Ser-536 in the presence of Tax in human T-cell lymphotropic virus type I-transformed cells. Finally, the ability of Tax to inhibit p53 is distinguished from the NF-kappaB transcription activation pathway. Our work, therefore, describes a novel Tax-NF-kappaB p65/RelA pathway that functions to inhibit p53 but does not require NF-kappaB transcription activity.

Protection of mitochondrial perturbation by human T-lymphotropic virus type 1 tax through induction of Bcl-xL expression

This study was designed to determine the inhibitory role of human T-lymphotropic virus type 1 (HTLV-1) tax against apoptotic cell death. We used JPX-9 cells, a Jurkat subclone generated by the stable introduction of a tax expression–plasmid vector, and induced tax expression in JPX-9 cells with CdCl 2. Expression of Bcl-2, Bcl-xL, and Bax in JPX-9 cells was assessed with Western blot analysis. Both tax-negative and tax-positive JPX-9 cells were incubated in the presence of several apoptogenic stimuli, and sensitivity to apoptogenic stimuli was also evaluated. Compared with tax-negative JPX-9 cells, Bcl-xL expression was clearly augmented in tax-positive JPX-9 cells. These cells were resistant to both receptor-mediated apoptosis (induced by anti-Fas IgM and tumor necrosis factor-related apoptosis–inducing ligand) and chemical-induced apoptosis (induced by pyrrolidine dithiocarbamate, etoposide, and staurosporine), as evidenced by the presence of hypodiploid DNA–positive cells, activation of caspase-3 and caspase-9, disruption of mitochondrial transmembrane potential (ΔΨm) and inhibition of cytochrome c release in tax-positive JPX-9 cells compared with tax-negative JPX-9 cells. Our results suggest that tax-mediated Bcl-xL expression inhibits apoptosis of activated T-cells in HTLV-1–seropositive subjects, which consequently promotes the onset of autoimmune disorders such as Sjögren's syndrome.

Inhibition of Caspase Cascade by HTLV-I Tax Through Induction of NF-κB Nuclear Translocation

AbstractNF-κB is required for prevention of apoptosis. We examined the importance of human T-cell leukemia virus–I (HTLV-I) Tax protein to stimulate NF-κB nuclear translocation, thus preventing apoptosis. Jurkat cells and JPX-9 cells in which the inducible Tax expression plasmid vector was stably transfected were used in the present study. Both Jurkat and Tax− JPX-9 cells had small amounts of basal nuclear NF-κB activity. The addition of NF-κB inhibitors suppressed NF-κB nuclear translocation of the cells, thus inducing apoptosis. Sequential activation of caspases from caspase-8 to caspase-3 was shown during this process. NF-κB nuclear translocation in JPX-9 cells was stimulated through Tax expression, and both the activation of caspases and apoptosis induced by NF-κB inhibitors were significantly suppressed in the Tax+ JPX-9 cells. The expression of Bcl-2, Bax, and Bcl-x was not changed among Jurkat, Tax− JPX-9, and Tax+ JPX-9 cells in the presence or absence of NF-κB inhibitors. X-chromosome–linked inhibitor of apoptosis (XIAP) protein expression in Tax−JPX-9 cells was significantly suppressed by NF-κB inhibitors, however, its expression in Tax+ JPX-9 cells was maintained even by the addition of NF-κB inhibitors. Our results suggest that the activation of NF-κB via Tax protein in HTLV-I infected cells renders the cells resistant to apoptosis. The expression of anti-apoptotic gene products such as XIAP to suppress caspase cascade, results in an increase of cytokine production and cell proliferation; one of the proposed mechanisms that promotes autoimmune disorders such as Sjögren's syndrome and rheumatoid arthritis found in HTLV-I seropositive subjects.

Inhibition of Caspase Cascade by HTLV-I Tax Through Induction of NF-κB Nuclear Translocation

NF-κB is required for prevention of apoptosis. We examined the importance of human T-cell leukemia virus–I (HTLV-I) Tax protein to stimulate NF-κB nuclear translocation, thus preventing apoptosis. Jurkat cells and JPX-9 cells in which the inducible Tax expression plasmid vector was stably transfected were used in the present study. Both Jurkat and Tax− JPX-9 cells had small amounts of basal nuclear NF-κB activity. The addition of NF-κB inhibitors suppressed NF-κB nuclear translocation of the cells, thus inducing apoptosis. Sequential activation of caspases from caspase-8 to caspase-3 was shown during this process. NF-κB nuclear translocation in JPX-9 cells was stimulated through Tax expression, and both the activation of caspases and apoptosis induced by NF-κB inhibitors were significantly suppressed in the Tax+ JPX-9 cells. The expression of Bcl-2, Bax, and Bcl-x was not changed among Jurkat, Tax− JPX-9, and Tax+ JPX-9 cells in the presence or absence of NF-κB inhibitors. X-chromosome–linked inhibitor of apoptosis (XIAP) protein expression in Tax−JPX-9 cells was significantly suppressed by NF-κB inhibitors, however, its expression in Tax+ JPX-9 cells was maintained even by the addition of NF-κB inhibitors. Our results suggest that the activation of NF-κB via Tax protein in HTLV-I infected cells renders the cells resistant to apoptosis. The expression of anti-apoptotic gene products such as XIAP to suppress caspase cascade, results in an increase of cytokine production and cell proliferation; one of the proposed mechanisms that promotes autoimmune disorders such as Sjögren’s syndrome and rheumatoid arthritis found in HTLV-I seropositive subjects.

Tax of the Human T-Lymphotropic Virus Type I Transactivates Promoter of theMDR-1Gene

Themdr-1gene has been shown to confer resistance to chemotherapy of multiple drugs which share no obvious structural similarities. We and others have previously reported that some virus-associated malignant cells express high levels of MDR-1 (1,2), probably regulated by some viral proteins. In this study we have examined the role of Tax, the key protein of HTLV-1. An excellent correlation was found between the existence of HTLV-1 and the expression of MDR-1 among seven human T-cell lines. In the second part of the study, a 1.76-kb DNA fragment representing the upstream regulatory elements of humanmdr-1gene was cloned into the CAT reporter plasmid. When the Tax expression plasmid was co-transfected with the MDR-1 reporter plasmid, a significant induction of CAT activity was observed. We conclude that Tax protein may up-regulate the expression of themdr-1gene.

Human T-Cell Leukemia Virus-1 Encoded Tax Protein Transactivates α1→3 Fucosyltransferase Fuc-T VII, Which Synthesizes Sialyl Lewis X, a Selectin Ligand Expressed on Adult T-Cell Leukemia Cells

Leukemia cells in patients with adult T-cell leukemia (ATL) and related cell lines strongly express the carbohydrate determinant sialyl Lewis X, a ligand for selectins. Its expression is thought to be related closely to the extravascular infiltration of the leukemia cells. Human T-cell leukemia virus type 1 (HTLV-1), the etiological agent of ATL, produces Tax protein, which is implicated in leukemogenesis through its transactivating effect on various cellular genes. In this study we investigated the transactivating effect of HTLV-1 Tax on the α1→3 fucosyltransferase Fuc-T VII, the putative rate-limiting enzyme in the synthesis of sialyl Lewis X in human leukocytes using JPX-9 cells. JPX-9 is a subclone of a non-ATL human lymphocytic leukemia cell line, Jurkat, and was established by introducing a metallothionein promoter-driven Tax expression plasmid. The JPX-9 cells as well as parental Jurkat cells did not express Fuc-T VII mRNA under normal culture conditions. When cultured in the presence of 10 μM CdCl2, Tax was induced and a significant amount of the Fuc-T VII message was ascertained by Northern blotting. The amount of the message was 24.5 times as much as was detected in non-treated cells, and was comparable to that which appeared by TPA stimulation of the cells, which is supposed to simulate the sequence of events occurring in normal activation of T lymphocytes activated by more physiological stimuli. Sialyl Lewis X determinant was expressed at the surface of CdCl2-treated cells, while the determinant was not detectable on either unstimulated JPX-9 or parental Jurkat cells. These results indicate that expression of sialyl Lewis X on leukemic cells in patients with ATL is at least partly due to the transactivation of the Fuc-T VII gene induced by the HTLV-1 Tax, and suggest that this leads to the accelerated extravascular infiltration of ATL cells.

Interleukin-10 Gene Expression in Adult T-Cell Leukemia

We studied the serum levels of interleukin-10 (IL-10), in patients with adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) infection. Elevated IL-10 levels were observed in 33 of 45 patients with ATL. Fresh leukemic cells from ATL patients as well as HTLV-I-infected T-cell lines MT-2, SLB-1, and C10/MJ expressed IL-10 mRNA by reverse transcription-polymerase chain reaction analysis, whereas IL-10 mRNA was not detected in normal peripheral mononuclear cells and an uninfected T-cell line Jurkat. IL-10 protein was also detected in the culture medium of leukemic cells from ATL patients as well as these HTLV-I-infected cell lines, and in the extracellular fluids of ATL patients. Interestingly, MT-4 cells, which did not express Tax although transformed by HTLV-I, did not express IL-10 at either the mRNA or protein level. To elucidate the role of the HTLV-I encoded transactivator Tax in IL-10 gene expression, Jurkat cells were transfected with a Tax expression plasmid. In transiently transfected Jurkat cells, endogenous IL-10 mRNA expression was induced by Tax. Stably transfected Jurkat cell lines expressed IL-10 mRNA and secreted IL-10 protein into the culture medium. The nuclear factor (NF)-kappa B pathway is a target for Tax transactivation. We treated MT-2 cells with phosphorothioate antisense oligonucleotides to the p65 subunit of NF-kappa B. A reduction in the expression of p65 was accompanied by a reduction in IL-10 gene expression and IL-10 production. We showed that the IL-10 kappa B-like sites ( kappa B1,-2,034 to -2,025; kappa B2, -1,961 to -1,952; kappa B3, -452 to -443) specifically formed a complex with NF-kappa B-containing nuclear extract from MT-2 cells and that NF-kappa B bound with the highest affinity to the kappa B2 element (kappa B2 > kappa B3 > kappa B1). These data suggest a general role for NF-kappa B activation in the induction of IL-10 gene transcription. Activation of IL-10 in HTLV-I-infected cells may contribute to the pathology associated with HTLV-I infection.

Interleukin-10 gene expression in adult T-cell leukemia

We studied the serum levels of interleukin-10 (IL-10), in patients with adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) infection. Elevated IL-10 levels were observed in 33 of 45 patients with ATL. Fresh leukemic cells from ATL patients as well as HTLV-I-infected T-cell lines MT-2, SLB-1, and C10/MJ expressed IL-10 mRNA by reverse transcription-polymerase chain reaction analysis, whereas IL-10 mRNA was not detected in normal peripheral mononuclear cells and an uninfected T-cell line Jurkat. IL-10 protein was also detected in the culture medium of leukemic cells from ATL patients as well as these HTLV-I-infected cell lines, and in the extracellular fluids of ATL patients. Interestingly, MT-4 cells, which did not express Tax although transformed by HTLV-I, did not express IL-10 at either the mRNA or protein level. To elucidate the role of the HTLV-I encoded transactivator Tax in IL-10 gene expression, Jurkat cells were transfected with a Tax expression plasmid. In transiently transfected Jurkat cells, endogenous IL-10 mRNA expression was induced by Tax. Stably transfected Jurkat cell lines expressed IL-10 mRNA and secreted IL-10 protein into the culture medium. The nuclear factor (NF)-kappa B pathway is a target for Tax transactivation. We treated MT-2 cells with phosphorothioate antisense oligonucleotides to the p65 subunit of NF- kappa B. A reduction in the expression of p65 was accompanied by a reduction in IL-10 gene expression and IL-10 production. We showed that the IL-10 kappa B-like sites ( kappa B1,-2,034 to -2,025; kappa B2, - 1,961 to -1,952; kappa B3, -452 to -443) specifically formed a complex with NF-kappa B-containing nuclear extract from MT-2 cells and that NF- kappa B bound with the highest affinity to the kappa B2 element (kappa B2 > kappa B3 > kappa B1). These data suggest a general role for NF- kappa B activation in the induction of IL-10 gene transcription. Activation of IL-10 in HTLV-I-infected cells may contribute to the pathology associated with HTLV-I infection.

Transcriptional regulation of the human interleukin-6 gene promoter in human T-cell leukemia virus type I-infected T-cell lines: evidence for the involvement of NF-kappa B

Freshly isolated leukemic cells from patients with adult T-cell leukemia (ATL) and human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of interleukin-6 (IL-6) protein and mRNA. To clarify the mechanisms that lead to the activation of IL-6 gene in HTLV-I-infected cells, we first studied the regulatory regions in the IL-6 gene transcription by transfection of chloramphenicol acetyltransferase (CAT) reporter plasmids containing the IL-6 promoter. When transfected into HTLV-I-infected T-cell lines MT-2 and HUT-102, IL-6 promoter/CAT plasmids were strongly activated without any stimulation. By deletion analysis of 52 upstream region of IL-6 promoter, the DNA region between -73 and -59 bp from the transcription start site of IL-6 gene was important in the expression of IL-6/CAT activities in HTLV-I-infected cells. This region contains nuclear factor (NF)-kappa B binding site. The site-directed mutation of the kappa B motif in IL-6/CAT plasmid resulted in the complete abrogation of IL-6 promoter activity in these cells. Furthermore, when IL-6 promoter/CAT plasmid was introduced into an HTLV-I-uninfected T- cell line, Jurkat, IL-6 promoter activity was silent in the basal level, but strongly increased by the cotransfection with an HTLV-I tax expression plasmid. However, tax expression plasmid showed no transactivation activity, when kappa B site was mutated in IL-6 promoter/CAT plasmid. We found that the IL-6 kappa B site specifically formed a complex with NF-kappa B-containing nuclear extracts from MT-2 and HUT-102 cells. Finally, transfection of HTLV-I tax into Jurkat cells resulted in induction of specific binding of nuclear extracts to the NF-kappa B sequence. These results strongly suggest that HTLV-I tax gene may transactivate IL-6 gene through kappa B site in HTLV-I- positive T-cell lines and activation of NF-kappa B may be crucial in HTLV-I-induced IL-6 gene activation in ATL.

Transcriptional Regulation of the Human Interleukin-6 Gene Promoter in Human T-Cell Leukemia Virus Type I-Infected T-Cell Lines: Evidence for the Involvement of NF-κB

Freshly isolated leukemic cells from patients with adult T-cell leukemia (ATL) and human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of interleukin-6 (IL-6) protein and mRNA. To clarify the mechanisms that lead to the activation of IL-6 gene in HTLV-I-infected cells, we first studied the regulatory regions in the IL-6 gene transcription by transfection of chloramphenicol acetyltransferase (CAT) reporter plasmids containing the IL-6 promoter. When transfected into HTLV-I-infected T-cell lines MT-2 and HUT-102, IL-6 promoter/CAT plasmids were strongly activated without any stimulation. By deletion analysis of 5' upstream region of IL-6 promoter, the DNA region between -73 and -59 bp from the transcription start site of IL-6 gene was important in the expression of IL-6/CAT activities in HTLV-I-infected cells. This region contains nuclear factor (NF)-kappa B binding site. The site-directed mutation of the kappa B motif in IL-6/CAT plasmid resulted in the complete abrogation of IL-6 promoter activity in these cells. Furthermore, when IL-6 promoter/CAT plasmid was introduced into an HTLV-I-uninfected T-cell line, Jurkat, IL-6 promoter activity was silent in the basal level, but strongly increased by the cotransfection with an HTLV-I tax expression plasmid. However, tax expression plasmid showed no transactivation activity, when kappa B site was mutated in IL-6 promoter/CAT plasmid. We found that the IL-6 kappa B site specifically formed a complex with NF-kappa B-containing nuclear extracts from MT-2 and HUT-102 cells. Finally, transfection of HTLV-I tax into Jurkat cells resulted in induction of specific binding of nuclear extracts to the NF-kappa B sequence. These results strongly suggest that HTLV-I tax gene may transactivate IL-6 gene through kappa B site in HTLV-I-positive T-cell lines and activation of NF-kappa B may be crucial in HTLV-I-induced IL-6 gene activation in ATL.

Transactivation of the Human Interleukin-6 Gene by Human T-Lymphotropic Virus Type 1 Tax Protein

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates both humoral and cellular immune responses. Accumulating evidence suggests that the infection of T cells and other cell types with human T-lymphotropic virus type 1 (HTLV-1) results in the constitutive expression of IL-6. However, the underlying molecular mechanisms are little understood. When a reporter plasmid, pIL6-CAT-E3, in which the human IL-6 enhancer/promoter region from -630 to +14 was linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, was transfected, HTLV-1-infected but not -uninfected T-cell lines activated the IL-6 promoter. This indicated the presence of a factor transactivating the IL-6 gene in the infected cells. To evaluate the involvement of the HTLV-1-encoded transacting factor (Tax) in this transactivation, we examined the effect of transient cotransfection with the Tax-expression plasmid, pMAX-Neo, on the transcription from the IL-6 promoter by use of COS1 cells. The cotransfected COS1 has about six-times greater the CAT activity than that transfected with pIL6-CAT-E3 alone. The analysis of a series of deletions of the IL-6 promoter suggested that the region (-105/-47) containing a NF kappa B site was crucial for the Tax responsiveness. We further examined the effect of Tax on endogenous IL-6 gene expression using the Jurkat clone, JPX-9, stably transfected with pMAX-Neo. JPX-9 accumulated steady state transcripts of the endogenous IL-6 gene in response to the induction of Tax expression. Our findings indicate an important role of the Tax protein in the expression of IL-6 in cells infected with HTLV-1.

Transactivation of the human interleukin-6 gene by human T-lymphotropic virus type 1 Tax protein

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates both humoral and cellular immune responses. Accumulating evidence suggests that the infection of T cells and other cell types with human T- lymphotropic virus type 1 (HTLV-1) results in the constitutive expression of IL-6. However, the underlying molecular mechanisms are little understood. When a reporter plasmid, pIL6-CAT-E3, in which the human IL-6 enhancer/promoter region from -630 to +14 was linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, was transfected, HTLV-1-infected but not -uninfected T-cell lines activated the IL-6 promoter. This indicated the presence of a factor transactivating the IL-6 gene in the infected cells. To evaluate the involvement of the HTLV-1-encoded transacting factor (Tax) in this transactivation, we examined the effect of transient cotransfection with the Tax-expression plasmid, pMAX-Neo, on the transcription from the IL-6 promoter by use of COS1 cells. The cotransfected COS1 has about six-times greater the CAT activity than that transfected with pIL6-CAT-E3 alone. The analysis of a series of deletions of the IL-6 promoter suggested that the region (-105/-47) containing a NF kappa B site was crucial for the Tax responsiveness. We further examined the effect of Tax on endogenous IL-6 gene expression using the Jurkat clone, JPX-9, stably transfected with pMAX-Neo. JPX-9 accumulated steady state transcripts of the endogenous IL-6 gene in response to the induction of Tax expression. Our findings indicate an important role of the Tax protein in the expression of IL-6 in cells infected with HTLV-1.

Overgrowth of human synovial cells driven by the human T cell leukemia virus type I tax gene.

One of the salient pathological features of rheumatoid arthritis is synovial cell proliferation with bone erosion. Despite extensive investigation, the factors essential for synovial cell proliferation remain to be identified. Recent studies suggest that human T cell leukemia virus type I (HTLV-I) may play an important role in synovial overgrowth observed in patients with one type of chronic inflammatory synovitis. In order to confirm and extend these observations, we have established synovial cell clones (SCCs) from three HTLV-I carriers who demonstrated synovial overgrowth but were otherwise asymptomatic. HTLV-I proviral DNA randomly integrated into the cellular genome was present in 20-30% of SCCs. The SCCs carrying HTLV-I proviral DNA and expressing the tax gene exhibited high levels of proliferative potential. HTLV-I was found to function as a transcriptional trans-activator in these SCCs. Moreover, transfection of the tax expression plasmid into SCCs resulted in the same phenotype of increased proliferation and cytokine expression as exhibited by HTLV-I provirus-carrying and tax-expressing SCCs. These data suggest that tax plays a critical role not only in leukemogenesis but also in synovial overgrowth in humans.

Human T-Lymphotropic Virus Type I tax Regulates the Expression of the Human Lymphotoxin Gene

Human T-lymphotropic virus type-I (HTLV-I)-infected T-cell lines constitutively produce high levels of lymphotoxin (LT). To analyze the mechanisms that lead to the expression of LT in HTLV-I-infected cell lines, we studied regulatory regions of the human LT promoter involved in the activation of the human LT gene. As determined by deletional analysis, sequences between +137 and -116 (relative to the transcription initiation site) are sufficient to direct expression of a reporter gene in the HTLV-I-infected cell line MT-2. Site-directed mutation of a of the single kappa B-like motif present in the LT promoter region (positions -99 to -89) completely abrogated LT promoter activity in MT-2 cells, suggesting that this site plays a critical role in the activation of the human LT gene. Transfection of LT constructs into HTLV-I-uninfected and -unstimulated Jurkat and U937 cell lines showed little to no activity of the LT promoter. Cotransfection of the same constructs with a tax expression plasmid into Jurkat cells led to detectable promoter activity, which could be significantly increased by stimulation of the cells with phorbol myristate acetate (PMA). Similarly, cotransfection of the LT promoter constructs and the tax expression plasmid into U937 cells led to significant promoter activity upon stimulation with PMA. These data suggest that HTLV-I tax is involved in the upregulation of LT gene expression in HTLV-I-infected cells.

Human T-lymphotropic virus type I tax regulates the expression of the human lymphotoxin gene

Human T-lymphotropic virus type-I (HTLV-I)-infected T-cell lines constitutively produce high levels of lymphotoxin (LT). To analyze the mechanisms that lead to the expression of LT in HTLV-I-infected cell lines, we studied regulatory regions of the human LT promoter involved in the activation of the human LT gene. As determined by deletional analysis, sequences between +137 and -116 (relative to the transcription initiation site) are sufficient to direct expression of a reporter gene in the HTLV-I-infected cell line MT-2. Site-directed mutation of a of the single kappa B-like motif present in the LT promoter region (positions -99 to -89) completely abrogated LT promoter activity in MT-2 cells, suggesting that this site plays a critical role in the activation of the human LT gene. Transfection of LT constructs into HTLV-I-uninfected and -unstimulated Jurkat and U937 cell lines showed little to no activity of the LT promoter. Cotransfection of the same constructs with a tax expression plasmid into Jurkat cells led to detectable promoter activity, which could be significantly increased by stimulation of the cells with phorbol myristate acetate (PMA). Similarly, cotransfection of the LT promoter constructs and the tax expression plasmid into U937 cells led to significant promoter activity upon stimulation with PMA. These data suggest that HTLV-I tax is involved in the upregulation of LT gene expression in HTLV-I-infected cells.

Effect of human T-cell leukemia virus type I tax protein on activation of the human vimentin gene

We report that the expression of the vimentin gene, a cytoskeletal growth-regulated gene, is activated in trans by the Tax (p40x) transactivator protein encoded by the human T-cell leukemia virus type I. Expression of the Tax protein activates a number of cellular genes, such as those coding for the alpha chain of the high-affinity interleukin-2 receptor and interleukin-2. These findings indicate that the Tax protein is involved in the unregulated T-cell growth associated with human T-cell leukemia virus type I infection. Higher levels of vimentin mRNA were expressed in two human T-cell leukemia virus type I-transformed T cell lines, C91/PL and C81-66/45, when compared with that in Jurkat T cells. We demonstrate that this activation is conferred by the vimentin upstream flanking sequences. Indeed, enhanced activity was detected when constructs with the vimentin promoter linked to the chloramphenicol acetyltransferase gene were transfected in HeLa cells and in two cell lines of hematopoietic origin (Jurkat T lymphoblastoid cells and U937 promonocytic cells) together with a Tax expression plasmid. By introducing a series of deletions in the vimentin promoter, we further restrict these sequences to 30 base pairs, located between 241 and 210 base pairs upstream of the mRNA cap site. A 40-base-pair oligonucleotide containing this regulatory region proved sufficient to confer Tax inducibility upon a heterologous promoter linked to chloramphenicol acetyltransferase. Importantly, this segment includes an 11-base-pair promoter segment that has homology with the binding site for the NF-kappa B transactivating factor. Our findings indicate that constitutive expression of the vimentin gene under the control of the Tax protein may be relevant in understanding the progression of the lymphoproliferative process associated with human T-cell leukemia virus type I infection.