Role of IL-21 in HTLV-1 infections with emphasis on HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP).

Interleukin-21 (IL-21) enhances the survival and cytotoxic properties of cytotoxic T cells (CTLs) and exhibits essential roles in controlling chronic viral infections. HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic progressive inflammatory disease of the nervous system. The main determinant of disease progression is efficiency of the CTL response to Human T lymphotropic virus types I (HTLV-1). In this study, the expression of host IL-21 and HTLV-I Tax and proviral load (PVL) was evaluated to understand the role and mechanism of IL-21 in HTLV-1 infections and the subsequent development of HAM/TSP. A cross-sectional study was carried out on 20 HAM/TSP patients, 20 asymptomatic HTLV-1 carriers (ACs) and 20 healthy controls (HCs) to evaluate the expression of IL-21 and Tax and PVL in non-activated and phorbol myristate acetate (PMA)-ionomycin-activated peripheral blood mononuclear cells (PBMCs). The mean mRNA expression of IL-21 in the non-activated and activated PBMCs was higher (by 5-13 times) in the HAM/TSP patients than in ACs and HCs (p < 0.05); however, there was no significant difference between ACs and HCs. In contrast to the IL-21 mRNA expression, the serum level of the IL-21 protein was significantly lower in the HAM/TSP patients than in ACs and HCs (p < 0.05). Furthermore, higher expression of Tax and PVL was observed in the HAM/TSP subjects than ACs (p < 0.05). In addition, Tax gene expression was positively correlated with PVL (R = 0.595, p = 0.000) and IL-21 gene expression (R = 0.395, p = 0.021) in the HTLV-1-infected subjects. In conclusion, the increase in IL-21 mRNA expression may reflect the attempt of infected T cells to induce an appropriate antiviral response, and the decrease in IL-21 protein expression may reflect the inhibition of IL-21 mRNA translation by viral factors in favour of virus evasion and dissemination.

SMYD3 interacts with HTLV-1 Tax and regulates subcellular localization of Tax.

HTLV-1 Tax deregulates signal transduction pathways, transcription of genes, and cell cycle regulation of host cells, which is mainly mediated by its protein-protein interactions with host cellular factors. We previously reported an interaction of Tax with a histone methyltransferase (HMTase), SUV39H1. As the interaction was mediated by the SUV39H1 SET domain that is shared among HMTases, we examined the possibility of Tax interaction with another HMTase, SMYD3, which methylates histone H3 lysine 4 and activates transcription of genes, and studied the functional effects. Expression of endogenous SMYD3 in T cell lines and primary T cells was confirmed by immunoblotting analysis. Co-immuno-precipitaion assays and in vitro pull-down assay indicated interaction between Tax and SMYD3. The interaction was largely dependent on the C-terminal 180 amino acids of SMYD3, whereas the interacting domain of Tax was not clearly defined, although the N-terminal 108 amino acids were dispensable for the interaction. In the cotransfected cells, colocalization of Tax and SMYD3 was indicated in the cytoplasm or nuclei. Studies using mutants of Tax and SMYD3 suggested that SMYD3 dominates the subcellular localization of Tax. Reporter gene assays showed that nuclear factor-κB activation promoted by cytoplasmic Tax was enhanced by the presence of SMYD3, and attenuated by shRNA-mediated knockdown of SMYD3, suggesting an increased level of Tax localization in the cytoplasm by SMYD3. Our study revealed for the first time Tax-SMYD3 direct interaction, as well as apparent tethering of Tax by SMYD3, influencing the subcellular localization of Tax. Results suggested that SMYD3-mediated nucleocytoplasmic shuttling of Tax provides one base for the pleiotropic effects of Tax, which are mediated by the interaction of cellular proteins localized in the cytoplasm or nucleus.

Detection of HTLV-1 by means of HBZ gene in situ hybridization in formalin-fixed and paraffin-embedded tissues.

Adult T-cell leukemia/lymphoma (ATLL) is a T-cell malignancy associated with HTLV-1. The HTLV-1 provirus genome has the pX region that encodes tax and HTLV-1 basic leucine zipper factor (HBZ). Previous studies have reported that the tax gene is expressed in few ATLL cases, but the HBZ gene in all ATLL cases. In this study, we used HBZ gene in situ hybridization (HBZ-ISH) for detection of the HBZ gene in formalin-fixed paraffin-embedded tissues. This method showed that all cases (n = 19) were positive for the ATLL cell line (MT-1, MT-2, and MT-4) and ATLL mouse model (HBZ-Tg mice and NOD/SCID/ß2-microglobulin(null) mice with ATLL transplanted), and the HBZ gene was also detected in all human ATLL cases (n = 16). The percentage of positive cells in HBZ-ISH was 5-70%. Immunohistochemical staining for Tax protein showed positivity in seven of 11 cases in NOD/SCID/ß2-microglobulin(null) mice with ATLL transplanted and in six of eight human ATLL cases, but the percentage of positive cells was very low (range, 1-5%). Although HBZ-ISH is unsuitable to detect HTLV-1 clonality, this method is convenient and can be useful for the histological diagnosis of ATLL in HTLV-1 sero-indeterminate patients.

Exclusion of Int-6 from PML nuclear bodies by binding to the HTLV-I Tax oncoprotein.

The Tax transactivator of the human T cell leukemia virus type I (HTLV-I) exhibits oncogenic properties. A screen for proteins interacting with Tax yielded a complementary DNA (cDNA) encoding the human Int-6 protein. In mice, the Int-6 gene can be converted into a putative dominant negative oncogene after retroviral insertion. Here, Int-6 was localized in the cell nucleus to give a speckled staining pattern superposed to that of the promyelocytic leukemia (PML) protein. The binding of Tax to Int-6 caused its redistribution from the nuclear domains to the cytoplasm. Thus, Int-6 is a component of the PML nuclear bodies and Tax disrupts its normal cellular localization by binding to it.

Flow cytometric methodology for the detection of de novo human T-cell leukemia virus -1 infection in vitro: A tool to study novel infection inhibitors.

Methodology to detect and study de novo human T-cell leukemia virus (HTLV)-1 infection is required to further our knowledge of the viruses' mechanisms of infection and to study potential therapeutic interventions. Whilst methodology currently exists, utilisation of an anti-Tax antibody to detect de novo Tax expression in permissive cells labelled with cell tracker allowing for the detection by flow cytometry of new infection after co-culture with donor cell lines productively infected with HTLV-1 is an alternative strategy. Using this methodology, we have been able to detect de novo infection of the T cell line HUT78 following co-culture with the productively infected HTLV-1 donor cell line MT-2 and to confirm that infection can be effectively blocked with well characterised infection inhibitors. This methodology will benefit experimental studies examining HTLV infection in vitro and may aid identification of therapeutic agents that block this process.

Human T-cell leukemia virus type I Tax associates with and is negatively regulated by the NF-kappa B2 p100 gene product: implications for viral latency.

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of the adult T-cell leukemia, an aggressive and often fatal malignancy of activated human CD4 T cells. HTLV-I encodes an essential 40-kDa protein termed Tax that not only transactivates the long terminal repeat of this retrovirus but also induces an array of cellular genes. Tax-mediated transformation of T cells likely involves the deregulated expression of various cellular genes that normally regulate lymphocyte growth produced by altered activity of various endogenous host transcription factors. In particular, Tax is capable of modulating the expression or activity of various host transcription factors, including members of the NF-kappa B/Rel and CREB/ATF families, as well as the cellular factors HEB-1 and p67SRF. An additional distinguishing characteristic of HTLV-I infection is the profound state of viral latency that is present in circulating primary leukemic T cells. In this study, we demonstrate that HTLV-I Tax can physically associate with p100, the product of the Rel-related NF-kappa B2 gene, both in transfected cells and in HTLV-I-infected leukemic T-cell lines. Furthermore, the physical interaction of Tax with p100 leads to the inhibition of Tax-induced activation of the HTLV-I and human immunodeficiency virus type 1 long terminal repeats, reflecting p100-mediated cytoplasmic sequestration of the normally nuclearly expressed Tax protein. In contrast, a mutant of Tax that selectively fails to activate nuclear NF-kappa B expression does not associate with p100. Together, these results suggest that the cytoplasmic interplay of Tax and p100 may play an important role in the initiation and maintenance of HTLV-1 latency observed in adult T-cell leukemia.

HTLV-I Tax induces and associates with Crk-associated substrate lymphocyte type (Cas-L).

Crk-associated substrate lymphocyte type (Cas-L) is a docking protein that is heavily tyrosine phosphorylated by the engagement of beta1 integrins in T cells. In the present study, we attempted to evaluate the role of Cas-L in the pathophysiology of adult T-cell leukemia (ATL). Examination of peripheral blood mononuclear cells from ATL patients as well as ATL-derived T cell lines showed an elevation of Cas-L in these cells. We showed that tyrosine phosphorylation as well as expression of Cas-L was markedly elevated through the induction of human T-lymphotropic virus type I (HTLV-I) Tax in JPX-9 cells, with these cells showing marked motile behavior on the ligands for integrins. We next performed yeast two-hybrid screening of cDNA library from an HTLV-I-transformed T cell line, which resulted in the identification of Tax as a putative binding partner for Cas-L. Co-precipitation experiments revealed that the serine-rich region of Cas-L might serve as the binding site with the highest affinity for Tax. Co-localization study showed that Tax and Cas-L partly merged in the cytoplasm. Finally, we showed that exogenous Cas-L inhibited Tax-mediated transactivation of nuclear factor kappaB (NF-kappaB), while Tax-independent activation of NF-kappaB remained intact, hence indicating that Cas-L might specifically regulate Tax-NF-kappaB pathway.

Quantification of the virus-host interaction in human T lymphotropic virus I infection.

BACKGROUND: HTLV-I causes the disabling inflammatory disease HAM/TSP: there is no vaccine, no satisfactory treatment and no means of assessing the risk of disease or prognosis in infected people. Like many immunopathological diseases with a viral etiology the outcome of infection is thought to depend on the virus-host immunology interaction. However the dynamic virus-host interaction is complex and current models of HAM/TSP pathogenesis are conflicting. The CD8+ cell response is thought to be a determinant of both HTLV-I proviral load and disease status but its effects can obscure other factors. RESULTS: We show here that in the absence of CD8+ cells, CD4+ lymphocytes from HAM/TSP patients expressed HTLV-I protein significantly more readily than lymphocytes from asymptomatic carriers of similar proviral load (P = 0.017). A high rate of viral protein expression was significantly associated with a large increase in the prevalence of HAM/TSP (P = 0.031, 89% of cases correctly classified). Additionally, a high rate of Tax expression and a low CD8+ cell efficiency were independently significantly associated with a high proviral load (P = 0.005, P = 0.003 respectively). CONCLUSION: These results disentangle the complex relationship between immune surveillance, proviral load, inflammatory disease and viral protein expression and indicate that increased protein expression may play an important role in HAM/TSP pathogenesis. This has important implications for therapy since it suggests that interventions should aim to reduce Tax expression rather than proviral load per se.

A balanced transcription between telomerase and the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 in resting, activated, HTLV-1-transformed and Tax-expressing human T lymphocytes.

BACKGROUND: The functional state of human telomeres is controlled by telomerase and by a protein complex named shelterin, including the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 involved in telomere capping functions. The expression of hTERT, encoding the catalytic subunit of telomerase, plays a crucial role in the control of lymphocyte proliferation by maintaining telomere homeostasis. It has been previously found that hTERT activity is down-regulated by the human T cell leukaemia virus type 1 (HTLV-1) Tax protein in HTLV-1 transformed T lymphocytes. In this study, we have examined the effects of Tax expression on the transcriptional profile of telomerase and of shelterin in human T lymphocytes. RESULTS: We first provide evidence that the up-regulation of hTERT transcription in activated CD4+ T lymphocytes is associated with a down-regulation of that of TERF1, TERF2 and POT1 genes. Next, the down-regulation of hTERT transcription by Tax in HTLV-1 transformed or in Tax-expressing T lymphocytes is found to correlate with a significant increase of TRF2 and/or Pot1 mRNAs. Finally, ectopic expression of hTERT in one HTLV-1 T cell line induces a marked decrease in the transcription of the POT1 gene. Collectively, these observations predict that the increased transcriptional expression of shelterin genes is minimizing the impact on telomere instability induced by the down-regulation of hTERT by Tax. CONCLUSION: These findings support the notion that Tax, telomerase and shelterin play a critical role in the proliferation of HTLV-1 transformed T lymphocytes.

Tumor necrosis factor-alpha and interferon-gamma, but not HTLV-I tax, are likely factors in the epidermotropism of cutaneous T-cell lymphoma via induction of interferon-inducible protein-10.

We have previously shown that Interferon-Inducible Protein-10 (IP-10), a cytokine chemotactic for CD4-positive lymphocytes, is overexpressed by lesional epidermal keratinocytes and probably accounts for the epidermotropism of cutaneous T-cell lymphoma (CTCL). The tax gene of human T-lymphotropic virus-I (HTLV-I) immortalizes CD4-positive lymphocytes, induces IFN-gamma, and has been detected in patients with classical CTCL who are seronegative for HTLV-I. TNF-alpha is synergistic with IFN-gamma for the induction of IP-10. We therefore decided to define the presence of tax, IFN-gamma, TNF-alpha, and IP-10 in lesions of 19 adults with classical CTCL who were seronegative for HTLV-I. Lesional mRNAs for actin, TNF-alpha, IFN-gamma, and tax were detected by reverse-transcriptase polymerase chain reaction (RT-PCR) amplification. In addition IP-10, TNF-alpha, and IFN-gamma were detected and localized with immunocytochemistry of frozen sections. In agreement with previous observations IP-10 was overexpressed in lesional keratinocytes of all 19 patients. By RT-PCR, mRNA for IFN-gamma was detected in lesions of 8, and for TNF-alpha in lesions of 13 patients. By immunocytochemistry, TNF-alpha was expressed by lesional keratinocytes in 10 of 13 tested patients, whereas IFN-gamma was focally expressed by lesional lymphocytes and faintly by lesional keratinocytes in 9 of 13 tested patients. tax mRNA was not detected in lesions of any patient, but was easily detectable in cutaneous lesions or peripheral blood of control patients who were seropositive for HTLV-I. We conclude that TNF-alpha and IFN-gamma may cause epidermotropism by inducing IP-10. However, the tax gene of HTLV-I does not appear to be involved in the pathogenesis of classical CTCL.

Detection of the tax gene of HTLV-I in labial salivary glands from patients with Sjögren's syndrome and other diseases of the oral cavity.

OBJECTIVE: To confirm a possible association between Sjögren's syndrome (SS) and the tax gene of human T lymphotropic virus type I (HTLV-I). METHODS: We studied by PCR labial salivary glands (LSG) from 50 patients with definite SS and from 58 controls including 32 patients with LSG involved by other inflammatory processes and 26 normal LSG. Antibodies to HTLV-I and antibodies to the Tax protein were searched for in serum. RESULTS: We detected the tax gene of HTLV-I in LSG from 15/50 (30%) of patients with SS but also in specimens from 9/32 (28%) patients with LSG involved by other inflammatory processes (3/9 graft-versus-host disease, 5/19 extra-vasated cysts, 1/4 sarcoidosis) and from only 1/26 (4%) normal LSG. A 652 bp region, sequenced in 2 SS patients, was 98-98.5% homologous to the canonic sequence of tax HTLV-I. The HTLV-I gag, pol and env genes were never detected. The serum of the SS patients did not contain antibodies to HTLV-I. However, anti-Tax antibodies were detected in the serum of 18/25 (72%) SS patients, 10/10 (100%) patients positive for tax DNA in their LSG and 8/15 (53%) patients negative for tax DNA in their LSG. CONCLUSION: Our observations raise the possibility that a very low number of copies of the tax gene may be harbored innocuously in cells within the oral cavity in some healthy individuals, but that this gene may play a role as a co-factor in the development of SS or other diseases of oral cavity.

Detection of px gene product of bovine leukemia virus in infected cells.

Bovine leukemia virus (BLV), like its closest relatives human T-cell leukemia virus-I and II, contain a 'px' gene, between the 'env' gene and the 3' long terminal repeat in its genome. A monoclonal antibody prepared against a synthetic oligopeptide whose sequence was deduced from highly conserved region of 'px' gene of BLV, was used to detect the presence of 'px' gene product in chronically BLV infected synchronised cells. By immunoperoxidase staining the 'px' gene product was detected maximum after 6-9 hr after synchronization in the nucleus of the cells which demonstrated the close interaction of it with viral DNA which is integrated with host cell genome.

In vivo T lymphocyte dynamics in humans and the impact of human T-lymphotropic virus 1 infection.

Human T-lymphotropic virus type 1 (HTLV-1) is a persistent CD4+ T-lymphotropic retrovirus. Most HTLV-1-infected individuals remain asymptomatic, but a proportion develop adult T cell leukemia or inflammatory disease. It is not fully understood how HTLV-1 persists despite a strong immune response or what determines the risk of HTLV-1-associated diseases. Until recently, it has been difficult to quantify lymphocyte kinetics in humans in vivo. Here, we used deuterated glucose labeling to quantify in vivo lymphocyte dynamics in HTLV-1-infected individuals. We then used these results to address four questions. (i) What is the impact of HTLV-1 infection on lymphocyte dynamics? (ii) How does HTLV-1 persist? (iii) What is the extent of HTLV-1 expression in vivo? (iv) What features of lymphocyte kinetics are associated with HTLV-1-associated myelopathy/tropical spastic paraparesis? We found that CD4+CD45RO+ and CD8+CD45RO+ T lymphocyte proliferation was elevated in HTLV-1-infected subjects compared with controls, with an extra 10(12) lymphocytes produced per year in an HTLV-1-infected subject. The in vivo proliferation rate of CD4+CD45RO+ cells also correlated with ex vivo viral expression. Finally, the inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis was associated with significantly increased CD4+CD45RO+ cell proliferation. We suggest that there is persistent viral gene expression in vivo, which is necessary for the maintenance of the proviral load and determines HTLV-1-associated myelopathy/tropical spastic paraparesis risk.

[Viral Tax protein expression in salivary glands of patients infected with human t-cell lymphotropic virus type I and Sicca Syndrome]. / Proteína Tax (HTLV-I), probable factor patogénico y marcador del síndrome sicca que se asocia a HAM/TSP.

BACKGROUND: Human T-cell lymphotropic virus type I (HTLV-I) is a retrovirus that influences cellular metabolism modifying biological responses. This results in oncogenic, degenerative or inflammatory changes. The myelopathy associated to HTLV-I or tropical spastic paraparesia (HAM/TSP) is a mainly degenerative response to the virus infection. On the other hand, Sjögren syndrome has an inflammatory appearance. The immunohistochemical study of CD-4, CD-8 and CD45 lymphocytes, metalloproteinase MMP-9 and viral Tax protein in pathological samples of salivary glands may help to differentiate primary from viral Sicca syndrome. AIM: To perform an immunohistochemical study of salivary glands of patients with HAM/TSP and Sicca syndrome and control subjects. MATERIAL AND METHODS: Pathological samples of salivary glands from 53 patients with HAM/TSP and Sicca syndrome and 10 control subjects, were studied. Immunohistochemistry was performed using antibodies against CD-4, CD-8 and CD-45 lymphocytes, metalloproteinase MMP-9 and viral Tax protein. RESULTS: Only in patients with HAM/TSP and Sicca syndrome, the presence of Tax protein was observed in CD-4 and CD-8 lymphocytes and in glandular acini. CONCLUSIONS: Patients infected with HTLV-I express Tax protein in salivary glands. This finding has diagnostic and pathogenic implications.

Human T-lymphotropic virus, type 1, tax protein triggers microtubule reorientation in the virological synapse.

We showed recently that the human T-lymphotropic virus, type 1 (HTLV-1), spreads directly from cell to cell via a virological synapse. The HTLV-1 virological synapse resembles the immunological synapse; each is a specialized contact between a lymphocyte and another cell that contains organized protein microdomains, and each involves repolarization of the T-cell microtubule cytoskeleton. However, formation of the virological synapse is not triggered by T-cell receptor-mediated antigen recognition. On the basis of our previous data, we postulated that formation of the viral synapse was triggered by a conjunction of two signals, one from HTLV-1 infection of the T-cell and one from cell-cell contact. We have recently identified ICAM-1 engagement as a cell-contact signal that causes the microtubule polarization associated with the virological synapse. Here we used confocal microscopy of T-lymphocytes naturally infected with HTLV-1 or transfected with individual HTLV-1 genes to investigate the role of the viral transcriptional transactivator protein Tax. Polarization of the microtubules was induced by cell-cell contact or by cross-linking T-cell surface molecules with monoclonal antibodies adsorbed to latex beads. We show that Tax, which is mainly found in the nucleus, is also present at two specific extranuclear sites as follows: around the microtubule organizing center in association with the cis-Golgi and in the cell-cell contact region. We show that expression of Tax provides an intracellular signal that synergizes with ICAM-1 engagement to cause the T-cell microtubule polarization observed at the virological synapse.

Transforming activity and the level of Tax protein: effect of one point mutation in HTLV-I tax gene.

The transcriptional trans-activator molecule of HTLV-I, Tax, is known to transform rodent fibroblasts. A revertant clone expressing Tax was obtained by treating transformed Rat-I cells harboring a single copy of the tax gene with a mutagen. Sequence analysis of the tax gene of the revertant clone revealed that it had one point mutation at codon 12(CTT----TTT), resulting in a change from Leu to Phe. The colony-forming efficiencies of the cells transfected with the mutant tax gene (mu71 tax) were significantly lower than those transfected with the wild-type by the soft-agar method. This difference was shown to be due to the instability of mu71 Tax.

Detection of human T-lymphotropic virus type I (HTLV-I) tax RNA in the central nervous system of HTLV-I-associated myelopathy/tropical spastic paraparesis patients by in situ hybridization.

Autopsy specimens from 3 patients with human T-lymphotropic virus (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) were examined for the presence of HTLV-I in the central nervous system (CNS). In situ hybridization using an HTLV-I tax RNA probe detected cells containing HTLV-I RNA in spinal cord and cerebellar sections. HTLV-I infected cells were located within the white matter and, in particular, within the anterior and lateral funiculi of the spinal cord. Consistent with previously described HAM/TSP pathology, there were perivascular infiltrates in these CNS specimens. Significantly, HTLV-I RNA was not localized to these infiltrates but was detected deeper within the neural tissue. Furthermore, phenotypic analysis demonstrated that at least some of the infected cells were astrocytes. While previous polymerase chain reaction studies have demonstrated the presence of proviral HTLV-I in CNS specimens, here we provide evidence for the in situ expression of HTLV-I RNA in the CNS of HAM/TSP patients.

The human T-cell leukemia virus type 1 transactivator protein Tax colocalizes in unique nuclear structures with NF-kappaB proteins.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent activator of viral transcription. Tax also activates the expression of specific cellular genes involved in the control of T-lymphocyte growth via effects on cellular transcription factors, including members of the NF-kappaB/cRel family. Immunocytochemistry and electron microscopy were used to characterize the intracellular localization of Tax and identify cellular factors which are the potential targets for its transcriptional activity. These studies indicated that Tax localizes in discrete nuclear foci in T lymphocytes transformed by HTLV-1 and in cells transduced with Tax expression vectors. The Tax-containing foci are complex nuclear structures comprising a central core in which Tax colocalizes with splicing factor Sm. In addition to splicing factors Sm and SC-35, the Tax-containing nuclear structures also contain transcriptional components, including the largest subunit of RNA polymerase II and cyclin-dependent kinase CDK8. The inclusion of the two subunits of NF-kappaB, p50 and RelA, and the presence of the mRNA from a gene specifically activated by Tax through NF-kappaB binding sites suggest that these unique nuclear structures participate in Tax-mediated activation of gene expression via the NF-kappaB pathway.

Subcellular redistribution of HTLV-1 Tax protein by NF-kappa B/Rel transcription factors.

The viral oncogene Tax derived from human T cell leukemia virus type I (HTLV-I) is a positive transcriptional activator of HTLV-1 gene expression. Tax is also able to indirectly stimulate transcription of several growth regulatory genes by an indirect mechanism via association with host transcription factors. One of the cellular targets of the trans-activating effects of Tax is the NF-kappa B/Rel family transcription factors, pleiotropic regulators of immunoregulatory, cytokine, and viral gene expression. Recent studies demonstrated that specific subunits of NF-kappa B (NFKB2(p 100) and c-Rel) were overexpressed in HTLV-I-infected and Tax-expressing cells. Furthermore, Tax physically associated with NFKB2(p 100). Monospecific antibodies directed against individual NF-kappa B subunits were generated and used to investigate the consequences of the interactions between Tax and NF-kappa B in a cotransfection-immunofluorescence assay. These studies demonstrate: (1) distinct compartmentalization of NF-kappa B precursors and products, (2) differential induction of the endogenous I kappa B alpha protein by transfected NF-kappa B subunits, (3) subcellular relocalization of Tax to the cytoplasm or nucleus depending on the coexpressed NF-kappa B subunit, and (4) Tax interaction with the Rel homology domain region of NFKB2. These studies indicate that the transcription modulatory influence of HTLV-I Tax may be significantly influenced by cytoplasmic-nuclear partitioning associated with the NF-kappa B proteins.

Identification of HTLV-I tax trans-activator mutants exhibiting novel transcriptional phenotypes.

The type I human T-cell leukemia virus (HTLV-I) encodes a 40-kD nuclear trans-regulatory protein termed Tax that transcriptionally activates the HTLV-I long terminal repeat (LTR), as well as select [corrected] cellular and heterologous viral promoters. Tax does not bind DNA specifically but, rather, acts in a more indirect manner. Tax activation of the HTLV-I LTR is mediated through constitutively expressed cellular factors that bind to cAMP response elements (CREs) present within the 21-bp enhancers of the LTR. In contrast, Tax transactivation of the interleukin-2 receptor-alpha gene (IL-2R alpha) and LTR of the type 1 human immunodeficiency virus (HIV-1) involves the induced nuclear expression of NF-kappa B. We now report the identification of missense mutations within the tax gene that functionally segregate these two pathways of trans-activation. Additionally, we demonstrate that the carboxyl terminus of the Tax protein, despite its acidic and predicted alpha-helical structure, is completely dispensable for trans-activation through either of these transcription factor pathways. Finally, we demonstrate that mutations within a putative zinc finger domain disrupt the nuclear localization of Tax and abolish trans-activation. These results demonstrate that Tax trans-activation of viral and cellular promoters involves at least two mechanisms of host transcription factor activation and suggest that this activation is likely mediated through distinct functional domains.