Activation of human T cell leukemia virus type 1 (HTLV-1) transcription is established through the formation of protein complexes on the viral promoter that are essentially composed of the cellular basic leucine zipper (bZIP) transcription factor cAMP-response element-binding protein (CREB (or certain other members of the ATF/CREB family), the HTLV-1-encoded transactivator Tax, and the pleiotropic cellular coactivators p300/CBP. HTLV-1 bZIP factor (HBZ) is a protein encoded by HTLV-1 that contains a bZIP domain and functions to repress HTLV-1 transcription. HBZ has been shown to repress viral transcription by dimerizing with CREB, which occurs specifically through the bZIP domain in each protein, and preventing CREB from binding to the DNA. However, we previously found that HBZ causes only partial removal of CREB from a chromosomally integrated viral promoter, and more importantly, an HBZ mutant lacking the COOH-terminal bZIP domain retains the ability to repress viral transcription. These results suggest that an additional mechanism contributes to HBZ-mediated repression of HTLV-1 transcription. In this study, we show that HBZ binds directly to the p300 and CBP coactivators. Two LXXLL-like motifs located within the NH 2 -terminal region of HBZ are important for this interaction and specifically mediate binding to the KIX domain of p300/CBP. We provide evidence that this interaction interferes with the ability of Tax to bind p300/CBP and thereby inhibits the association of the coactivators with the viral promoter. Our findings demonstrate that HBZ utilizes a bipartite mechanism to repress viral transcription.Human T-cell leukemia virus type 1 (HTLV-1) 5 is a retrovirus that is the causative agent of adult T-cell leukemia and a neurodegenerative disorder termed tropical spastic paraparesis/HTLV-1-associated myelopathy (1, 2). Following its integration, the provirus utilizes the cellular RNA polymerase II transcription machinery for replication of the viral genome and expression of viral genes. These processes are dependent on the viral transactivator Tax, which is essential for strong activation of HTLV-1 transcription. Tax alone lacks DNA binding activity and is therefore recruited to the viral promoter as part of a complex with the cellular transcription factor CREB or other members of the activating transcription factor/cyclic AMP-responsive element (CRE)-binding protein (ATF/CREB) family (3-6). These proteins carry a basic leucine zipper (bZIP) domain that stimulates protein dimerization and subsequent DNA binding. Dimer formation is specifically mediated through the leucine zipper (ZIP) domain, whereas DNA binding involves the basic region of each binding partner directly contacting the DNA. The Tax-CREB complexes associate with three Tax-responsive elements called viral CREs (vCREs) within the viral promoter that encompasses the U3 region of the 5Ј-long terminal repeat of the provirus. Each vCRE contains a central sequence similar to that of a cellular CRE that is recognized by CREB and flanking GC-rich s...

The complex human T-cell leukemia virus type 1 (HTLV-1) retrovirus encodes several proteins that are unique to the virus within its 3-end region. Among them, the viral transactivator Tax and posttranscriptional regulator Rex are well characterized, and both positively regulate HTLV-1 viral expression. Less is known about the other regulatory proteins encoded in this region of the provirus, including the recently discovered HBZ protein. HBZ has been shown to negatively regulate basal and Tax-dependent HTLV-1 transcription through its ability to interact with specific basic-leucine zipper (bZIP) proteins. In the present study, we found that HBZ reduces HTLV-1 transcription and virion production. We then characterized the interaction between HBZ and the cellular transcription factor CREB. CREB plays a critical role in Tax-mediated HTLV-1 transcription by forming a complex with Tax that binds to viral cyclic AMP-response elements (CREs) located within the viral promoter. We found that HBZ and CREB interact in vivo and directly in vitro, and this interaction occurs through the bZIP domain of each protein. We also found that CREM-Ia and ATF-1, which share significant homology in their bZIP domains with the bZIP domain of CREB, interact with HBZ-bZIP. The interaction between CREB and HBZ prevents CREB binding to the viral CRE elements in vitro and in vivo, suggesting that the reduction in HTLV-1 transcription by HBZ is partly due to the loss of CREB at the promoter. We also found that HBZ displaces CREB from a cellular CRE, suggesting that HBZ may deregulate CREB-dependent cellular gene expression.Human T-cell leukemia virus type 1 (HTLV-1) is a human retrovirus that is associated with two distinct diseases: adult T-cell leukemia (ATL), an abnormal proliferation of infected CD4 ϩ T lymphocytes, and HTLV-1-associated myelopathy and/or tropical spastic paraparesis, a neurodegenerative disorder (19,48,49). The molecular mechanisms leading to the development of both diseases are unclear, although the viral protein Tax is postulated to play an important role in these processes. Tax functions as a transcription factor and is essential for strong HTLV-1 transcription. Tax activates transcription through three 21-bp repeats that contain imperfect cyclic AMP responsive elements (called viral CREs) situated within the long terminal repeat of the HTLV-1 genome (1,6,14,15,22,27). Tax does not bind DNA alone but interacts with cellular transcription factors from the ATF/CREB family to form complexes that associate with the DNA. Within these complexes, Tax contacts the GC-rich sequences flanking the CRE core (31,37,38,41). The formation of Tax/CREB/DNA complexes is critical for the recruitment of the cellular coactivators CBP/p300 and subsequent high transcriptional activation of the virus (18,21,32,39,58).A number of cellular factors containing basic leucine zipper (bZIP) motifs have been shown to bind the viral CREs in HTLV-1-infected T cells. These factors included the ATF/ CREB family members (ATF-1, ATF-2, CREB, CREB-2, and CRE...

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The human T-cell leukemia virus (HTLV-I)-encodedTax protein is a potent transcriptional activator that stimulates expression of the integrated provirus. Biochemical studies indicate that Tax, together with cellular transcription factors, interacts with viral cAMP-response element enhancer elements to recruit the pleiotropic coactivators CREB-binding protein and p300. Histone acetylation by these coactivators has been shown to play a major role in activating HTLV-I transcription from chromatin templates in vitro. However, the extent of histone modification and the precise identity of the cellular regulatory proteins bound at the HTLV-I promoter in vivo is not known. Chromatin immunoprecipitation analysis was used to investigate factor binding and histone modification at the integrated HTLV-I provirus in infected T-cells (SLB-1). These studies reveal the presence of Tax, a variety of ATF/CREB and AP-1 family members (CREB, CREB-2, ATF-1, ATF-2, c-Fos, and c-Jun), and both p300 and CREB-binding protein at the HTLV-I promoter. Consistent with the binding of these coactivators, we observed histone H3 and H4 acetylation at three regions within the proviral genome. Histone deacetylases were also present at the viral promoter and, following their inhibition, we observe an increase in histone H4 acetylation on the HTLV-I promoter and a concomitant increase in viral RNA. Together, these results suggest that a variety of transcriptional activators, coactivators, and histone deacetylases participate in the regulation of HTLV-I transcription in infected T-cells.

The human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that integrates randomly into the T-cell genome. Two long terminal repeats (LTRs) flank the integrated provirus. The upstream and downstream LTRs carry identical promoter sequences. Studies with other retroviruses suggest that the downstream promoter is silent and that RNA polymerases initiating at the upstream promoter proceed through the 3 LTR. In this study, we used the chromatin immunoprecipitation assay to compare the binding of transcription regulatory proteins at both the upstream and downstream promoters in HTLV-1-infected cell lines and adult T-cell leukemia-lymphoma cells. Unexpectedly, we detected a nearly equal distribution of activator (Tax, CREB, ATF-1, ATF-2, c-Fos, and c-Jun) and regulatory protein (CBP, p300, TAF II 250, and polymerase II) binding at both the upstream and downstream promoters. Consistent with this observation, we found that the downstream promoter was transcriptionally active, suggesting that the two promoters are functionally equivalent. We also detected asymmetrical binding of histone deacetylases (HDAC-1, -2, and -3) at both promoters. All three HDACs strongly repressed Tax transactivation, and this repression correlated with displacement of Tax from the HTLV-1 promoter. These effects were reciprocal, as Tax expression reversed HDAC repression and displaced HDACs from the HTLV-1 promoter. These data suggest that HTLV-1 transcriptional regulation at both the 5 and 3 LTRs is mediated, in part, through the mutually exclusive binding of Tax and HDACs at the proviral promoters.The human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for an aggressive and fatal malignancy called adult T-cell leukemia-lymphoma (ATLL) (37, 46). Following infection, HTLV-1 integrates randomly into the host cell chromosome (40). During this event, the 5Ј and 3Ј ends of the retrovirus are duplicated, forming long terminal repeats (LTRs). The LTRs of the integrated provirus carry two identical U3 regions containing two identical promoters. The 5Ј promoter directs synthesis of the genomic RNA, whereas the 3Ј promoter, if active, synthesizes RNAs that extend into the adjacent host cell genome. Previous studies with other retroviruses have suggested that RNA polymerase II (Pol II) initiates at the upstream U3 promoter and transcription proceeds through the provirus and downstream U3 promoter, as the polyadenylation signal is located adjacent to the TATA sequence and the polyadenylation site is downstream of the U3 region (13).The U3 promoter regions of HTLV-1 each carry three highly conserved 21-bp enhancer elements that are critical for Tax-activated transcription. These 21-bp elements are referred to as viral CREs. The viral CREs carry a central binding site for the cellular transcription factor CREB and/or other ATF/ CREB family members (reviewed in reference 11). The CREs are flanked by GC-rich DNA sequences that are conserved in all HTLV family members. The virally encoded Tax oncoprotein is the regulatory protein respon...

(R)-PCEP (3-amino-3-carboxypropyl-2'-carboxyethyl phosphinic acid, 1), a new metabotropic glutamate receptor 4 (mGlu4R) agonist, was discovered in a previously reported virtual screening. The (S)-enantiomer and a series of derivatives were synthesized and tested on recombinant mGlu4 receptors. A large number of derivatives activated this receptor but was not able to discriminate between mGlu4 and mGlu8 receptors. The most potent ones 6 and 12 displayed an EC(50) of 1.0 +/- 0.2 microM at mGlu4R. Interestingly these agonists with longer alkyl chains revealed a new binding pocket adjacent to the glutamate binding site, which is lined with residues that differ among the mGluR subtypes and that will allow the design of more selective compounds. Additionally 6 was able to activate mGlu7 receptor with an EC(50) of 43 +/- 16 microM and is thus significantly more potent than L-AP4 (EC(50) of 249 +/- 106 microM).

Infection with the human T-cell leukemia virus type 1 (HTLV-1) results in a variety of diseases including adult T-cell leukemia (ATL), a fatal malignancy characterized by the uncontrolled proliferation of virally infected CD4 + T cells. The HTLV-1 basic leucine zipper factor (HBZ) is believed to contribute to development and maintenance of ATL. Unlike the other HTLV-1 genes, the hbz gene is encoded on the complementary strand of the provirus and therefore is not under direct control of the promoter within the 5′ long terminal repeat (LTR) of the provirus. This promoter can undergo inactivating genetic or epigenetic changes during the course of ATL that eliminates expression of all viral genes except that of hbz . In contrast, repressive modifications are not known to occur on the hbz promoter located in the 3′ LTR, and hbz expression has been consistently detected in all ATL patient samples. Although Sp1 regulates basal transcription from the HBZ promoter, other factors that activate transcription remain undefined. In this study, we used a proviral reporter construct deleted of the 5′ LTR to show that HBZ upregulates its own expression through cooperation with JunD. Activation of antisense transcription was apparent in serum-deprived cells in which the level of JunD was elevated, and elimination of JunD expression by gene knockout or shRNA-mediated knockdown abrogated this effect. Activation through HBZ and JunD additionally required Sp1 binding at the hbz promoter. These data favor a model in which JunD is recruited to the promoter through Sp1, where it heterodimerizes with HBZ thereby enhancing its activity. Separately, hbz gene expression led to an increase in JunD abundance, and this effect correlated with emergence of features of transformed cells in immortalized fibroblasts. Overall, our results suggest that JunD represents a novel therapeutic target for the treatment of ATL.

The complex retrovirus Human T-cell Leukemia Virus type I (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). Deregulation of cellular transcription is thought to be an important step for T-cell transformation caused by viral infection. HTLV-1 basic leucine zipper factor (HBZ) is one of the viral proteins believed to be involved in this process as it deregulates the expression of numerous cellular genes. In the context of the provirus, HBZ represses HTLV-1 transcription, in part, by binding to the homologous cellular coactivators p300 and CBP. These coactivators play a central role in transcriptional regulation. In this study we determined that HBZ binds with high affinity to the KIX domain of p300/CBP. This domain contains two binding surfaces that are differentially targeted by multiple cellular factors. We show that two ϕXXϕϕ motifs in the activation domain of HBZ mediate binding to a single surface of the KIX domain, the mixed-lineage leukemia (MLL)-binding surface. Formation of this interaction inhibits binding of MLL to the KIX domain while enhancing the binding of the transcription factor, c-Myb to the opposite surface of KIX. Consequently, HBZ inhibits transcriptional activation mediated by MLL and enhances activation mediated by c-Myb. CREB, which binds the same surface of KIX as c-Myb, also exhibited an increase in activity through HBZ. These results indicate that HBZ is able to alter gene expression by competing with transcription factors for occupancy of one surface of KIX while enhancing the binding of factors to the other surface.