The RNA genome of the human T-cell leukemia virus type 1 (HTLV-1) codes for proteins involved in infectivity, replication, and transformation. We report in this study the characterization of a novel viral protein encoded by the complementary strand of the HTLV-1 RNA genome. This protein, designated HBZ (for HTLV-1 bZIP factor), contains a N-terminal transcriptional activation domain and a leucine zipper motif in its C terminus. We show here that HBZ is able to interact with the bZIP transcription factor CREB-2 (also called ATF-4), known to activate the HTLV-1 transcription by recruiting the viral trans -activator Tax on the Tax-responsive elements (TxREs). However, we demonstrate that the HBZ/CREB-2 heterodimers are no more able to bind to the TxRE and cyclic AMP response element sites. Taking these findings together, the functional inactivation of CREB-2 by HBZ is suggested to contribute to regulation of the HTLV-1 transcription. Moreover, the characterization of a minus-strand gene protein encoded by HTLV-1 has never been reported until now.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of a fatal malignancy known as adult T-cell leukemia (ATL). One way to address the pathology of the disease lies on conducting research with a molecular approach. In addition to the analysis of ATL-relevant signaling pathways, understanding the regulation of important and relevant transcription factors allows researchers to reach this fundamental objective. HTLV-1 encodes for two oncoproteins, Tax and HTLV-1 basic leucine-zipper factor, which play significant roles in the cellular transformation and the activation of the host’s immune responses. Activating protein-1 (AP-1) transcription factor has been linked to cancer and neoplastic transformation ever since the first representative members of the Jun and Fos gene family were cloned and shown to be cellular homologs of viral oncogenes. AP-1 is a dimeric transcription factor composed of proteins belonging to the Jun (c-Jun, JunB, and JunD), Fos (c-Fos, FosB, Fra1, and Fra2), and activating transcription factor protein families. Activation of AP-1 transcription factor family by different stimuli, such as inflammatory cytokines, stress inducers, or pathogens, results in innate and adaptive immunity. AP-1 is also involved in various cellular events including differentiation, proliferation, survival, and apoptosis. Deregulated expression of AP-1 transcription factors is implicated in various lymphomas such as classical Hodgkin lymphomas, anaplastic large cell lymphomas, diffuse large B-cell lymphomas, and adult T-cell leukemia. Here, we review the current thinking behind deregulation of the AP-1 pathway and its contribution to HTLV-induced cellular transformation.

The human T-cell leukemia virus type I (HTLV-I)-encoded Tax protein activates transcription from the viral promoter via association with the cellular basic leucine zipper factor cAMP-response element-binding protein-2. Tax is also able to induce cellular transformation of T lymphocytes probably by modulating transcriptional activity of cellular factors, including nuclear factor-κB, E2F, activator protein-1 (AP-1), and p53. Recently, we characterized in HTLV-I-infected cells the presence of a novel viral protein, HBZ, encoded by the complementary strand of the HTLV-I RNA genome (Gaudray, G., Gachon, F., Basbous, J., Biard-Piechaczyk, M., Devaux, C., and Mesnard, J.-M. (2002) J. Virol. 76, 12813–12822). HBZ is a nuclear basic leucine zipper protein that down-regulates Tax-dependent viral transcription by inhibiting the binding of cAMP-response element-binding protein-2 to the HTLV-I promoter. In searching for other cellular targets of HBZ, we identified two members of the Jun family, JunB and c-Jun. Co-immunoprecipitation and cellular colocalization confirmed that HBZ interacts in vivo with JunB and c-Jun. When transiently introduced into CEM cells with a reporter gene containing the AP-1 site from the collagenase promoter, HBZ suppressed transactivation by c-Jun. On the other hand, the combination of HBZ with Jun-B had higher transcriptional activity than JunB alone. Consistent with the structure of its basic domain, we demonstrate that HBZ decreases the DNA-binding activity of c-Jun and JunB. Last, we show that c-Jun is no longer capable of activating the basal expression of the HTLV-I promoter in the presence of HBZ in vivo. Our results support the hypothesis that HBZ could be a negative modulator of the Tax effect by controlling Tax expression at the transcriptional level and by attenuating activation of AP-1 by Tax.

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.

Background: Antisense transcription in retroviruses has been suggested for both HIV-1 and HTLV-I, although the existence and coding potential of these transcripts remain controversial. Thorough characterization is required to demonstrate the existence of these transcripts and gain insight into their role in retrovirus biology. Results: This report provides the first complete characterization of an antisense retroviral transcript that encodes the previously described HTLV-I HBZ protein. In this study, we show that HBZ-encoding transcripts initiate in the 3' long terminal repeat (LTR) at several positions and consist of two alternatively spliced variants (SP1 and SP2). Expression of the most abundant HBZ spliced variant (SP1) could be detected in different HTLV-I-infected cell lines and importantly in cellular clones isolated from HTLV-I-infected patients. Polyadenylation of HBZ RNA occurred at a distance of 1450 nucleotides downstream of the HBZ stop codon in close proximity of a typical polyA signal. We have also determined that translation mostly initiates from the first exon located in the 3' LTR and that the HBZ isoform produced from the SP1 spliced variant demonstrated inhibition of Tax and c-Jun-dependent transcriptional activation. Conclusion: These results conclusively demonstrate the existence of antisense transcription in retroviruses, which likely plays a role in HTLV-I-associated pathogenesis through HBZ protein synthesis.

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 NH₂-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.