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 Taxresponsive 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), the etiologic agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis, is a T-cell-tropic virus that belongs to the HTLV-bovine leukemia virus (HTLV-BLV) group of the retrovirus family. Other group members include HTLV-2, BLV, and the simian T-cell leukemia viruses. All retroviruses share similarity in their genome organization, with four genes encoding proteins necessary for the production of infectious virions, the order of these genes being invariably gag, pro, pol, env (from 5Ј to 3Ј) (Fig. 1A). gag codes for internal structural proteins of the virion, pro codes for the viral protease, pol codes for reverse transcriptase, and env codes for envelope glycoproteins of the virion. The expression of the HTLV-1 genome is controlled by two regulatory genes, the rex and tax genes, located in the 3Ј region of the viral genome. Tax is a potent transactivator of viral transcription (5, 30), and Rex acts at the posttranscriptional level by modulating the transport of the viral RNAs (14). Moreover, the HTLV-1 genome carries between the env gene and the 3Ј long terminal repeat (LTR) other open reading frames encoding proteins with unknown functions. All these viral proteins are encoded by the viral plus-strand RNA.The trans-activation of the viral transcription by Tax operates through three 21-bp repeats (6, 28) located in the U3 region of the LTR. These conserved repeats, called Tax-responsive elements (TxREs), contain an imperfect cyclic AMP response element (CRE) (17) that is recognized by CREB (4, 37, 38) and CREM (29), two members of the activating transcription factor/CRE-binding protein (ATF/CREB) family. These factors are characterized by basic leucine zipper (bZIP) C-terminal structures required for DNA binding and protein dimerization. To activate transcription of the HTLV-1 genome, Tax interacts with CREB or CREM bound to the TxREs (4, 38) and then can recruit the transcriptional coactivator CR...