Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax BLV decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267 LTaxSN 5-LTR compared with the L267 5-LTR. Interestingly, DNA methylation inhibitors and Tax BLV synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the ؊154 or ؊129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at ؊129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency. Bovine leukemia virus (BLV)12 is a B-lymphotropic oncogenic retrovirus that infects cattle and is associated with enzo-

Efficient bovine leukemia virus (BLV) transcription requires the virus-encoded transactivatorBovine leukemia virus (BLV) 1 is a B-lymphotropic retrovirus associated with enzootic bovine leukosis, a disease characterized by an increased number of B-lymphocytes and, in some cases, after a long latency period, by the subsequent development of B-cell leukemia or lymphosarcoma (1). BLV is closely related structurally and biologically to the human T-lymphotropic viruses HTLV-I and HTLV-II (1). Expression of BLV is regulated at the transcriptional level by the virus-encoded transactivator Tax BLV (2, 3). The molecular mechanism by which Tax BLV activates viral transcription is not fully understood. Transactivation by Tax BLV requires three 21-bp imperfect repeats located in the U3 region of the 5Ј long terminal repeat (LTR) (2, 4). These Tax BLV -responsive elements (called TxREs) contain a core octanucleotide sequence with similarity to the cAMP-responsive element (CRE) consensus (TGACGTCA) (5). The BLV CRE-like motifs located in the middle of each TxRE have been shown to serve as binding sites for three members of the basic leucine zipper (bZIP) family of cellular transcription factors: the CRE-binding protein (CREB) and the activating transcription factors-1 and Ϫ2 (ATF-1 and ATF-2) (4, 6). Because there is no evidence for direct binding of Tax BLV to DNA, it has been proposed that Tax BLV transactivation of the BLV promoter could be mediated, as reported for the HTLV-I system, through protein-protein interactions with CREB/ATF (4, 6, 7). The formation of this promoter-bound Tax BLV -CREB/ATF complex could then serve for the recruitment of the multifunctional cellular coactivators CBP (CREB-binding protein) and p300.There is now strong evidence that both transcriptional activation and silencing are mediated through the recruitment of enzymes that control protein acetylation: the histone deacetylases (HDACs) and the histone acetyltransferases (HATs). Acetylation of specific lysine residues within the amino-terminal tails of nucleosomal histones is generally linked to chroma-