DNA Cytosine Methylation in the Bovine Leukemia Virus Promoter Is Associated with Latency in a Lymphoma-derived B-cell Line

Abstract

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-

Chromatin disruption in the promoter of Bovine Leukemia Virus during transcriptional activation

“…Accordingly, we have previously reported the absence of DNA methylation in the U3/R region of the YR2 provirus, in opposition to the hypermethylated state of the L267 provirus corresponding region (36). The R/U5 region was partially accessible to nucleases in both models.…”

A detailed molecular analysis of complete Bovine Leukemia Virus genomes isolated from B-cell lymphosarcomas

“…It is thought that silencing of viral expression is a multi-step process leading to the uncontrolled growth of a transformed B-cell clone and the onset of disease [5] and is critical for tumor progression and proliferation of BLV-infected tumor cells [5], as well as escaping recognition by the host immune response [4]. In that sense, the substitution found in the GRE site of the 5′LTR of all BLV strains isolated from the lymphosarcomas might contribute to these factors, since previous studies have shown that substitutions in GRE site significantly reduces basal LTR transcription activity [52] (see Figure 1).…”

“…This is thought to be related to the transcriptional repression of viral expression in vivo [3]. Latency is likely a viral strategy to evade the host immune response, thereby allowing tumor development [4,5]. In fact, B lymphocytes harboring an integrated provirus do not produce detectable levels of viral RNA or proteins [6].…”

Characterization of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome

“…Indeed, we have previously reported that 5′LTR hypermethylation is associated with the BLV repression in the true latent L267 cell line but not in the defective latency state present in the YR2 cell line25. Here, we showed by bisulfite genome sequencing that the DNA encoding the BLV miRNA cluster, which is located within a CpG island, was not methylated.…”

“…Indeed, we have previously demonstrated that the 5′LTR RNA polymerase II-driven transcriptional repression is due to the epigenetic state of the 5′LTR characterized by weak histone acetylation and DNA CpG hypermethylation associated to closed chromatin in a lymphoma-derived BLV-infected L267 ovine cell line harboring a fully competent provirus192125.…”