Maxime Bellefroid

Study of the intragenic cis-regulatory region of HIV-1

Abstract

Bovineleukemia virus (BLV) is a B-lymphotropic oncogenic retrovirus infecting cattle and associated asthe etiologic agentof theenzootic bovine leucosis(EBL). The major feature of the BLV infection is the viral latency inducedby a genetic or an epigenetic repressionofthe5'LTR-driven viral gene expression, allowingthe escape from the host immune system and thetumor development. However, the complexity of the BLV transcriptional regulation has been recently increased by the discovery by several laboratories, including ours, of two new active promoters despite the transcriptional repression of the 5'LTR: an RNAPIII-dependent promoter responsible for the transcription of a clustercoding for 10 viral miRNAs and an RNAPII-dependent promoter responsible for the expression of LTR3'-driven antisensetranscripts. In the first part of this master thesis, we studied the potential transcriptional interference between the three characterized BLV promoters by creatinga double reporter episomal plasmid allowing us to study simultaneously both the RNAPII-and the RNAPIII-dependent transcriptional activities. By transient transfection experiments followed by luciferase assays and multiplexed RT-qPCR, we first demonstrated the functionality of the double reporter plasmidfor reporting the different BLV transcriptional activities. By generating a double reporter episomal plasmid mutated in the 3'LTR cis-regulatory elements MTE/DPE and BRE, we also showed that the repression of 3'LTR-driven antisensetranscriptioncould positively affect the expression of the viral miRNAs and the 5'LTR-driven sensetranscription. These results suggest a potential transcriptional interference mechanism between the three RNA polymerase complexes that could contribute to the establishment of BLVviral latency andtumor development. In the second part of this master thesis, we studied the implication of the cellulartranscriptionfactor CTCF in BLV both epigenetic andtranscriptional regulations. By ChIP experiments in infected cell linesand primary cells, we demonstrated the in vivorecruitment of CTCF to the 3' ends of bothLTRsand,toalesser extent,tothe Tax/Rex E2 region. Furthermore, we observedthat the recruitment profile of CTCF along the BLV genome changed aftertransactivation of viral gene expressionby the viral transactivator Tax. By additional ChIP assays, we showed an important co-recruitment of the Rad21 subunitof the cohesin complex at bothLTRs, independently of the presence of Tax, suggesting the implication of theCTCF binding sites in the formation of chromatin loops. Taken together, our results contribute to an improved comprehension of the BLV complex transcriptional network and further suggest new ways of understanding the BLV-induced tumor development.