Tristan Marray

Study of the intragenic cis-regulatory region of human immunodeficiency virus type 1 (HIV-1)

Abstract

The replication rate of human immunodeficiency virus type 1 (HIV-1) is directly proportional to the progression of acquired immunodeficiency syndrome (AIDS) and is primarily controlled at the level of retroviral gene transcription by cis-regulatory elements located at the 5′ LTR and the leader region. In addition to these elements, the host laboratory has identified an intragenic cis-regulatory region (IRR) composed of fragments 5103 and 5105 that exhibit PMA-inducible enhancer activity on the viral promoter and flank the nuclease hypersensitive HSVII site, identified only in the myeloid lineage, associated with promoter activity within the IRR. The existence of such a region would correspond to an adaptive strategy of HIV-1 allowing the regulation of viral gene expression in different environments. However, the mechanisms responsible for the enhancer activity of the IRR on the viral promoter and the establishment of a specific cellular chromatin environment are still poorly understood. In this context, this master thesis focuses on intragenic binding sites for the myeloid-specific factor PU.1 and the ubiquitous factor YY1. Two new intragenic binding sites for the transcription factor PU.1 were identified and added to the three sites previously characterized by the laboratory for their activating effect on IRR enhancer activity and HIV-1 replication. The PU.1 protein also has a pioneering activity, capable of binding nucleosomal DNA and inducing chromatin opening. The first part of this master thesis focuses on the in vitro characterization of the two new PU.1 binding sites by EMSAs. Furthermore, at the level of the viral promoter, the impact of PU.1 overexpression, in a heterologous context, induces a decrease in the transcriptional activity of 5’ LTR. This effect is independent of the binding of NF-κB factors at the 5' LTR. Furthermore, the use of a PU.1 protein truncated from its activation domain induces, conversely, an increase of the promoter activity. ATAC-seq experiments revealed chromatin remodeling at the 3' LTR after inhibition of intragenic PU.1 recruitment. YY1 has ten binding sites at the IRR, three of which are uncharacterized. YY1 could also be involved in the formation of chromatin loops that allow an enhancer region to be brought together with its target promoter. The study of the first seven intragenic YY1 binding sites by the laboratory revealed its activating effect on the IRR enhancer activity. In the second part of this master thesis, the three new intragenic YY1 binding sites were characterized in vitro by EMSAs. Transient transfection experiments demonstrated complete inhibition of IRR enhancer activity following mutation of all intragenic YY1 binding sites. T-lymphocyte and myeloid cell lines infected with HIV-1 mutated for intragenic YY1 binding sites show a significant decrease in viral replication. Overall, these results contribute to a better understanding of the molecular mechanisms regulating the expression of retroviral genes, which may serve as targets for new therapeutic strategies.