Colomban Coene

tudy of the impact of PU.1 binding sites of the intragenic cis-regulatory region on the transcriptional activity of human immunodeficiency virus type 1 (HIV-1)

Abstract

The replication rate of the human immunodeficiency virus type 1 (HIV-1), directly related to the progression of acquired immunodeficiency syndrome (AIDS), is mainly regulated at the transcription of retroviral genes. Therefore, understanding the mechanisms regulating the expression of viral genes is essential for a better understanding of the pathogenicity of the virus and the implementation of treatments aimed at its eradication. In addition to the cis-regulatory region present in the 5'LTR of the provirus, our laboratory has highlighted an Intragenic cis-Regulatory Region (IRR), located between nucleotides 4079 and 6026 (where the nt + 1 corresponds to the first nucleotide of the 5’LTR). IRR exhibits two distinct transcriptional activities: promoter and enhancer. It is composed of three functional domains: fragments 5103 and 5105, flanking the hypersensitive site to DNAse I (HSVII) only present in the infected promonocytic line. Within IRR, several binding sites have been identified for ubiquitous and myeloid-specific transcription factors such as factor PU.1. In addition to its transcription factor activity, PU.1 exhibits pioneering activity by binding to heterochromatin and inducing its opening. Knowing this, the first part of my master’s thesis focuses on the involvement of the three intragenic binding sites for the factor PU.1 in enhancer activity of the IRR and in the control of the viral replicative cycle in promonocytic lines. We have shown that these sites are important for enhancer activity and viral replication because inhibition of PU.1 binding to one or more of its sites induces a total loss of IRR enhancer activity and a statistically significant decrease in the viral replication. In the second part, we studied the mechanistic allowing factor PU.1 to regulate the transcriptional activity of HIV-1 by ChIP and FAIRE in chronically infected lines. Our experiments showed recruitment of PU.1 and a chromatin opening at the level of the IRR, suggesting that PU.1 could be at the origin of chromatin opening. Finally, we used a pharmacological approach to inhibit the binding of PU.1 to IRR using a dicationic heterocyclic molecule named DB2115. Our results showed a significant decrease in viral replication, indicating that this molecule could be used in anti-HIV treatment. These results are presented in detail in my master’s thesis and contribute to improve the understanding of the molecular mechanisms involved in the transcriptional regulation of HIV-1 in promonocytic lines and offer new paths to develop new therapeutic approaches.