Olivier Hernalsteens

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

Abstract

The replication rate of the Human Immunodeficiency Virus type 1 (HIV-1), which is directly correlated to the disease progression, is controlled primarily at the transcription level of the viral genes. Understanding the molecular mechanisms underlying the HIV-1 gene expression is therefore crucial for the understanding of its pathogenesis. In addition to the cis-regulatory elements located at 5’LTR, our laboratory has identified an Intragenic cis-Regulatory Region (IRR), located within the coding sequence of HIV-1 and spanning from nt 4079 to nt 6026 (nt1 corresponding to the first nucleotide of 5’LTR). The IRR shows a double transcriptional activity, both promoter and enhancer on the 5’LTR. Moreover, a site which is hypersensitive to nucleases has been identified in the IRR, specifically in promonocytic HIV-1-infected cell lines but not in CD4+ T-lymphocytic cell lines, suggesting a cellular-specific role of the IRR in HIV-1 gene expression. Several binding sites for ubiquitous transcription factor (AP-1, Sp1, Oct1,...) and myeloid-specific factors, such as PU.1, have been identified in the IRR. In this context, the first part of this Master’s thesis studies the involvement of the three intragenic sites for the transcription factor PU.1 in the control of the replicative cycle of HIV-1, as well as in the transcriptional, cellular-specific activity of the IRR. We have shown that these sites are crucial for the replication of the virus and for the enhancer activity of the IRR, including in T-lymphocyte lines where the factor PU.1 is not expressed. Subsequently, we our results put in evidence the dierential occupation of the “PU.1” sites of the intragenic region of HIV-1 according to the cellular context. In the future, we will determine the nature of these complexes. In a second step, we studied the involvement of the PU.1 factor in the latency of HIV-1 in myeloid lineage by a functional approach of depletion by an pharmacological inhibitor : the pomalidomide. Our experiments have shown that the inhibition of PU.1 causes reactivations of HIV-1 genes, which points to the involvement of the PU.1 factor in maintaining the viral latency. Finally, we established a CRISPR interference system that allows to study the importance of several transcriptional activities of HIV-1. These keys results are discussed in this work and are shown to contribute to a better understanding of specific molecular mechanisms governing the transcription of HIV-1 genes, which will in turn lead to a better understanding of the pathogenesis of the virus.