Role of the polgene enhancer in HIV-1 transcription and replication in myeloid infected cells

Title

Role of the polgene enhancer in HIV-1 transcription and replication in myeloid infected cells

Presenter

Carine Van Lint

Authors

C. Van Lint * (1), O. Hernalsteens (1), R. Verdikt (1), C. Vanhulle (1), A. Dutilleul (1), S. Cristinelli (2), T. Marray (1), L. Nestola (1), V. Monceaux (3), A. David (3), B. Verhasselt (4), H. Galons (5), A. Ciuffi (2), A. Saez-Cirion (3), O. Rohr (6)

Institutions

(1) Université Libre de Bruxelles (ULB), Gosselies, Belgium, (2) Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland, (3) Institut Pasteur de Paris, Paris, France, (4) Ghent University, Ghent, Belgium, (5) CNRS UMR 8258 - U 1267 INSERM, Paris, France, (6) IUT Louis Pasteur de Schiltigheim, Strasbourg, France

BACKGROUND: There is increasing evidence for the physiological relevance of myeloid HIV-1 reservoirs such as brain microglia and urethral macrophages. However, the molecular mechanisms of HIV-1 expression in myeloid infected cells are still poorly understood. The HIV-1 intragenic cis-regulatory region (IRR) located in the pol gene exhibits an enhancer activity on the 5'LTR promoter. The IRR possesses multiple binding sites for various cellular transcription factors (TF). Here, we characterized several of these binding sites and their functional involvement in the IRR-mediated control of HIV-1 gene expression in monocytes/macrophages. Special emphasis was put on studying several binding sites for the myeloid-specific PU.1 TF, known to be a pioneer factor inducing the opening of heterochromatin in enhancers.
METHODS: Electrophoretic Mobility Shift Assays (EMSAs), chromatin immunoprecipitations, infection studies, ATAC-seq (Assay for Transposase-Accessible Chromatin sequencing).
RESULTS: We demonstrated the in vivo recruitment of PU.1 to the HIV-1 intragenic enhancer in latently-infected cell lines from myeloid origin. We physically characterized in vitro PU.1 binding to the different intragenic PU-boxes by EMSAs and identified mutations abolishing PU.1 binding without altering the underlying amino acid sequence of the polgene. We demonstrated the role of the PU-boxes in the IRR enhancer activity, its chromatin remodeling and HIV-1 epigenetic regulation, in concert with other IRR transcription factor binding sites. We showed the importance of intragenic TF binding sites in HIV-1 replication using mutated viral particles in single-round infection experiments using primary monocytes-derived macrophages isolated from uninfected individuals. To overcome HIV- 1 persistence, targeted approaches for each specific cellular reservoir are needed. As a proof-of-concept, we revealed the potential therapeutic application of a specific inhibitor interfering with PU.1 binding as a new anti-HIV-1 approach.
CONCLUSIONS: The HIV-1 intragenic enhancer brings an additional factor in an already complex network of regulators affecting the level of HIV-1 transcription. Such complexity could allow a finer-tuned regulation that might find its purpose when HIV-1 transcription needs to be moderately or transiently modified within different cellular and chromatin environments. The cell specificity of the IRR in HIV-1 gene expression regulation opens new avenues for HIV cure approaches targeting the heterogeneous cellular and tissue latent reservoirs of virus.

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