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Abstract

The establishment of HIV-1 latent reservoirs, residing primarily in T cells of memory phenotype, represent the main barrier to an HIV-1 cure. However, also cells of the central nervous system, including astrocytes and microglia, may contribute to the reservoir, and are particularly important sanctuaries of latent HIV-1 due to low penetration of antiretroviral drugs, lack of resident T cells, and permanent viral integration. Moreover, as many as half of the people living with HIV-1 display mild to moderate neurocognitive disorders, even under antiretroviral therapy. The aim of this PhD thesis was to determine HIV-1 integration sites in the microglia genome, and to profile global genomic and cell type specific chromatin signatures that may play an important role in the establishment of productive versus latent infections in the brain microglia model. In the first part of the thesis, I established a linker-mediated PCR based method to create a first map of HIV-1 integration patterns in microglia. Subsequently, I generated multi-omics data sets to profile HIV-1 insertions on different scales together with transcriptome by RNA-Seq, epigenome by ChIP-Seq and chromatin accessibility by ATAC-Seq in uninfected microglia cellular model. Comparison of the ISs with insertions of the main cellular targets of HIV-1 CD4+ T cells and macrophages revealed similar genomic feature distribution and shared gene repertoires. Intronic targeting of highly transcribed genes, demarcated by H3K36me3 and genic enhancers along with cellular cofactors requirements were found to be universal integration properties within different HIV-1 reservoirs. The second aim of my thesis focused on the investigation of potential differences in transcriptional networks between cells harboring actively replicating virus versus cells with latent HIV-1 by performing ATAC-Seq. Transcription factor footprinting as well as biochemical fractionations revealed the architectural protein CCCTC-binding factor (CTCF) as a dynamic mark of infection in microglia cellular model. Given CTCF's significant role in determining 3D chromatin structures, the thesis explored the possibility of a connection between HIV-1 integration sites and topologically associated domains (TADs) by mapping HIV-1 insertion sites onto previously published Hi-C maps of microglia-containing tissues and T cells. The analysis uncovered TAD boundaries delimited with H3K36me3 chromatin mark as a new 3D genome determinant of HIV-1 integration in microglia and CD4+ T cells. Moreover, CTCF was found to interact with viral integrase in a LEDGF/p75 dependent manner, which is an essential integration host factor and integrase interactor. To explore the potential involvement of CTCF in HIV-1 integration, I transiently depleted CTCF in HIV-1 target cells and observed a reduction in HIV-1 integration efficiency and redistributed insertion sites, indicating that the 3D arrangement of chromatin may play a significant role in HIV-1 infection. In conclusion, this thesis provides valuable insights into the role of chromatin shaping proteins and 3D genome architecture in viral integration and latency, which holds increasing importance in HIV-1 research. Furthermore, it demonstrated that integration patterns and chromatin characteristics in microglia, a central nervous system HIV-1 target cell type, resemble those found in blood reservoirs. This underscores the potential of using common therapeutic strategies for different HIV-1 reservoirs. Nonetheless, future research using primary tissues could unveil new, cell-type-specific factors as possible targets for effective and safe brain-targeting therapeutics.