Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY / ABSTRACT Establishment of the long-lasting latent reservoir and the low level of detectable HIV replication remain stumbling blocks to cure from HIV infection. Long non-coding RNAs (lncRNAs), novel regulators of gene expression, hold promise for development of HIV cure strategies, because they are more tissue and cell-type specific than protein coding genes. Our long-term goal is to develop more potent strategies with higher specificity than are currently available to target the latent HIV reservoir for reactivation or silencing. The overall objective of this application is to evaluate the merit of using lncRNA-based approaches for HIV cure. Our central hypothesis is that targeting single lncRNAs or their combinations will result in more robust effects on HIV expression compared to existing strategies that use latency reversing (LRAs) or latency promoting agents (LPAs). To test our central hypothesis, two specific aims will be pursued: (1) Identify lncRNAs with a role in the regulation of HIV expression by conducting a CRISPR/Cas9 screen; (2) Identify overlapping functions of lncRNAs using the RNA interactome of the HIV repressor zeste 2 polycomb repressive complex 2 subunit (EZH2). For the CRISPR/Cas9 screen, JLat cell lines constitutively expressing Cas9 will be generated and transduced with the sgRNA library to target lncRNA splice sites. To identify lncRNAs that act as HIV repressors, cells will be screened in the resting state. When repressive lncRNAs are knocked out, sgRNAs are expected to be enriched in the population of HIV-producing cells that express GFP reporter protein. To identify lncRNAs that act as HIV activators, the fraction of cells that remain GFP-negative after initial lncRNA knockout will be treated with tumor necrosis factor (TNF) alpha. When lncRNAs that contribute to HIV activation are knocked out, sgRNAs are expected to be enriched in the population of cells that remain GFP-negative following treatment with TNF alpha. For identification of overlapping functions of lncRNAs, EZH2 has been selected because it is an HIV repressor that has several known lncRNA interacting partners. We will identify the entire lncRNA interactome of EZH2 using the enhanced UV cross-linking and immunoprecipitation (eCLIP). Selected lncRNAs from the screen and the EZH2 interactome will be knocked down individually and in combinations in JLat and primary CD4+ T cells, and effect on HIV expression will be compared to that induced by LRAs and LPAs. We will further conduct chromatin immunoprecipitation to determine whether knockdown of identified lncRNAs affects EZH2 occupancy of the HIV promoter. Because eCLIP will result in identification of specific sequences of the lncRNAs that interact with EZH2, we will mutate these sequences to determine whether interaction with EZH2 is responsible for the observed activator or repressor phenotype. These results will be significant because they will provide strong scientific justification for development of lncRNA-based therapeutic interventions to reactivate latent HIV or enhance viral suppression on antiretroviral therapy. For example, small molecules can be used to specifically disrupt interactions between lncRNAs and proteins.
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