The Role of LEDGF in establishing HIV latency in primary CD4 T cells

Award amount: 40,000.00

One of the most promising strategies for curing HIV infection is referred to as “shock and kill,” whereby latently infected cells are induced to express the virus and subsequently cleared from the body. Understanding how certain cells become latently infected, however, has been a major hurdle towards realizing this strategy. It is known that certain host factors, such as the chromatin bound protein LEDGF, can influence latency by dictating the site of HIV integration and recruiting epigenetic factors. In primary CD4+ T cells, a major reservoir for HIV in vivo, the role of LEDGF in dictating the site of HIV integration and in establishing latency has been poorly characterized due to the significant technical limitations these cells impose. Recently, we have developed a novel platform for high-throughput CRISPR/Cas9 editing in primary T cells that enables the efficient generation of knock-out cell populations for functional interrogation. Using this technology, we found that LEDGF is critical, but not absolutely necessary, for HIV integration in primary cells. Furthermore, viruses integrated in the absence of LEDGF were more likely to be transcriptionally silent. We therefore hypothesize that HIV can utilize LEDGF-independent pathways for integration in primary T cells and that these pathways may be important for establishing latent infection. To test this hypothesis, we aim to knock-out LEDGF in primary CD4+ T cells and quantify the contribution of this protein to both active and latent infection using a state-of the-art primary model of latency. We will also use this model system to measure viral reactivation and map HIV integration sites in the presence and absence of LEDGF. Second, we aim to identify alternate chromatin receptors for HIV integration by performing affinity-purification mass spectrometry on HIV Integrase. The impact of candidate receptors on HIV infection will be tested by double knock-out experiments and infection.