HIV persists for many years during therapy and normally rebounds to pretreatment levels when therapy is stopped. However, a small number of individuals have been able to control their infection, despite the presence of infectious virus, fo many years after stopping therapy. Understanding the factors that lead to virus control under these circumstances is critical to our ability to achieve this end in others as well as for an eventual cure of infection. We are learning that where the virus integrates its genome into the infected cell genome is of significance to the maintenance of HIV reservoirs. However, during suppressive therapy, the rarity of cells with replication competent HIV and the lack of expression of viral proteins by these cells have hampered the study of HIV reservoirs. We propose novel methods to overcome the problem of cell rarity by the use of a novel method to enrich for and expand single infected cells for study of structure and RNA expression of the integrated proviral genome and surrounding sequences within the host cell genome. To address the problem of the lack of protein expression, we will use additional novel methods to purify expanded infected cells, as lysates, allowing analysis of the entire cellular transcriptome and proteome. Lastly, we will recreate the proviral structures we observe in vivo in an in vitro model to discern the global impact of provirus integration on cell functionality.
We propose to understand the role HIV has, as it exists at different sites in the infected cell genome, to the persistence of HIV reservoirs in different cell types and tissues, and the ability to control HIV infection after interruption of ART. We propose 3 Aims to address these issues, each building on the previous, and each providing heretofore unprecedented windows into reservoir cell structure, function and virologic control.
