Post-integration viral latency is a major barrier to eradicating HIV-1 infection. The current theoretical paradigm for eliminating the viral reservoir is known as `Shock and Kill', reactivation of latent virus using non-targeted small molecules. Key hurdles to this approach have recently emerged such as inefficient and/or stochastic viral reactivation, avoidance of global T-cell activation, and limited cellular death upon re-activation. It is also becoming apparent that our understanding of the molecular mechanisms of HIV latency are fragmentary and in particular the relevance of drugs of abuse on the initiation and maintenance of HIV latency. Here, we propose to explore on a genomic scale the landscape of cellular factors that regulate HIV latency establishment and maintenance and their relationship to drugs of abuse. Our exploration will be based on validating a recently completed genome scale shRNA screen for genes that control latency maintenance and reactivation. We propose to expand this screen using the novel CRISPR-Cas9 technology based on targeting via a guide RNA fusion proteins that either activate (CRISRa) or inhibit (CRISPRi) gene expression. These screens will focus on the mTOR pathway and on methamphetamine as we have recently uncovered evidence that they may act independently or together to modulate the reactivation of latent HIV. We will validate and mechanistically explore both pathways and other top hits in primary CD4 T cells and in cells from HIV-infected patients. We also propose to further develop and exploit new dual-fluorescence reporter HIV-1 genomes to identify, quantify, and purify latently infected cells in their native state, without inducing viral reactivation. This new latency model will allow us to study the effect of drugs of abuse, particularly methamphetamine, on the establishment and maintenance of latency in primary human lymphoid cells and to study the very earliest mechanisms of HIV-1 latency establishment. By combining the power of our dual-labeled latency model with high-resolution single-cell systems-biology techniques, we are uniquely suited to map out the cellular regulatory networks that control HIV latency and the role of drugs of abuse in this process.
HIV-1 persists in spite of antiretroviral therapy due to a reservoir of long-lived resting memory CD4+ T-cells. This process, called viral latency, is a major barrier to eradicating HIV-1 infection. While significant progress has been made in our understanding of the molecular mechanisms of HIV latency, our understanding is incomplete. In particular, the relevance of drugs of abuse on this process has not been studied in details. Given the fact that a significant fraction of HIV-infected patients are also drug abusers, there isa need to better understand the role of drugs of abuse on the establishment, the maintenance and reactivation of latent HIV. Here, we propose to explore on a genomic scale the landscape of cellular factors that regulate HIV latency establishment and maintenance and their relationship to drugs of abuse. We will use genome scale screens to identify cellular genes that control latency maintenance and reactivation and their relationship to drugs of abuse with a particular focus on methamphetamine. We also propose to further develop new HIV-1 reporter viruses to study latency in vitro. Using these new tools, we can study the role of drugs of abuse in the establishment, maintenance and reactivation of latent HIV. By combining the power of our new latency model with high-resolution systems-biology techniques, we are uniquely suited to map out the cellular regulatory networks that control HIV latency and the role of drugs of abuse in this process.
| Battivelli, Emilie; Dahabieh, Matthew S; Abdel-Mohsen, Mohamed et al. (2018) Distinct chromatin functional states correlate with HIV latency reactivation in infected primary CD4+ T cells. Elife 7: |
| Besnard, Emilie; Hakre, Shweta; Kampmann, Martin et al. (2016) The mTOR Complex Controls HIV Latency. Cell Host Microbe 20:785-797 |
