HIV-1 cannot be cured by antiretroviral therapies alone. The major obstacle to curing HIV-1 is the ability of the virus to persist in a latent but activatable form within cellular reservoirs. The best-characterized cellular reservoir of HIV-1 is the resting memory CD4+ T-cell compartment of peripheral organ systems. However, the brain becomes colonized with HIV-1 very early during infection, and resident cell types within the brain including astrocytes and macrophage-lineage cells (perivascular macrophages and microglia) are frequently infected and can harbor latent forms of the virus. Understanding the mechanisms of HIV-1 latency in astrocyte and macrophage reservoirs of the brain is therefore critical for HIV-1 cure strategies. Our broad hypothesis is that latent HIV-1 long terminal repeat sequences (LTRs) persisting in astrocytes and a non-productively infected subset of macrophages in the brain are distinct to those detected in productively infected brain macrophages and peripheral blood mononuclear cells (PBMC) of the same subjects, and are defined by alterations associated with the Sp1 transcription factor binding motif. Furthermore, Sp1 binding alterations in these latent LTRs result in rearrangements in chromatin complexes and reduced and/or altered ability of the LTR to be activated by the HIV-1 Tat protein and other transcriptional activators. These hypotheses will be tested in a novel and clinically relevant setting by ex vivo assays using LTRs derived from populations of cells purified from brain tissues of cART-treated patients, and from matched PBMC. First, we will utilize cutting edge lase capture microdissection technology and highly sensitive PCR and cloning protocols to generate a large bank of LTR sequences obtained from purified populations of astrocytes, and of macrophages that are either productively infected or non-productively infected. LTRs will also be generated from matched patient PBMC for comparison. These LTRs will be sequenced and characterized functionally for their transcriptional activity. From our preliminary data, we expect that the LTRs from astrocytes and non- productively infected macrophages will have comparatively reduced transcriptional activity characterized by sequence alterations in the Sp-1 binding motif. Second, we will elucidate the mechanisms of altered transcriptional activity using electrophoretic mobility shift assays to characterize Sp-1 protein binding, and by chromatin immunoprecipitation assays to determine the contributing chromatin remodeling factors and transcriptional regulators, using relevant cellular models. Finally, we will test a panel of HDAC inhibitors for their ability to activate latent LTRs derived from both astrocytes and non-productively infected macrophages in relevant cellular models. These experiments will identify the compounds that have either cell specific or broad activity that may inform future strategic trials aimed at eradicating HIV-1 from latent reservoirs of the central nervous system.
Curing HIV-1 infection would mean infected subjects would no longer have to take anti-HIV medication and therefore, would have a highly significant impact on public health. A significant hurdle to curing HIV-1 is that the virus cannot presently be flushe out of certain long-lived cells in the body, which include astrocytes and macrophages in the brain. This study will determine why HIV-1 can persist in these brain cell types, and how it might be purged. Therefore, the proposed study will make a significant contribution to the present global effort that aims to cure HIV-1 infection.
|Churchill, Melissa J; Cowley, Daniel J; Wesselingh, Steve L et al. (2015) HIV-1 transcriptional regulation in the central nervous system and implications for HIV cure research. J Neurovirol 21:290-300|
|Gray, Lachlan R; Roche, Michael; Flynn, Jacqueline K et al. (2014) Is the central nervous system a reservoir of HIV-1? Curr Opin HIV AIDS 9:552-8|