The existence of latent reservoirs of HIV-infected cells constitutes the major impediment towards viral eradication. HIV-1 latent reservoirs are small, but extremely long-lived. Latent infection is associated with undetectable levels of viral gene expression and appears to be non-cytopathic. However, upon reactivation, latent viruses enter an active mode of replication in which they are fully competent for spread and induction of disease. The current thinking in the field is that a combination of hypothetical drugs that will reactivate latent viruss (``anti-latency''drugs), with present-day antiretroviral drugs, will be an effective approach towad viral eradication. We have found that benzotriazole derivatives can reactivate latent HIV-1 in an in vitro primary cell model of latency and in cells isolated from aviremic patients by potentiating ?c cytokine signaling. This reactivation is achieved in an unusual way in that it is independent on cellular activation or proliferation. We therefore propose to explore this novel mechanism of viral reactivation as a novel therapeutic target in the context of reactivation of latent HIV. In he R21 phase, we will study how benzotriazole derivatives mediate reactivation of latent HIV and what are the effects of these compounds on global transcription, both viral and cellular.
In Aim 1, we plan to characterize in detail the mechanism of action of benzotriazole derivatives as well as identify the target for these compounds. Preliminary data suggest that these compounds suppress a negative feed-back loop that normally resets STAT5 to an inactive, baseline state after cytokine signaling.
In Aim 2, we will determine the effects on global transcription as well a HIV-1 transcription using both RNAseq and ChIPseq in primary CD4 T cells. We will evaluate RNApol-II binding to the HIV-1 LTR as well as cellular promoters in the presence of benzotriazole derivatives. We will complement this study with an analysis of STAT5 occupancy across host and HIV genomes. These studies will be complemented with the optimization of benzotriazole derivatives via medicinal chemistry. This will be done in collaboration with GlaxoSmithKline. Finally, we plan to test the ability of benzotriazole derivatives to reactivate latent HIV-1 in cells isolated from aviremic patients in the presence of different ?c-cytokines. These studies will set the stage for testing of these compounds in humanized mice during the R33 phase of the grant. The R33 phase will be undertaken only if well-defined milestones are achieved. The goal of Aim 3 is to mirror a "shock and kill" strategy in humanized mice, with the following primary goals of (a) establishing a latent HIV reservoir and then to measure changes in HIV transcription upon administration of benzotriazole derivatives found in Aim 1;(b) measuring changes in reservoir size that may result from viral reactivation;and (c) evaluating an effector arm consisting of engineered cytotoxic T lymphocytes.
The existence of latent reservoirs of HIV-infected cells constitutes the major impediment towards viral eradication. We have found that benzotriazole derivatives are able to induce viral reactivation from latency in central memory CD4 T cells as well as in cells isolated from aviremic patients. This application seeks to characterize how benzotriazole derivatives induce viral reactivation from latency, with the ultimate goal of using this pathway as a target for viral eradication strategies.
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|Spivak, Adam M; Bosque, Alberto; Balch, Alfred H et al. (2015) Ex Vivo Bioactivity and HIV-1 Latency Reversal by Ingenol Dibenzoate and Panobinostat in Resting CD4(+) T Cells from Aviremic Patients. Antimicrob Agents Chemother 59:5984-91|
|Ramirez, Peter W; DePaula-Silva, Ana Beatriz; Szaniawski, Matt et al. (2015) HIV-1 Vpu utilizes both cullin-RING ligase (CRL) dependent and independent mechanisms to downmodulate host proteins. Retrovirology 12:65|