The Role of Gene-Expression Noise in Regulating HIV-1 Proviral Latency
Leor Weinberger, University of California San Diego
Basic Biomedical Sciences
Innovative, Developmental, Exploratory Award (IDEA)
HIV-1 post-integration latency remains the most problematic obstacle facing viral eradication from a patient (Han et al. 2007). Despite extensive knowledge of the molecular components involved in HIV-1 transcriptional regulation, there is a fundamental gap in our understanding of the kinetics of how these molecules interact to form a regulatory “switch” that can establish, maintain, and break HIV-1 post-integration latency in single cells. Specifically, it is now clear that at the single cell level, "noise" (random fluctuations in biochemical molecules) is inescapable and can critically influence developmental decisions mediated by auto-regulatory circuits; how biochemical noise influences entry into and exit from proviral latency is not understood.
The long-term goal of this project is to understand the fundamental molecular mechanisms governing HIV-1 latency in vivo and to identify therapeutic strategies to manipulate HIV-1’s regulatory circuit and therapeutically alter HIV-1’s ability to enter, or reactivate from, HIV-1 proviral latency in the patient. The specific goal of this CHRP IDEA proposal is a pilot study to understand the molecular mechanisms regulating HIV-1 latency in primary CD4+ T cells.
Based on preliminary data, the hypothesis is that HIV-1 utilizes Tat transcriptional positive feedback to amplify stochastic intracellular noise (random fluctuations in biochemical molecules that are inescapable at the single-cell level) and establish a probabilistic molecular “switch” between proviral-latency and active-infection. We have established the feasibility of this hypothesis using minimal Tat-circuit lentiviral constructs in Jurkat cells and showing that (i) gene expression noise in a minimal a HIV Tat circuit can influence a “switch” between active-replication and proviral-latency (Weinberger et al. Cell 2005), (ii) that HIV encodes a novel feedback-resistor motif to regulate this Tat noise (Weinberger et al., PLoS Biology 2007), and (iii) that altering host SirT1 expression modulates Tat noise to significantly influence a single infected cell’s entry into latency (Weinberger et al. Nature Genetics, 2008).
Preliminary evidence suggests that this hypothesis of noise-mediated fate-determination may hold true in full-length HIV-1 in primary CD4+ T-cells.
To test this central hypothesis and determine if Tat transcriptional noise is driving a molecular switch in primary CD4+ T cells, we will directly image gene-expression noise in real-time in single cells during infection and reactivation using fluorescent proteins encoded within recombinant HIV-1 viruses. We will employ an integrated wet-lab single-cell imaging and dry-lab computational approach to accomplish the following two specific aims: (1) Determine, in primary CD4+ T cells, if stochastic noise in minimal Tat circuits is sufficient to reproduce a latency “decision” (2) Determine, in primary CD4+ T cells, if stochastic noise in full-length HIV-1 infection is necessary to generate latency.
This project’s expected outcome is the identification and characterization of potential gene-therapy candidates that can interfere with transcriptional regulation in full-length HIV-1 and bias the molecular decision for HIV-1 post-integration latency. This project is innovative because it will capitalize on recent advances in single-cell microscopy, genetically encoded fluorescent probes, and automated image-analyses to quantify HIV feedback kinetics in live single cells.