Leor S. Weinberger

Stochastic Gene Expression in a Lentiviral Positive-Feedback Loop: HIV-1 Tat Fluctuations Drive Phenotypic Diversity

HIV-1 Tat transactivation is vital for completion of the viral life cycle and has been implicated in determining proviral latency. We present an extensive experimental/computational study of an HIV-1 model vector (LTR-GFP-IRES-Tat) and show that stochastic fluctuations in Tat influence the viral latency decision. Low GFP/Tat expression was found to generate bifurcating phenotypes with clonal populations derived from single proviral integrations simultaneously exhibiting very high and near zero GFP expression. Although phenotypic bifurcation (PheB) was correlated with distinct genomic integration patterns, neither these patterns nor other extrinsic cellular factors (cell cycle/size, aneuploidy, chromatin silencing, etc.) explained PheB. Stochastic computational modeling successfully accounted for PheB and correctly predicted the dynamics of a Tat mutant that were subsequently confirmed by experiment. Thus, Tat stochastics appear sufficient to generate PheB (and potentially proviral latency), illustrating the importance of stochastic fluctuations in gene expression in a mammalian system.

Transcriptional burst frequency and burst size are equally modulated across the human genome

Gene expression occurs either as an episodic process, characterized by pulsatile bursts, or as a constitutive process, characterized by a Poisson-like accumulation of gene products. It is not clear which mode of gene expression (constitutive versus bursty) predominates across a genome or how transcriptional dynamics are influenced by genomic position and promoter sequence. Here, we use time-lapse fluorescence microscopy to analyze 8,000 individual human genomic loci and find that at virtually all loci, episodic bursting—as opposed to constitutive expression—is the predominant mode of expression. Quantitative analysis of the expression dynamics at these 8,000 loci indicates that both the frequency and size of the transcriptional bursts varies equally across the human genome, independent of promoter sequence. Strikingly, weaker expression loci modulate burst frequency to increase activity, whereas stronger expression loci modulate burst size to increase activity. Transcriptional activators such as trichostatin A (TSA) and tumor necrosis factor α (TNF) only modulate burst size and frequency along a constrained trend line governed by the promoter. In summary, transcriptional bursting dominates across the human genome, both burst frequency and burst size vary by chromosomal location, and transcriptional activators alter burst frequency and burst size, depending on the expression level of the locus.

BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism

The therapeutic potential of pharmacologic inhibition of bromodomain and extraterminal (BET) proteins has recently emerged in hematological malignancies and chronic inflammation. We find that BET inhibitor compounds (JQ1, I-Bet, I-Bet151 and MS417) reactivate HIV from latency. This is evident in polyclonal Jurkat cell populations containing latent infectious HIV, as well as in a primary T-cell model of HIV latency. Importantly, we show that this activation is dependent on the positive transcription elongation factor p-TEFb but independent from the viral Tat protein, arguing against the possibility that removal of the BET protein BRD4, which functions as a cellular competitor for Tat, serves as a primary mechanism for BET inhibitor action. Instead, we find that the related BET protein, BRD2, enforces HIV latency in the absence of Tat, pointing to a new target for BET inhibitor treatment in HIV infection. In shRNA-mediated knockdown experiments, knockdown of BRD2 activates HIV transcription to the same extent as JQ1 treatment, while a lesser effect is observed with BRD4. In single-cell time-lapse fluorescence microscopy, quantitative analyses across ~2,000 viral integration sites confirm the Tat-independent effect of JQ1 and point to positive effects of JQ1 on transcription elongation, while delaying re-initiation of the polymerase complex at the viral promoter. Collectively, our results identify BRD2 as a new Tat-independent suppressor of HIV transcription in latently infected cells and underscore the therapeutic potential of BET inhibitors in the reversal of HIV latency.

Transcriptional Bursting from the HIV-1 Promoter is a Significant Source of Stochastic Noise in HIV-1 Gene Expression

Analysis of noise in gene expression has proven a powerful approach for analyzing gene regulatory architecture. To probe the regulatory mechanisms controlling expression of HIV-1, we analyze noise in gene-expression from HIV-1s long terminal repeat (LTR) promoter at different HIV-1 integration sites across the human genome. Flow cytometry analysis of GFP expression from the HIV-1 LTR shows high variability (noise) at each integration site. Notably, the measured noise levels are inconsistent with constitutive gene expression models. Instead, quantification of expression noise indicates that HIV-1 gene expression occurs through randomly timed bursts of activity from the LTR and that each burst generates an average of 2-10 mRNA transcripts before the promoter returns to an inactive state. These data indicate that transcriptional bursting can generate high variability in HIV-1 early gene products, which may critically influence the viral fate-decision between active replication and proviral latency.

Screening for noise in gene expression identifies drug synergies

Noisy genes flush HIV out of hiding HIV can hide in the body, making it hard to kill with drugs. Increasing variation or “noise” in the viruss gene expression turns out to be an effective strategy for reactivating latent HIV. Once reawakened, the virus is more sensitive to antiviral drugs. Dar et al. screened for agents that increased variation in the expression of HIV genes. In a model system with HIV-infected human cells, the noise enhancers worked with existing compounds used to reactivate latent HIV and helped eradicate the virus. Science, this issue p. 1392 Turning up the noise in HIV gene expression can help HIV drugs be more effective. Stochastic fluctuations are inherent to gene expression and can drive cell-fate specification. We used such fluctuations to modulate reactivation of HIV from latency—a quiescent state that is a major barrier to an HIV cure. By screening a diverse library of bioactive small molecules, we identified more than 80 compounds that modulated HIV gene–expression fluctuations (i.e., “noise”), without changing mean expression. These noise-modulating compounds would be neglected in conventional screens, and yet, they synergized with conventional transcriptional activators. Noise enhancers reactivated latent cells significantly better than existing best-in-class reactivation drug combinations (and with reduced off-target cytotoxicity), whereas noise suppressors stabilized latency. Noise-modulating chemicals may provide novel probes for the physiological consequences of noise and an unexplored axis for drug discovery, allowing enhanced control over diverse cell-fate decisions.

A Hardwired HIV Latency Program

Biological circuits can be controlled by two general schemes: environmental sensing or autonomous programs. For viruses such as HIV, the prevailing hypothesis is that latent infection is controlled by cellular state (i.e., environment), with latency simply an epiphenomenon of infected cells transitioning from an activated to resting state. However, we find that HIV expression persists despite the activated-to-resting cellular transition. Mathematical modeling indicates that HIVs Tat positive-feedback circuitry enables this persistence and strongly controls latency. To overcome the inherent crosstalk between viral circuitry and cellular activation and to directly test this hypothesis, we synthetically decouple viral dependence on cellular environment from viral transcription. These circuits enable control of viral transcription without cellular activation and show that Tat feedback is sufficient to regulate latency independent of cellular activation. Overall, synthetic reconstruction demonstrates that a largely autonomous, viral-encoded program underlies HIV latency—potentially explaining why cell-targeted latency-reversing agents exhibit incomplete penetrance.

Dynamics of protein noise can distinguish between alternate sources of gene-expression variability.

Within individual cells, two molecular processes have been implicated as sources of noise in gene expression: (i) Poisson fluctuations in mRNA abundance arising from random birth and death of individual mRNA transcripts or (ii) promoter fluctuations arising from stochastic promoter transitions between different transcriptional states. Steady‐state measurements of variance in protein levels are insufficient to discriminate between these two mechanisms, and mRNA single‐molecule fluorescence in situ hybridization (smFISH) is challenging when cellular mRNA concentrations are high. Here, we present a perturbation method that discriminates mRNA birth/death fluctuations from promoter fluctuations by measuring transient changes in protein variance and that can operate in the regime of high molecular numbers. Conceptually, the method exploits the fact that transcriptional blockage results in more rapid increases in protein variability when mRNA birth/death fluctuations dominate over promoter fluctuations. We experimentally demonstrate the utility of this perturbation approach in the HIV‐1 model system. Our results support promoter fluctuations as the primary noise source in HIV‐1 expression. This study illustrates a relatively simple method that complements mRNA smFISH hybridization and can be used with existing GFP‐tagged libraries to include or exclude alternate sources of noise in gene expression.

Theoretical Design of a Gene Therapy To Prevent AIDS but Not Human Immunodeficiency Virus Type 1 Infection

ABSTRACT Recent reports confirm that, due to the presence of long-lived, latently infected cell populations, eradication of human immunodeficiency virus type 1 (HIV-1) from infected patients by using antiretroviral drugs will be exceedingly difficult. An alternative to virus eradication may be to use gene therapy to induce a pseudo-latent state in virus-producing cells, thus transforming HIV-1 into a lifelong, but manageable, virus. Conditionally replicating HIV-1 (crHIV-1) gene therapy vectors provide an avenue for subduing HIV-1 expression in infected cells (by creating a parasite, crHIV-1, of the parasite HIV-1), potentially reducing the HIV-1 set point and delaying AIDS onset. Development of crHIV-1 vectors has proceeded in vitro, but the requirements for a crHIV-1 vector to proliferate and persist in vivo have not been explored. We expand a widely accepted mathematical model of HIV-1 in vivo dynamics to include a crHIV-1 gene therapy virus and derive a simple criterion for designing crHIV-1 viruses that will persist in vivo. The model introduces only two new parameters—HIV-1 inhibition and crHIV-1 production—and both can be experimentally engineered and controlled. Analysis demonstrates that crHIV-1 gene therapy can indefinitely reduce HIV-1 set point to levels comparable to those achieved with highly active antiretroviral therapy, provided crHIV-1 production is more efficient than HIV-1. Paradoxically, highly efficient therapeutic inhibition of HIV-1 was found to be disadvantageous. Thus, the field may benefit by shifting the search for more potent antiviral genes toward engineering optimized therapy viruses that package ultraefficiently while downregulating viral production moderately.

Early hepatitis C viral kinetics correlate with long-term outcome in patients receiving high dose induction followed by combination interferon and ribavirin therapy

BACKGROUND/AIMS The majority of patients with genotype 1 do not respond to interferon (IFN) plus ribavirin. Limited data exist on the use of induction followed by combination therapy. METHODS In this prospective study of 28 patients infected with genotype 1, randomization involved either daily or twice daily high dose IFN for 6 weeks, followed by standard therapy of 3 million units three times a week in combination with ribavirin for an additional 42 weeks. Hepatitis C virus (HCV) RNA was quantitated before and frequently during treatment. RESULTS The best correlate of response was delta (the infected cell loss rate). Sixteen patients continued on the study because they had at least a 2 log drop in their HCV RNA levels by week 12; all but one were PCR negative for HCV RNA at 48 weeks, and 14 of these 16 patients continued to be PCR negative at 72 weeks. Both African-Americans in our trial failed to respond to therapy, and differences were evident during the induction phase. CONCLUSIONS This randomized study of induction IFN therapy followed by combination IFN plus ribavirin yielded the highest rate of sustained response (50%) reported to date in chronically HCV-infected patients with genotype 1. The predictive value of the infected cell loss rate needs to be evaluated prospectively in larger studies, particularly in patients receiving pegylated IFN.

An Endogenous Accelerator for Viral Gene Expression Confers a Fitness Advantage

Many signaling circuits face a fundamental tradeoff between accelerating their response speed while maintaining final levels below a cytotoxic threshold. Here, we describe a transcriptional circuitry that dynamically converts signaling inputs into faster rates without amplifying final equilibrium levels. Using time-lapse microscopy, we find that transcriptional activators accelerate human cytomegalovirus (CMV) gene expression in single cells without amplifying steady-state expression levels, and this acceleration generates a significant replication advantage. We map the accelerator to a highly self-cooperative transcriptional negative-feedback loop (Hill coefficient ∼7) generated by homomultimerization of the viruss essential transactivator protein IE2 at nuclear PML bodies. Eliminating the IE2-accelerator circuit reduces transcriptional strength through mislocalization of incoming viral genomes away from PML bodies and carries a heavy fitness cost. In general, accelerators may provide a mechanism for signal-transduction circuits to respond quickly to external signals without increasing steady-state levels of potentially cytotoxic molecules.