Control of stochastic gene expression by host factors at the HIV promoter.
ABSTRACT: The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-kappaB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-kappaB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-kappaB sites exhibit distinct properties, with kappaB site I serving a stronger activating role than kappaB site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-kappaB site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally "dampen" transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency.
Project description:Cells latently infected with HIV represent a currently insurmountable barrier to viral eradication in infected patients. Using the J-Lat human T-cell model of HIV latency, we have investigated the role of host factor binding to the kappaB enhancer elements of the HIV long terminal repeat (LTR) in the maintenance of viral latency. We show that NF-kappaB p50-HDAC1 complexes constitutively bind the latent HIV LTR and induce histone deacetylation and repressive changes in chromatin structure of the HIV LTR, changes that impair recruitment of RNA polymerase II and transcriptional initiation. Knockdown of p50 expression with specific small hairpin RNAs reduces HDAC1 binding to the latent HIV LTR and induces RNA polymerase II recruitment. Similarly, inhibition of histone deacetylase (HDAC) activity with trichostatin A promotes binding of RNA polymerase II to the latent HIV LTR. This bound polymerase complex, however, remains non-processive, generating only short viral transcripts. Synthesis of full-length viral transcripts can be rescued under these conditions by expression of Tat. The combination of HDAC inhibitors and Tat merits consideration as a new strategy for purging latent HIV proviruses from their cellular reservoirs.
Project description:The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1-72aa) is sufficient for viral transcript elongation and second exon (73-101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-kappaB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat-Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells.
Project description:Human Immune Deficiency Virus-1 (HIV-1) infection can induce severe and debilitating neurological problems, including behavioral abnormalities, motor dysfunction, and dementia. HIV can persistently infect astrocytes, during which viral accessory proteins are produced that are unaffected by current antiretroviral therapy. The effect of these proteins on astrocyte function remains unknown. Astrocytes are the predominant cells within the brain; thus, disruption of astrocyte function could influence the neuropathogenesis of HIV infection. To explore further these effects, we constitutively expressed HIV-Tat protein in astrocytes. Since the nuclear presence of Tat protein leads to alteration of host gene expression, we further analyzed the effects of Tat on host gene transcripts. Endothelin-1 (ET-1) was a significantly elevated transcript as verified by reverse transcription-polymerase chain reaction (RT-PCR), and it was subsequently released extracellularly in Tat-expressing and HIV-infected astrocytes. ET-1 expression was also prominent in reactive astrocytes and neurons in brain tissues from basal ganglia and frontal lobes of HIV encephalitic patients. HIV-Tat regulated ET-1 at the transcriptional level through NF-kappaB (NF-kappaB)-responsive sites in the ET-1 promoter. Intriguingly, simvastatin (10 microM) down-regulated HIV-Tat-induced ET-1 and also inhibited activation of NF-kappaB in astrocytes. Our findings suggest that ET-1 may be critical in mediating the neuropathogenesis of HIV dementia and that statins may have therapeutic potential in these patients.
Project description:The sequence of a promoter within a genome does not uniquely determine gene expression levels and their variability; rather, promoter sequence can additionally interact with its location in the genome, or genomic context, to shape eukaryotic gene expression. Retroviruses, such as human immunodeficiency virus-1 (HIV), integrate their genomes into those of their host and thereby provide a biomedically-relevant model system to quantitatively explore the relationship between promoter sequence, genomic context, and noise-driven variability on viral gene expression. Using an in vitro model of the HIV Tat-mediated positive-feedback loop, we previously demonstrated that fluctuations in viral Tat-transactivating protein levels generate integration-site-dependent, stochastically-driven phenotypes, in which infected cells randomly 'switch' between high and low expressing states in a manner that may be related to viral latency. Here we extended this model and designed a forward genetic screen to systematically identify genetic elements in the HIV LTR promoter that modulate the fraction of genomic integrations that specify 'Switching' phenotypes. Our screen identified mutations in core promoter regions, including Sp1 and TATA transcription factor binding sites, which increased the Switching fraction several fold. By integrating single-cell experiments with computational modeling, we further investigated the mechanism of Switching-fraction enhancement for a selected Sp1 mutation. Our experimental observations demonstrated that the Sp1 mutation both impaired Tat-transactivated expression and also altered basal expression in the absence of Tat. Computational analysis demonstrated that the observed change in basal expression could contribute significantly to the observed increase in viral integrations that specify a Switching phenotype, provided that the selected mutation affected Tat-mediated noise amplification differentially across genomic contexts. Our study thus demonstrates a methodology to identify and characterize promoter elements that affect the distribution of stochastic phenotypes over genomic contexts, and advances our understanding of how promoter mutations may control the frequency of latent HIV infection.
Project description:Multiple human immunodeficiency virus type 1 (HIV-1) sequences with deletions of NF-kappaB binding sites at both the 5' and 3' long terminal repeats (LTRs) were identified in serial samples collected from an infected individual. The effect of this deletion on the level of transcription was studied by transient transfection of an LTR-driven luciferase reporter gene and by infection with a full-length recombinant HIV-1 containing a luciferase reporter (HIVHXBluc). Detectable levels of gene expression were found in both systems, in the presence or absence of the viral transactivator Tat. Interestingly, a duplication of a putative TCF-1alpha motif was found in place of the NF-kappaB elements in these viruses. Higher transcriptional activity was observed with HXBLTR (NF-kappaB intact) than with the patient's LTR (NF-kappaB deleted), suggesting that the NF-kappaB binding sites may promote optimal levels of viral gene transcription. The ability of these viruses with NF-kappaB deleted to replicate and cause substantial decline in CD4 cell counts demonstrates that the NF-kappaB binding sites are not absolutely required for viral replication or pathogenicity in vivo. These results are consistent with the notion that the HIV-1 LTR possesses functional redundancy which allows it to interact with multiple transcription factors, thereby ensuring viral replication in a variety of cell types.
Project description:Current antiretroviral therapy (ART) efficiently controls HIV-1 replication but fails to eradicate the virus. Even after years of successful ART, HIV-1 can conceal itself in a latent state in long-lived CD4(+) memory T cells. From this latent reservoir, HIV-1 rebounds during treatment interruptions. Attempts to therapeutically eradicate this viral reservoir have yielded disappointing results. A major problem with previously utilized activating agents is that at the concentrations required for efficient HIV-1 reactivation, these stimuli trigger high-level cytokine gene expression (hypercytokinemia). Therapeutically relevant HIV-1-reactivating agents will have to trigger HIV-1 reactivation without the induction of cytokine expression. We present here a proof-of-principle study showing that this is a possibility. In a high-throughput screening effort, we identified an HIV-1-reactivating protein factor (HRF) secreted by the nonpathogenic bacterium Massilia timonae. In primary T cells and T-cell lines, HRF triggered a high but nonsustained peak of nuclear factor kappa B (NF-kappaB) activity. While this short NF-kappaB peak potently reactivated latent HIV-1 infection, it failed to induce gene expression of several proinflammatory NF-kappaB-dependent cellular genes, such as those for tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8), and gamma interferon (IFN-gamma). Dissociation of cellular and viral gene induction was achievable, as minimum amounts of Tat protein, synthesized following application of a short NF-kappaB pulse, triggered HIV-1 transactivation and subsequent self-perpetuated HIV-1 expression. In the absence of such a positive feedback mechanism, cellular gene expression was not sustained, suggesting that strategies modulating the NF-kappaB activity profile could be used to selectively trigger HIV-1 reactivation.
Project description:Symptoms of T cell hyperactivation shape the course and outcome of HIV-1 infection, but the mechanism(s) underlying this chronic immune activation are not well understood. We find that the viral transactivator Tat promotes hyperactivation of T cells by blocking the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase SIRT1. Tat directly interacts with the deacetylase domain of SIRT1 and blocks the ability of SIRT1 to deacetylate lysine 310 in the p65 subunit of NF-kappaB. Because acetylated p65 is more active as a transcription factor, Tat hyperactivates the expression of NF-kappaB-responsive genes, a function lost in SIRT1-/- cells. These results support a model where the normal function of SIRT1 as a negative regulator of T cell activation is suppressed by Tat during HIV infection. These events likely contribute to the state of immune cell hyperactivation found in HIV-infected individuals.
Project description:The magnitude of transcription factor binding site variation emerging in HIV-1 subtype C (HIV-1C), especially the addition of NF-?B motifs by sequence duplication, makes the examination of transcriptional silence challenging. How can HIV-1 establish and maintain latency despite having a strong long terminal repeat (LTR)? We constructed panels of subgenomic reporter viral vectors with varying copy numbers of NF-?B motifs (0 to 4 copies) and examined the profile of latency establishment in Jurkat cells. Surprisingly, we found that the stronger the viral promoter, the faster the latency establishment. Importantly, at the time of commitment to latency and subsequent points, Tat levels in the cell were not limiting. Using highly sensitive strategies, we demonstrate the presence of Tat in the latent cell, recruited to the latent LTR. Our data allude, for the first time, to Tat establishing a negative feedback loop during the late phases of viral infection, leading to the rapid silencing of the viral promoter.<b>IMPORTANCE</b> Over the past 10 to 15?years, HIV-1 subtype C (HIV-1C) has been evolving rapidly toward gaining stronger transcriptional activity by sequence duplication of major transcription factor binding sites. The duplication of NF-?B motifs is unique and exclusive to HIV-1C, a property not shared with any of the other eight HIV-1 genetic families. What mechanism(s) does HIV-1C employ to establish and maintain transcriptional silence despite the presence of a strong promoter and concomitant strong, positive transcriptional feedback is the primary question that we attempted to address in the present manuscript. The role that Tat plays in latency reversal is well established. Our work with the most common HIV-1 subtype, HIV-1C, offers crucial leads toward Tat possessing a dual role in serving as both a transcriptional activator and repressor at different phases of viral infection of the cell. The leads that we offer through the present work have significant implications for HIV-1 cure research.
Project description:The activation and latency of human immunodeficiency virus type 1 (HIV-1) are tightly controlled by the transcriptional activity of its long terminal repeat (LTR) region. The LTR is regulated by viral proteins as well as host factors, including the nuclear factor kappaB (NF-kappaB) that becomes activated in virus-infected cells. The two tandem NF-kappaB sites of the LTR are among the most highly conserved sequence elements of the HIV-1 genome. Puzzlingly, these sites are arranged in a manner that seems to preclude simultaneous binding of both sites by NF-kappaB, although previous biochemical work suggests otherwise. Here, we have determined the crystal structure of p50:RelA bound to the tandem kappaB element of the HIV-1 LTR as a dimeric dimer, providing direct structural evidence that NF-kappaB can occupy both sites simultaneously. The two p50:RelA dimers bind the adjacent kappaB sites and interact through a protein contact that is accommodated by DNA bending. The two dimers clamp DNA from opposite faces of the double helix and form a topological trap of the bound DNA. Consistent with these structural features, our biochemical analyses indicate that p50:RelA binds the HIV-1 LTR tandem kappaB sites with an apparent anti-cooperativity but enhanced kinetic stability. The slow on and off rates we observe may be relevant to viral latency because viral activation requires sustained NF-kappaB activation. Furthermore, our work demonstrates that the specific arrangement of the two kappaB sites on the HIV-1 LTR can modulate the assembly kinetics of the higher-order NF-kappaB complex on the viral promoter. This phenomenon is unlikely restricted to the HIV-1 LTR but probably represents a general mechanism for the function of composite DNA elements in transcription.
Project description:Human immunodeficiency virus type 1 (HIV-1) Tat binds the viral RNA structure transactivation-responsive element (TAR) and recruits transcriptional cofactors, amplifying viral mRNA expression. The Tat inhibitor didehydro-cortistatin A (dCA) promotes a state of persistent latency, refractory to viral reactivation. Here we investigated mechanisms of HIV-1 resistance to dCA in vitro Mutations in Tat and TAR were not identified, consistent with the high level of conservation of these elements. Instead, viruses resistant to dCA developed higher Tat-independent basal transcription. We identified a combination of mutations in the HIV-1 promoter that increased basal transcriptional activity and modifications in viral Nef and Vpr proteins that increased NF-?B activity. Importantly, these variants are unlikely to enter latency due to accrued transcriptional fitness and loss of sensitivity to Tat feedback loop regulation. Furthermore, cells infected with these variants become more susceptible to cytopathic effects and immune-mediated clearance. This is the first report of viral escape to a Tat inhibitor resulting in heightened Tat-independent activity, all while maintaining wild-type Tat and TAR.IMPORTANCE HIV-1 Tat enhances viral RNA transcription by binding to TAR and recruiting activating factors. Tat enhances its own transcription via a positive-feedback loop. Didehydro-cortistatin A (dCA) is a potent Tat inhibitor, reducing HIV-1 transcription and preventing viral rebound. dCA activity demonstrates the potential of the "block-and-lock" functional cure approaches. We investigated the viral genetic barrier to dCA resistance in vitro While mutations in Tat and TAR were not identified, mutations in the promoter and in the Nef and Vpr proteins promoted high Tat-independent activity. Promoter mutations increased the basal transcription, while Nef and Vpr mutations increased NF-?B nuclear translocation. This heightened transcriptional activity renders CD4+ T cells infected with these viruses more susceptible to cytotoxic T cell-mediated killing and to cell death by cytopathic effects. Results provide insights on drug resistance to a novel class of antiretrovirals and reveal novel aspects of viral transcriptional regulation.