Genome-wide shRNA screen to identify cellular regulators of the maintenance of HIV-1 latency
ABSTRACT: Latent HIV-1 infection represents a barrier to virus eradication as latent HIV-1 is impervious to the effects of antiretroviral drugs and can avoid detection by the host immune system. Strategies to clear latent HIV-1 infection in patients have so far failed in clinical trials to increase the decay rate of the latent reservoir underscoring the need for continued study of HIV-1 latency. In this study, a genome-wide RNAi screen was performed to probe cellular factors involved in maintaining HIV-1 latency in HeLa cells latently infected with an HIV-1 reporter virus. Overall design: HeLa cells that were latently infected with an HIV-1 reporter virus (referred to as the experimental sample) and uninfected, parental HeLa cells (referred to as the reference control sample) were transduced with the GeneNet Genome-wide Human 50K Lentiviral shRNA Library (System Biosciences (SBI), Mountain View, CA). The library consists of approximately 200,000 shRNA constructs targeting about 38,000 human transcripts. It includes 1-4 shRNA constructs per gene, each targeting a different sequence within a specific gene. Transduced cells were selected with puromycin and cultured for a total of 17 days, which was experimentally determined to provide ample time for shRNA expression and knockdown of the target gene, as well as viral reactivation and viral protein-mediated cell death in the experimental sample. In other words, a negative selection protocol was used to identify shRNAs that activated latent virus by taking advantage of the cytotoxic effects of the HIV-1 viral protein Vpr. Following the culture period, cells were lysed and total RNA was isolated, reverse transcribed, amplified, and hybridized to an Affymetrix GeneChip Array (HG-U133+ 2.0) for identification. As the functional assay employed in this screen involved viral protein-mediated cell death upon the re-activation of latent HIV-1, signals from the reference control sample microarray that were at least three times greater than the corresponding signals from the experimental sample microarray were scored as “hits” and statistically analyzed further.
INSTRUMENT(S): [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
Project description:Antiretroviral therapy (ART) inhibits HIV-1 replication, but the virus persists in latently infected resting memory CD4(+) T cells susceptible to viral reactivation. The virus-encoded early gene product Tat activates transcription of the viral genome and promotes exponential viral production. Here we show that the Tat inhibitor didehydro-cortistatin A (dCA), unlike other antiretrovirals, reduces residual levels of viral transcription in several models of HIV latency, breaks the Tat-mediated transcriptional feedback loop, and establishes a nearly permanent state of latency, which greatly diminishes the capacity for virus reactivation. Importantly, treatment with dCA induces inactivation of viral transcription even after its removal, suggesting that the HIV promoter is epigenetically repressed. Critically, dCA inhibits viral reactivation upon CD3/CD28 or prostratin stimulation of latently infected CD4(+) T cells from HIV-infected subjects receiving suppressive ART. Our results suggest that inclusion of a Tat inhibitor in current ART regimens may contribute to a functional HIV-1 cure by reducing low-level viremia and preventing viral reactivation from latent reservoirs.Antiretroviral therapy (ART) reduces HIV-1 replication to very low levels, but the virus persists in latently infected memory CD4(+) T cells, representing a long-lasting source of resurgent virus upon ART interruption. Based on the mode of action of didehydro-cortistatin A (dCA), a Tat-dependent transcription inhibitor, our work highlights an alternative approach to current HIV-1 eradication strategies to decrease the latent reservoir. In our model, dCA blocks the Tat feedback loop initiated after low-level basal reactivation, blocking transcriptional elongation and hence viral production from latently infected cells. Therefore, dCA combined with ART would be aimed at delaying or halting ongoing viral replication, reactivation, and replenishment of the latent viral reservoir. Thus, the latent pool of cells in an infected individual would be stabilized, and death of the long-lived infected memory T cells would result in a continuous decay of this pool over time, possibly culminating in the long-awaited sterilizing cure.
Project description:HIV infection requires lifelong antiretroviral therapy because of the persistence of latently infected CD4+ T cells. The induction of virus expression from latently infected cells occurs following T cell receptor (TCR) activation, but not all latently infected cells respond to TCR stimulation. We compared two models of latently infected cells using an enhanced green fluorescent protein (EGFP) reporter virus to infect CCL19-treated resting CD4+ (rCD4+) T cells (preactivation latency) or activated CD4+ T cells that returned to a resting state (postactivation latency). We isolated latently infected cells by sorting for EGFP-negative (EGFP-) cells after infection. These cells were cultured with antivirals and stimulated with anti-CD3/anti-CD28, mitogens, and latency-reversing agents (LRAs) and cocultured with monocytes and anti-CD3. Spontaneous EGFP expression was more frequent in postactivation than in preactivation latency. Stimulation of latently infected cells with monocytes/anti-CD3 resulted in an increase in EGFP expression compared to that for unstimulated controls using the preactivation latency model but led to a reduction in EGFP expression in the postactivation latency model. The reduced EGFP expression was not associated with reductions in the levels of viral DNA or T cell proliferation but depended on direct contact between monocytes and T cells. Monocytes added to the postactivation latency model during the establishment of latency reduced spontaneous virus expression, suggesting that monocyte-T cell interactions at an early time point postinfection can maintain HIV latency. This direct comparison of pre- and postactivation latency suggests that effective strategies needed to reverse latency will depend on how latency is established.IMPORTANCE One strategy being evaluated to eliminate latently infected cells that persist in HIV-infected individuals on antiretroviral therapy (ART) is to activate HIV expression or production with the goal of inducing virus-mediated cytolysis or immune-mediated clearance of infected cells. The gold standard for the activation of latent virus is T cell receptor stimulation with anti-CD3/anti-CD28. However, this stimulus activates only a small proportion of latently infected cells. We show clear differences in the responses of latently infected cells to activating stimuli based on how latent infection is established, an observation that may potentially explain the persistence of noninduced intact proviruses in HIV-infected individuals on ART.
Project description:A long-lived reservoir of latently infected T cells prevents antiretroviral therapy from eliminating HIV-1 infection. Furthering our understanding of the dynamics of latency generation and maintenance is therefore vital to improve treatment outcome. Using mathematical models and experiments, we suggest that the death of latently infected cells brought about by pyroptosis, or to a lesser extent by superinfection, might be key mechanisms to account for the size and composition of the latent reservoir. Pyroptosis is a form of cell death that occurs in a resting (and thus latently infected) T cell when a productively infected cell attempts cell-to-cell transmission of virus. Superinfection of latently infected cells by productive virus could similarly remove those cells through active virus replication and resulting cytopathicity. The mathematical models presented can explain a number of previously published clinical observations including latent reservoir size and the relationships to viral load in acute HIV infection, measurements of the latent reservoir in chronic infection, and the replacement of wild-type virus by CTL escape mutants within the latent reservoir. Basic virus dynamics models of latency that do not take into account pyroptosis, superinfection, or other potential complexities cannot account for the data.
Project description:The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency in vivo are also complicated by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (TCM), full-length virus infection (HIVNL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.
Project description:The C-promoter binding factor-1 (CBF-1) is a potent and specific inhibitor of the human immunodeficiency virus (HIV)-1 LTR promoter. Here, we demonstrate that the knockdown of endogenous CBF-1 in latently infected primary CD4+ T cells, using specific small hairpin RNAs (shRNA), resulted in the reactivation of latent HIV proviruses. Chromatin immunoprecipitation (ChIP) assays using latently infected primary T cells and Jurkat T-cell lines demonstrated that CBF-1 induces the establishment and maintenance of HIV latency by recruiting polycomb group (PcG/PRC) corepressor complexes or polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Knockdown of CBF-1 resulted in the dissociation of PRCs corepressor complexes enhancing the recruitment of RNA polymerase II (RNAP II) at HIV LTR. Knockdown of certain components of PRC1 and PRC2 also led to the reactivation of latent proviruses. Similarly, the treatment of latently infected primary CD4+ T cells with the PRC2/EZH2 inhibitor, 3-deazaneplanocin A (DZNep), led to their reactivation.
Project description:Human T lymphotropic virus type I (HTLV-I) infection is largely latent in infected persons. How HTLV-1 establishes latency and reactivates is unclear. Here we show that most HTLV-1-infected HeLa cells become senescent. By contrast, when NF-?B activity is blocked, senescence is averted, and infected cells continue to divide and chronically produce viral proteins. A small population of infected NF-?B-normal HeLa cells expresses low but detectable levels of Tax and Rex, albeit not Gag or Env. In these "latently" infected cells, HTLV-1 LTR trans-activation by Tax persists, but NF-?B trans-activation is attenuated due to inhibition by HBZ, the HTLV-1 antisense protein. Furthermore, Gag-Pol mRNA localizes primarily in the nuclei of these cells. Importantly, HBZ was found to inhibit Rex-mediated export of intron-containing mRNAs. Over-expression of Rex or shRNA-mediated silencing of HBZ led to viral reactivation. Importantly, strong NF-?B inhibition also reactivates HTLV-1. Hence, during HTLV-1 infection, when Tax/Rex expression is robust and dominant over HBZ, productive infection ensues with expression of structural proteins and NF-?B hyper-activation, which induces senescence. When Tax/Rex expression is muted and HBZ is dominant, latent infection is established with expression of regulatory (Tax/Rex/HBZ) but not structural proteins. HBZ maintains viral latency by down-regulating Tax-induced NF-?B activation and senescence, and by inhibiting Rex-mediated expression of viral structural proteins.
Project description:HIV-1 persists in a latent state in resting CD4(+) T lymphocytes of infected adults despite prolonged highly active antiretroviral therapy (HAART). To determine whether a latent reservoir for HIV-1 exists in infected children, we performed a quantitative viral culture assay on highly purified resting CD4(+) T cells from 21 children with perinatally acquired infection. Replication-competent HIV-1 was recovered from all 18 children from whom sufficient cells were obtained. The frequency of latently infected resting CD4(+) T cells directly correlated with plasma virus levels, suggesting that in children with ongoing viral replication, most latently infected cells are in the labile preintegration state of latency. However, in each of 7 children who had suppression of viral replication to undetectable levels for 1-3 years on HAART, latent replication-competent HIV-1 persisted with little decay, owing to a stable reservoir of infected cells in the postintegration stage of latency. Drug-resistance mutations generated by previous nonsuppressive regimens persisted in this compartment despite more than 1 year of fully suppressive HAART, rendering untenable the idea of recycling drugs that were part of failed regimens. Thus the latent reservoir for HIV-1 in resting CD4(+) T cells will be a major obstacle to HIV-1 eradication in children.
Project description:Despite the high success rate, antiretroviral therapy does not cure the disease completely due to presence of latent viral reservoirs. Although several studies have addressed this issue earlier, the role of serum starvation/deprivation in HIV-1 latency has not been studied. So, we investigated the role of serum starvation in regulating HIV-1 latency. The impact of serum starvation on HIV-1 latency was assessed in latently infected monocytes U1 and T-cells J1.1. Serum starvation breaks HIV-1 latency in U1 cells. Under similar conditions, J1.1 cells failed to show reactivation of virus. We investigated the involvement of cell death pathway and autophagy during the serum starvation in viral reactivation. Inhibition of these pathways did not affect viral reactivation. Furthermore, other crucial factors like NF-?B, SP1 and AKT did not play any role in regulating viral latency. Here, we report that serum deprivation up-regulates ERK/JNK pathway. This leads to phosphorylation of c-Jun which plays an important role in viral reactivation. Treatment of cells with U0126, an ERK kinase inhibitor, potently inhibited viral replication. In summary, we show that serum starvation leads to reactivation of HIV-1 in latently infected monocytes through the ERK/JNK pathway.
Project description:Human immunodeficiency virus type 1 (HIV-1) infection is a chronic condition, where viral DNA integrates into the genome. Latently infected cells form a persistent, heterogeneous reservoir that at any time can reactivate the integrated HIV-1. Here we confirmed that latently infected cells from HIV-1 positive study participants exhibited active HIV-1 transcription but without production of mature spliced mRNAs. To elucidate the mechanisms behind this we employed primary HIV-1 latency models to study latency establishment and maintenance. We characterized proviral transcription and chromatin development in cultures of resting primary CD4+ T-cells for four months after ex vivo HIV-1 infection. As heterochromatin (marked with H3K9me3 or H3K27me3) gradually stabilized, the provirus became less accessible with reduced activation potential. In a subset of infected cells, active marks (e.g. H3K27ac) and elongating RNAPII remained detectable at the latent provirus, despite prolonged proviral silencing. In many aspects, latent HIV-1 resembled an active enhancer in a subset of resting cells. The enhancer chromatin actively promoted latency and the enhancer-specific CBP/P300-inhibitor GNE049 was identified as a new latency reversal agent. The division of the latent reservoir according to distinct chromatin compositions with different reactivation potential enforces the notion that even though a relatively large set of cells contains the HIV-1 provirus, only a discrete subset is readily able to reactivate the provirus and spread the infection.
Project description:Despite significant advances in the treatment of human immunodeficiency virus type-1 (HIV) infection, antiretroviral therapy only suppresses viral replication but is unable to eliminate infection. Thus, discontinuation of antiretrovirals results in viral reactivation and disease progression. A major reservoir of HIV latent infection resides in resting central memory CD4+ T cells (TCM) that escape clearance by current therapeutic regimens and will require novel strategies for elimination. Here, we evaluated the therapeutic potential of autophagy-inducing peptides, Tat-Beclin 1 and Tat-vFLIP-α2, which can induce a novel Na+/K+-ATPase dependent form of cell death (autosis), to kill latently HIV-infected TCM while preventing virologic rebound. In this study, we encapsulated autophagy inducing peptides into biodegradable lipid-coated hybrid PLGA (poly lactic-co-glycolic acid) nanoparticles for controlled intracellular delivery. A single dose of nanopeptides was found to eliminate latent HIV infection in an in vitro primary model of HIV latency and ex vivo using resting CD4+ T cells obtained from peripheral blood mononuclear cells of HIV-infected patients on antiretroviral with fully suppressed virus for greater than 12 months. Notably, increased LC3B lipidation, SQSTM1/p62 degradation and Na+/K+-ATPase activity characteristic of autosis, were detected in nanopeptide treated latently HIV-infected cells compared to untreated uninfected or infected cells. Nanopeptide-induced cell death could be reversed by knockdown of autophagy proteins, ATG5 and ATG7, and inhibition or knockdown of Na+/K+-ATPase. Importantly, viral rebound was not detected following the induction of the Na+/K+-ATPase dependent form of cell death induced by the Tat-Beclin 1 and Tat-vFLIP-α2 nanopeptides. These findings provide a novel strategy to eradicate HIV latently infected resting memory CD4+ T cells, the major reservoir of HIV latency, through the induction of Na+/K+-ATPase dependent autophagy, while preventing reactivation of virus and new infection of uninfected bystander cells.