Long noncoding RNA NRON contributes to HIV-1 latency by specifically inducing tat protein degradation.
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ABSTRACT:
SUBMITTER: Li J
PROVIDER: S-EPMC4909936 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Although combined antiretroviral therapy (cART) successfully decreases plasma viremia to undetectable levels, the complete eradication of human immunodeficiency virus type 1 (HIV-1) remains impractical because of the existence of a viral reservoir, mainly in resting memory CD4(+) T cells. Various cytokines, protein kinase C activators, and histone deacetylase inhibitors (HDACi) have been used as latency-reversing agents (LRAs), but their unacceptable side effects or low efficiencies limit their clinical use. Here, by a mutation accumulation strategy, we generated an attenuated HIV-1 Tat protein named Tat-R5M4, which has significantly reduced cytotoxicity and immunogenicity, yet retaining potent transactivation and membrane-penetration activity. Combined with HDACi, Tat-R5M4 activates highly genetically diverse and replication-competent viruses from resting CD4(+) T lymphocytes isolated from HIV-1-infected individuals receiving suppressive cART. Thus, Tat-R5M4 has promising potential as a safe, efficient, and specific LRA in HIV-1 treatment.
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Project description:Long noncoding RNAs are widely implicated in diverse disease processes. Nonetheless, their regulatory roles in bone resorption are undefined. Here, we identify lncRNA Nron as a critical suppressor of bone resorption. We demonstrate that osteoclastic Nron knockout mice exhibit an osteopenia phenotype with elevated bone resorption activity. Conversely, osteoclastic Nron transgenic mice exhibit lower bone resorption and higher bone mass. Furthermore, the pharmacological overexpression of Nron inhibits bone resorption, while caused apparent side effects in mice. To minimize the side effects, we further identify a functional motif of Nron. The delivery of Nron functional motif to osteoclasts effectively reverses bone loss without obvious side effects. Mechanistically, the functional motif of Nron interacts with E3 ubiquitin ligase CUL4B to regulate ERα stability. These results indicate that Nron is a key bone resorption suppressor, and the lncRNA functional motif could potentially be utilized to treat diseases with less risk of side effects.
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Project description:Replication of HIV-1 requires the viral Tat protein, which increases the extent of transcription elongation by RNA polymerase II after activation at the single viral long terminal repeat (LTR) promoter. This effect of Tat on transcription requires Tat interactions with a 5' region (TAR) in nascent transcripts as well as Tat-specific cofactors. The present study identifies a cellular protein, TIP30, that interacts with Tat and with an SRB-containing RNA polymerase II complex both in vivo and in vitro. Coexpression of TIP30 specifically enhances transactivation by Tat in transfected cells, and immunodepletion of TIP30 from nuclear extracts abolishes Tat-activated transcription without affecting Tat-independent transcription. These results implicate TIP30 as a specific coactivator that may enhance formation of a Tat-RNA polymerase II holoenzyme complex.
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Project description:A major barrier to HIV-1 cure is caused by the pool of latently infected CD4 T-cells that persist under combination antiretroviral therapy (cART). This latent reservoir is capable of producing replication-competent infectious viruses once prolonged suppressive cART is withdrawn. Inducing the reactivation of HIV-1 gene expression in T-cells harboring a latent provirus in people living with HIV-1 under cART may result in depletion of this latent reservoir due to cytopathic effects or immune clearance. Studies have investigated molecules that reactivate HIV-1 gene expression, but to date, no latency reversal agent has been identified to eliminate latently infected cells harboring replication-competent HIV in cART-treated individuals. Stochastic fluctuations in HIV-1 tat gene expression have been described and hypothesized to allow the progression into proviral latency. We hypothesized that exposing latently infected CD4+ T-cells to Tat would result in effective latency reversal. Our results indicate the capacity of a truncated Tat protein and mRNA to reactivate HIV-1 in latently infected T-cells ex vivo to a similar degree as the protein kinase C agonist: phorbol 12-myristate 13-acetate, without T-cell activation or any significant transcriptome perturbation.
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Project description:Different classes of latency reversing agents (LRAs) are being evaluated to measure their effects in reactivating HIV replication from latently infected cells. A limited number of studies have demonstrated additive effects of LRAs with the viral protein Tat in initiating transcription, but less is known about how LRAs interact with Tat, particularly through basic residues that may be post-translationally modified to alter the behaviour of Tat for processive transcription and co-transcriptional RNA processing.Here we show that various lysine and arginine mutations reduce the capacity of Tat to induce both transcription and mRNA splicing. The lysine 28 and lysine 50 residues of Tat, or the acetylation and methylation modifications of these basic amino acids, were essential for Tat transcriptional control, and also for the proviral expression effects elicited by histone deacetylase inhibitors (HDACi) or the bromodomain inhibitor JQ1. We also found that JQ1 was the only LRA tested that could induce HIV mRNA splicing in the absence of Tat, or rescue splicing for Tat lysine mutants in a BRD4-dependent manner.Our data provide evidence that Tat activities in both co-transcriptional RNA processing together with transcriptional initiation and processivity are crucial during reactivation of latent HIV infection. The HDACi and JQ1 LRAs act with Tat to increase transcription, but JQ1 also enables post-transcriptional mRNA splicing. Tat residues K28 and K50, or their modifications through acetylation or methylation, are critical for LRAs that function in conjunction with Tat.
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Project description:Human immunodeficiency virus type 1 (HIV-1) Tat is a virus-encoded trans-activator that plays a central role in viral transcription. We used our recently developed parallel analysis of in vitro translated open reading frames (ORFs) (PLATO) approach to identify host proteins that associate with HIV-1 Tat. From this proteomic assay, we identify 89 Tat-associated proteins (TAPs). We combine our results with other datasets of Tat or long terminal repeat (LTR)-associated proteins. For some of these proteins (NAT10, TINP1, XRCC5, SIN3A), we confirm their strong association with Tat. These TAPs also suppress Tat-mediated HIV-1 transcription. Removing suppression of HIV-1 transcription benefits the reversal of post-integrated, latent HIV-1 proviruses. We demonstrate that these transcriptionally suppressing TAPs contribute to HIV-1 latency in Jurkat latency (J-LAT) cells. Therefore, our proteomic analysis highlights the previously unappreciated TAPs that play a role in maintaining HIV-1 latency and can be further studied as potential pharmacological targets for the "shock and kill" HIV-1 cure strategy.
| S-EPMC5408673 | biostudies-literature
Project description:The reservoir of HIV latently infected cells is the major obstacle for eradication of HIV infection. The "shock-and-kill" strategy proposed earlier aims to reduce the reservoir by activating cells out of latency. While the intracellular HIV Tat gene circuit is known to play important roles in controlling latency and its transactivation in HIV-infected cells, the detailed control mechanisms are not well understood. Here we study the mechanism of probabilistic control of the latent and the transactivated cell phenotypes of HIV-infected cells. We reconstructed the probability landscape, which is the probability distribution of the Tat gene circuit states, by directly computing the exact solution of the underlying chemical master equation. Results show that the Tat circuit exhibits a clear bimodal probability landscape (i.e., there are two distinct probability peaks, one associated with the latent cell phenotype and the other with the transactivated cell phenotype). We explore potential modifications to reactions in the Tat gene circuit for more effective transactivation of latent cells (i.e., the shock-and-kill strategy). Our results suggest that enhancing Tat acetylation can dramatically increase Tat and viral production, while increasing the Tat-transactivation response binding affinity can transactivate latent cells more rapidly than other manipulations. Our results further explored the "block and lock" strategy toward a functional cure for HIV. Overall, our study demonstrates a general approach toward discovery of effective therapeutic strategies and druggable targets by examining control mechanisms of cell phenotype switching via exactly computed probability landscapes of reaction networks.
| S-EPMC6298123 | biostudies-literature
Project description:UnlabelledAntiretroviral 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.ImportanceAntiretroviral 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.
| S-EPMC4495168 | biostudies-literature
Project description:BackgroundEpithelial ovarian cancer (EOC) is a highly prevalent disease that rapidly metastasizes and has poor prognosis. Most women are in the middle or late stages when diagnosed and have low survival rates. Recently, long non-coding RNAs (lncRNAs) were recognized to play pivotal roles in the development of EOC.MethodsThe expression of SLC25A21 antisense RNA 1 (SLC25A21-AS1) and Polypyrimidine Tract Binding Protein 3 (PTBP3) in EOC cells was assessed via qPCR. The proliferation activity of these cells was detected by EdU and Cell counting kit-8 (CCK8) assays, while the death rate of apoptotic cells and the cell cycle were detected by flow cytometry. Detection of cell transfer rate by transwell assay. Protein expression was measured through western blotting. Interactions between SLC25A21-AS1 and PTBP3 were detected through RNA immunoprecipitation (RIP), IF-FISH co-localization experiments and electrophoretic mobility shift assay (EMSA). The in vivo importance of SLC25A21-AS1 as a tumor suppressor modulator was assessed using murine xenograft models.ResultsThe lncRNA SLC25A21-AS1 has negligible expression in ovarian cancer tissues compared with that in normal ovarian tissues. A series of functional experiments revealed that the upregulation of SLC25A21-AS1 markedly blocked the proliferation and metastasis of EOC cells in vitro, while its downregulation had the opposite effect. Overexpression of SLC25A21-AS1 in a nude mouse model of EOC in vivo resulted in slower tumor growth and weakened metastatic potential. Moreover, SLC25A21-AS1 reduced the protein stability of PTBP3 and promoted its degradation. A series of subsequent experiments found that SLC25A21-AS1 inhibits EOC cell proliferation and metastasis by modulating PTBP3 through the ubiquitin-proteasome pathway and that the combination of SLC25A21-AS1 and PTBP3 provides the necessary conditions for the for the function to be realized.ConclusionsOur research reveals the effect of SLC25A21-AS1 in EOC development and suggests SLC25A21-AS1 can serve as a prognostic target by promoting the degradation of PTBP3 to improve patient survival.
| S-EPMC9885650 | biostudies-literature
Project description:The barrier to HIV-1 functional cure is caused by a small pool of latently infected resting CD4 T-cells that persist under antiretroviral therapy. Notably this latent reservoir of infected cells will produce replication-competent infectious virus once prolonged suppressive HAART is withdrawn. The reactivation of HIV-1 gene expression in T-cells harboring latent provirus in HIV-1 patients under HAART will likely result in depletion of this latent reservoir due to cytopathic effects and immune clearance. Many studies have investigated small molecules that reactivate HIV-1 gene expression but to date no latency reversal agent (LRA) has been identified to be specific, non-toxic, and effective in primary T-cells isolated from HIV-1 infected individuals undergoing long-term HAART. Stochastic fluctuations in HIV-1 tat gene expression have been attributed to be essential in the viral progression to latency. We hypothesized that exposing Tat to latently infected CD4 T-cells will result in potent latency reversal. Our results indicate the capacity of an engineered Tat to reactivate HIV-1 in latently infected cells from patients to a similar degree as the protein kinase C agonist PMA (Phorbol 12-Myristate 13-Acetate) while showing no T-cell activation nor any significant transcriptome perturbation in primary CD4 T-cells.
2023-09-05 | GSE235229 | GEO