RESUMO
Antiretroviral therapy reduces circulating HIV-1 to undetectable amounts but does not eliminate the virus due to the persistence of a stable reservoir of latently infected cells. The reservoir is maintained both by proliferation of latently infected cells and by reseeding from reactivated cells. A major challenge for the field is to find safe and effective methods to eliminate this source of rebounding HIV-1. Studies on the molecular mechanisms leading to HIV-1 latency and reactivation are being transformed using latency models in primary and patient CD4+ T cells. These studies have revealed the central role played by the biogenesis of the transcription elongation factor P-TEFb (Positive Transcription Elongation Factor b) and its recruitment to proviral HIV-1, for the maintenance of viral latency and the control of viral reactivation.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Transcrição Gênica , Latência Viral , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Linfócitos T CD4-Positivos/metabolismoRESUMO
HIV-1 latency is a major obstacle to achieving a functional cure for AIDS. Reactivation of HIV-1-infected cells followed by their elimination via immune surveillance is one proposed strategy for eradicating the viral reservoir. However, current latency-reversing agents (LRAs) show high toxicity and low efficiency, and new targets are needed to develop more promising LRAs. Here, we found that the histone chaperone CAF-1 (chromatin assembly factor 1) is enriched on the HIV-1 long terminal repeat (LTR) and forms nuclear bodies with liquid-liquid phase separation (LLPS) properties. CAF-1 recruits epigenetic modifiers and histone chaperones to the nuclear bodies to establish and maintain HIV-1 latency in different latency models and primary CD4+ T cells. Three disordered regions of the CHAF1A subunit are important for phase-separated CAF-1 nuclear body formation and play a key role in maintaining HIV-1 latency. Disruption of phase-separated CAF-1 bodies could be a potential strategy to reactivate latent HIV-1.
Assuntos
HIV-1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Epigênese Genética/genética , Epigênese Genética/fisiologia , Células HEK293 , Humanos , Regiões Promotoras Genéticas/genéticaRESUMO
The human immunodeficiency virus type 1 (HIV-1) reservoir consists of latently infected cells which present a major obstacle to achieving a functional cure for HIV-1. The formation and maintenance of HIV-1 latency have been extensively studied, and latency-reversing agents (LRAs) that can reactivate latent HIV-1 by targeting the involved host factors are developed; however, their clinical efficacies remain unsatisfactory. Therefore, it is imperative to identify novel targets for more potential candidates or better combinations for LRAs. In this study, we utilized CRISPR affinity purification in situ of regulatory elements system to screen for host factors associated with the HIV-1 long terminal repeat region that could potentially be involved in HIV-1 latency. We successfully identified that origin recognition complex 1 (ORC1), the largest subunit of the origin recognition complex, contributes to HIV-1 latency in addition to its function in DNA replication initiation. Notably, ORC1 is enriched on the HIV-1 promoter and recruits a series of repressive epigenetic elements, including DNMT1 and HDAC1/2, and histone modifiers, such as H3K9me3 and H3K27me3, thereby facilitating the establishment and maintenance of HIV-1 latency. Moreover, the reactivation of latent HIV-1 through ORC1 depletion has been confirmed across various latency cell models and primary CD4+ T cells from people living with HIV-1. Additionally, we comprehensively validated the properties of liquid-liquid phase separation (LLPS) of ORC1 from multiple perspectives and identified the key regions that promote the formation of LLPS. This property is important for the recruitment of ORC1 to the HIV-1 promoter. Collectively, these findings highlight ORC1 as a potential novel target implicated in HIV-1 latency and position it as a promising candidate for the development of novel LRAs. IMPORTANCE: Identifying host factors involved in maintaining human immunodeficiency virus type 1 (HIV-1) latency and understanding their mechanisms prepares the groundwork to discover novel targets for HIV-1 latent infection and provides further options for the selection of latency-reversing agents in the "shock" strategy. In this study, we identified a novel role of the DNA replication factor origin recognition complex 1 (ORC1) in maintaining repressive chromatin structures surrounding the HIV-1 promoter region, thereby contributing to HIV-1 latency. This discovery expands our understanding of the non-replicative functions of the ORC complex and provides a potential therapeutic strategy for HIV-1 cure.
Assuntos
Epigênese Genética , Infecções por HIV , Repetição Terminal Longa de HIV , HIV-1 , Complexo de Reconhecimento de Origem , Regiões Promotoras Genéticas , Latência Viral , Latência Viral/genética , Humanos , HIV-1/genética , HIV-1/fisiologia , Repetição Terminal Longa de HIV/genética , Infecções por HIV/virologia , Infecções por HIV/genética , Infecções por HIV/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Complexo de Reconhecimento de Origem/genética , Linfócitos T CD4-Positivos/virologia , Células HEK293 , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/genética , Regulação Viral da Expressão Gênica , Replicação Viral , Histonas/metabolismo , Histonas/genéticaRESUMO
Despite potent suppression of HIV-1 viral replication in the central nervous system (CNS) by antiretroviral therapy (ART), between 15% and 60% of HIV-1-infected patients receiving ART exhibit neuroinflammation and symptoms of HIV-1-associated neurocognitive disorder (HAND) - a significant unmet challenge. We propose that the emergence of HIV-1 from latency in microglia underlies both neuroinflammation in the CNS and the progression of HAND. Recent molecular studies of cellular silencing mechanisms of HIV-1 in microglia show that HIV-1 latency can be reversed both by proinflammatory cytokines and by signals from damaged neurons, potentially creating intermittent cycles of HIV-1 reactivation and silencing in the brain. We posit that anti-inflammatory agents that also block HIV-1 reactivation, such as nuclear receptor agonists, might provide new putative therapeutic avenues for the treatment of HAND.
Assuntos
Infecções por HIV , HIV-1 , Infecções por HIV/tratamento farmacológico , Humanos , Microglia , Transtornos Neurocognitivos/complicações , Doenças Neuroinflamatórias , Latência ViralRESUMO
Transcriptionally latent forms of replication-competent proviruses, present primarily in a small subset of memory CD4+ T cells, pose the primary barrier to a cure for HIV-1 infection because they are the source of the viral rebound that almost inevitably follows the interruption of antiretroviral therapy. Over the last 30 years, many of the factors essential for initiating HIV-1 transcription have been identified in studies performed using transformed cell lines, such as the Jurkat T-cell model. However, as highlighted in this review, several poorly understood mechanisms still need to be elucidated, including the molecular basis for promoter-proximal pausing of the transcribing complex and the detailed mechanism of the delivery of P-TEFb from 7SK snRNP. Furthermore, the central paradox of HIV-1 transcription remains unsolved: how are the initial rounds of transcription achieved in the absence of Tat? A critical limitation of the transformed cell models is that they do not recapitulate the transitions between active effector cells and quiescent memory T cells. Therefore, investigation of the molecular mechanisms of HIV-1 latency reversal and LRA efficacy in a proper physiological context requires the utilization of primary cell models. Recent mechanistic studies of HIV-1 transcription using latently infected cells recovered from donors and ex vivo cellular models of viral latency have demonstrated that the primary blocks to HIV-1 transcription in memory CD4+ T cells are restrictive epigenetic features at the proviral promoter, the cytoplasmic sequestration of key transcription initiation factors such as NFAT and NF-κB, and the vanishingly low expression of the cellular transcription elongation factor P-TEFb. One of the foremost schemes to eliminate the residual reservoir is to deliberately reactivate latent HIV-1 proviruses to enable clearance of persisting latently infected cells-the "Shock and Kill" strategy. For "Shock and Kill" to become efficient, effective, non-toxic latency-reversing agents (LRAs) must be discovered. Since multiple restrictions limit viral reactivation in primary cells, understanding the T-cell signaling mechanisms that are essential for stimulating P-TEFb biogenesis, initiation factor activation, and reversing the proviral epigenetic restrictions have become a prerequisite for the development of more effective LRAs.
Assuntos
Infecções por HIV , HIV-1 , Humanos , HIV-1/fisiologia , Latência Viral , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Linfócitos T CD4-Positivos , Provírus/metabolismo , Ativação ViralRESUMO
HIV-1 persists in a latent form during antiretroviral therapy, mainly in CD4+ T cells, thus hampering efforts for a cure. HIV-1 infection is accompanied by metabolic alterations, such as oxidative stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replication, both in vitro and in vivo in SIVmac-infected macaques, by upregulating antioxidant pathways and the intertwined iron import pathway. These changes are associated with remodeling of promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV-1 latency. We found that PML NBs are hyper-SUMOylated and that PML protein is degraded via the ubiquitin-proteasome pathway in productively infected cells, before latency establishment and after reactivation. Conversely, normal numbers of PML NBs were restored upon transition to latency or by decreasing oxidative stress or iron content. Our results highlight antioxidant and iron import pathways as determinants of HIV-1 latency and support their pharmacologic inhibition as tools to regulate PML stability and impair latency establishment.
Assuntos
Redes Reguladoras de Genes , Infecções por HIV/virologia , HIV-1/fisiologia , Ferro/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Infecções por HIV/genética , Infecções por HIV/metabolismo , Humanos , Macaca , Oxirredução , Proteólise , Análise de Sequência de RNA , Sumoilação , Regulação para Cima , Latência ViralRESUMO
Stochastic fluctuations in gene expression emanating from the HIV-1 long terminal repeat (LTR), amplified by the Tat positive feedback circuit, determine the choice between viral infection fates: active transcription (ON) or transcriptional silence (OFF). The emergence of several transcription factor binding site (TFBS) variant strains in HIV-1 subtype C (HIV-1C), especially those containing the duplication of the NF-κB motif, mandates the evaluation of the effect of enhanced transcriptional strength on gene expression noise and its influence on viral fate selection switch. Using a panel of subgenomic LTR-variant strains containing different copy numbers of the NF-κB motif (ranging from 0 to 4), we used flow cytometry, mRNA quantification, and pharmacological perturbations to demonstrate an inverse correlation between promoter strength and gene expression noise in Jurkat T cells and primary CD4+ T cells. The inverse correlation is consistent in clonal cell populations at constant intracellular concentrations of Tat and when NF-κB levels were regulated pharmacologically. Further, we show that strong LTRs containing at least two copies of the NF-κB motif in the enhancer establish a more stable latent state and demonstrate more rapid latency reversal than weak LTRs containing fewer motifs. We also demonstrate a cooperative binding of NF-κB to the motif cluster in HIV-1C LTRs containing two, three, or four NF-κB motifs (Hill coefficient [H] = 2.61, 3.56, and 3.75, respectively). The present work alludes to a possible evolution of the HIV-1C LTR toward gaining transcriptional strength associated with attenuated gene expression noise with implications for viral latency. IMPORTANCE Over the past two consecutive decades, HIV-1 subtype C (HIV-1C) has been undergoing directional evolution toward augmenting the transcriptional strength of the long terminal repeat (LTR) by adding more copies of the existing transcription factor binding site (TFBS) by sequence duplication. Additionally, the duplicated elements are genetically diverse, suggesting broader-range signal receptivity by variant LTRs. The HIV-1 promoter is inherently noisy, and the stochastic fluctuations in gene expression of variant LTRs may influence the active transcription (ON)/transcriptional silence (OFF) latency decisions. The evolving NF-κB motif variations of HIV-1C offer a powerful opportunity to examine how the transcriptional strength of the LTR might influence gene expression noise. Our work here shows that the augmented transcriptional strength of the HIV-1C LTR leads to concomitantly reduced gene expression noise, consequently leading to stabler latency maintenance and rapid latency reversal. The present work offers a novel lead toward appreciating the molecular mechanisms governing HIV-1 latency.
Assuntos
Infecções por HIV , HIV-1 , Latência Viral , Humanos , Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , Repetição Terminal Longa de HIV , HIV-1/genética , HIV-1/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Transcrição Gênica , Latência Viral/genéticaRESUMO
IMPORTANCE: The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.
Assuntos
Núcleo Celular , Eucromatina , HIV-1 , Código das Histonas , Histonas , Empacotamento do Genoma Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Humanos , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Elementos Facilitadores Genéticos/genética , Eucromatina/genética , Eucromatina/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Células HeLa , Histonas/metabolismo , HIV-1/genética , HIV-1/crescimento & desenvolvimento , HIV-1/metabolismo , Regiões Promotoras Genéticas/genética , Linfócitos T/virologia , Transcrição Gênica , Ativação ViralRESUMO
The retrovirus HIV-1 integrates into the host genome and establishes a latent viral reservoir that escapes immune surveillance. Molecular mechanisms of HIV-1 latency have been studied extensively to achieve a cure for the acquired immunodeficiency syndrome (AIDS). Latency-reversing agents (LRAs) have been developed to reactivate and eliminate the latent reservoir by the immune system. To develop more promising LRAs, it is essential to evaluate new therapeutic targets. Here, we find that CBX4, a component of the Polycomb Repressive Complex 1 (PRC1), contributes to HIV-1 latency in seven latency models and primary CD4+ T cells. CBX4 forms nuclear bodies with liquid-liquid phase separation (LLPS) properties on the HIV-1 long terminal repeat (LTR) and recruits EZH2, the catalytic subunit of PRC2. CBX4 SUMOylates EZH2 utilizing its SUMO E3 ligase activity, thereby enhancing the H3K27 methyltransferase activity of EZH2. Our results indicate that CBX4 acts as a bridge between the repressor complexes PRC1 and PRC2 that act synergistically to maintain HIV-1 latency. Dissolution of phase-separated CBX4 bodies could be a potential intervention to reactivate latent HIV-1.
Assuntos
Infecções por HIV , HIV-1 , Linfócitos T CD4-Positivos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , HIV-1/genética , Humanos , Ligases , Corpos Nucleares , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb/genética , Latência Viral/genéticaRESUMO
BACKGROUND: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus. RESULTS: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes. CONCLUSIONS: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies.
Assuntos
HIV-1 , Humanos , HIV-1/genética , Guanina , Fatores de Transcrição/genética , Cromatina , Repetição Terminal Longa de HIV/genética , Transcrição GênicaRESUMO
The presence of latent HIV-1 reservoirs in the periphery and brain represents a major obstacle to curing HIV-1 infection. As an essential protein for HIV-1 viral replication, HIV-1 Tat, mostly intracellular, has been implicated in latent HIV-1 infection. From HIV-1 infected cells, HIV-1 Tat is actively secreted and bystander cells uptake the released Tat whereupon it is endocytosed and internalized into endolysosomes. However, to activate the HIV-1 LTR promoter and increase HIV-1 replication, HIV-1 Tat must first escape from the endolysosomes and then enter the nucleus. Here, we tested the hypothesis that HIV-1 Tat can accumulate in endolysosomes and contribute to the activation of latent HIV-1 in astrocytes. Using U87MG astrocytoma cells expressing HIV-1 LTR-driven luciferase and primary human astrocytes we found that exogenous HIV-1 Tat enters endolysosomes, resides in endolysosomes for extended periods of time, and induces endolysosome de-acidification as well as enlargement. The weak base chloroquine promoted the release of HIV-1 Tat from endolysosomes and induced HIV-1 LTR transactivation. Similar results were observed by activating endolysosome Toll-like receptor 3 (TLR3) and TLR7/8. Conversely, pharmacological block of TLRs and knocking down expression levels of TLR3 and TLR7, but not TLR8, prevented endolysosome leakage and attenuated HIV-1 Tat-mediated HIV-1 LTR transactivation. Our findings suggest that HIV-1 Tat accumulation in endolysosomes may play an important role in controlling HIV-1 transactivation.
Assuntos
Astrócitos/virologia , Endocitose/genética , Endossomos/genética , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Lisossomos/genética , Ativação Transcricional/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica/genética , Infecções por HIV/genética , Infecções por HIV/virologia , Humanos , Regiões Promotoras Genéticas/genética , Latência Viral/genética , Replicação Viral/genéticaRESUMO
Reversing HIV-1 latency promotes the killing of infected cells and is essential for cure strategies. However, current latency-reversing agents (LRAs) are not entirely effective and safe in activating latent viruses in patients. In this study, we investigated whether Scopoletin (6-Methoxy-7-hydroxycoumarin), an important coumarin phytoalexin found in plants with multiple pharmacological activities, can reactivate HIV-1 latency and elucidated its underlying mechanism. Using the Jurkat T cell model of HIV-1 latency, we found that Scopoletin can reactivate latent HIV-1 replication with a similar potency to Prostratin and did so in a dose- and time-dependent manner. Moreover, we provide evidence indicating that Scopoletin-induced HIV-1 reactivation involves the nuclear factor kappa B (NF-κB) signaling pathway. Importantly, Scopoletin did not have a stimulatory effect on T lymphocyte receptors or HIV-1 receptors. In conclusion, our study suggests that Scopoletin has the potential to reactivate latent HIV-1 without causing global T-cell activation, making it a promising treatment option for anti-HIV-1 latency strategies.
Assuntos
Infecções por HIV , HIV-1 , Humanos , NF-kappa B , Escopoletina/farmacologia , Latência ViralRESUMO
The existence of latent viral reservoirs (LVRs), also called latent cells, has long been an acknowledged stubborn hurdle for effective treatment of HIV-1/AIDS. This stable and heterogeneous reservoir, which mainly exists in resting memory CD4+ T cells, is not only resistant to highly active antiretroviral therapy (HAART) but cannot be detected by the immune system, leading to rapid drug resistance and viral rebound once antiviral treatment is interrupted. Accordingly, various functional cure strategies have been proposed to combat this barrier, among which one of the widely accepted and utilized protocols is the so-called 'shock-and-kill' regimen. The protocol begins with latency-reversing agents (LRAs), either alone or in combination, to reactivate the latent HIV-1 proviruses, then eliminates them by viral cytopathic mechanisms (e.g., currently available antiviral drugs) or by the immune killing function of the immune system (e.g., NK and CD8+ T cells). In this review, we focuse on the currently explored small molecular LRAs, with emphasis on their mechanism-directed drug targets, binding modes and structure-relationship activity (SAR) profiles, aiming to provide safer and more effective remedies for treating HIV-1 infection.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , HIV-1 , Latência Viral , Humanos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos , Química Farmacêutica , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Ativação Viral , Latência Viral/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Romidepsin (RMD) is a histone deacetylase inhibitor reported to reverse HIV-1 latency. We sought to identify doses of RMD that were safe and induced HIV-1 expression. METHODS: Enrollees had HIV-1 RNA <40 copies/mL on antiretroviral therapy. Measurements included RMD levels, plasma viremia by single-copy HIV-1 RNA assay, HIV-1 DNA, cell-associated unspliced HIV-1 RNA (CA-RNA), acetylation of histone H3-lysine-9 (H3K9ac+), and phosphorylation of transcription factor P-TEFb. Wilcoxon tests were used for comparison. RESULTS: In the single-dose cohorts 1-3, 43 participants enrolled (36 participants 0.5, 2, 5 mg/m 2 RMD; 7 placebo) and 16 enrolled in the multidose cohort 4 (13 participants 5 mg/m 2 RMD; 3 placebo). One grade 3 event (neutropenia) was possibly treatment related. No significant changes in viremia were observed in cohorts 1-4 compared to placebo. In cohort 4, pharmacodynamic effects of RMD were reduced proportions of CD4+ T cells 24 hours after infusions 2-4 (median, -3.5% to -4.5%) vs placebo (median, 0.5% to 1%; P ≤ .022), and increased H3K9ac+ and phosphorylated P-TEFb in CD4 + T cells vs placebo (P ≤ .02). CONCLUSIONS: RMD infusions were safe but did not increase plasma viremia or unspliced CA-RNA despite pharmacodynamic effects on CD4 + T cells. CLINICAL TRIALS REGISTRATION: NCT01933594.
Assuntos
Depsipeptídeos/uso terapêutico , Infecções por HIV , Soropositividade para HIV , Inibidores de Histona Desacetilases/uso terapêutico , Fármacos Anti-HIV/uso terapêutico , Contagem de Linfócito CD4 , Infecções por HIV/tratamento farmacológico , Humanos , Fator B de Elongação Transcricional Positiva , RNA Viral , Viremia/tratamento farmacológico , Latência Viral/efeitos dos fármacosRESUMO
Individuals infected with human immunodeficiency virus (HIV) 1 have increased inflammation, which has been associated with age-associated diseases. Plasma markers, cell-associated virus levels, and ability to stimulate RNA transcription in latently infected cell lines was examined in younger and older HIV-1-infected individuals with suppressed virus. Cell-associated RNA, but not intact provirus level, had positive correlation with plasma D-dimer levels. Compared with the younger group, the older group had higher D-dimer levels and a trend toward more cell-associated RNA but similar levels of intact proviruses. Even though all measured inflammatory markers were relatively higher in the older group, this greater inflammation did not induce more HIV-1 transcription in latently infected cell lines. Inflammation and HIV-1 RNA expression increase with age despite similar levels of intact infectious HIV DNA. While plasma inflammation is correlated with HIV-1 RNA expression in peripheral blood mononuclear cells, it does not induce HIV-1 transcription in latently infected cell lines.
Assuntos
Infecções por HIV , HIV-1 , Inflamação , Provírus , Produtos de Degradação da Fibrina e do Fibrinogênio , HIV-1/genética , Humanos , Inflamação/virologia , Leucócitos Mononucleares , Provírus/genética , RNA Viral , Latência ViralRESUMO
Although substantial progress has been made in depicting the molecular pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection, the comprehensive mechanism of HIV-1 latency and the most promising therapeutic strategies to effectively reactivate the HIV-1 latent reservoir to achieve a functional cure for AIDS remain to be systematically illuminated. Here, we demonstrated that piwi (P element-induced Wimpy)-like RNA-mediated gene silencing 4 (PIWIL4) played an important role in suppressing HIV-1 transcription and contributed to the latency state in HIV-1-infected cells through its recruitment of various suppressive factors, including heterochromatin protein 1α/ß/γ, SETDB1, and HDAC4. The knockdown of PIWIL4 enhanced HIV-1 transcription and reversed HIV-1 latency in both HIV-1 latently infected Jurkat T cells and primary CD4+ T lymphocytes and resting CD4+ T lymphocytes from HIV-1-infected individuals on suppressive combined antiretroviral therapy (cART). Furthermore, in the absence of PIWIL4, HIV-1 latently infected Jurkat T cells were more sensitive to reactivation with vorinostat (suberoylanilide hydroxamic acid, or SAHA), JQ1, or prostratin. These findings indicated that PIWIL4 promotes HIV-1 latency by imposing repressive marks at the HIV-1 5' long terminal repeat. Thus, the manipulation of PIWIL4 could be a novel strategy for developing promising latency-reversing agents (LRAs).IMPORTANCE HIV-1 latency is systematically modulated by host factors and viral proteins. During this process, the suppression of HIV-1 transcription plays an essential role in promoting HIV-1 latency. In this study, we found that PIWIL4 repressed HIV-1 promoter activity and maintained HIV-1 latency. In particular, we report that PIWIL4 can regulate gene expression through its association with the suppressive activity of HDAC4. Therefore, we have identified a new function for PIWIL4: it is not only a suppressor of endogenous retrotransposons but also plays an important role in inhibiting transcription and leading to latent infection of HIV-1, a well-known exogenous retrovirus. Our results also indicate a novel therapeutic target to reactivate the HIV-1 latent reservoir.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas Argonautas/farmacologia , Epigênese Genética , Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/fisiologia , Latência Viral/efeitos dos fármacos , Antirretrovirais/uso terapêutico , Proteínas Argonautas/genética , Linfócitos T CD4-Positivos/virologia , Células HEK293 , Infecções por HIV/virologia , HIV-1/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Células Jurkat , Proteínas de Ligação a RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Virais/metabolismo , Latência Viral/genética , Replicação Viral/efeitos dos fármacosRESUMO
NF-κB-interacting long noncoding RNA (NKILA) was recently identified as a negative regulator of NF-κB signaling and plays an important role in the development of various cancers. It is well known that NF-κB-mediated activation of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-driven gene expression is required for HIV-1 transcription and reactivation of latency. However, whether NKILA plays essential roles in HIV-1 replication and latency is unclear. Here, by ectopic expression and silencing experiments, we demonstrate that NKILA potently inhibits HIV-1 replication in an NF-κB-dependent manner by suppressing HIV-1 LTR promoter activity. Moreover, NKILA showed broad-spectrum inhibition on the replication of HIV-1 clones with different coreceptor tropisms as well as on LTR activity of various HIV-1 clinical subtypes. Chromatin immunoprecipitation (ChIP) assays revealed that NKILA expression abolishes the recruitment of p65 to the duplicated κB binding sites in the HIV-1 LTR. NKILA mutants disrupting NF-κB inhibition also lost the ability to inhibit HIV-1 replication. Notably, HIV-1 infection or reactivation significantly downregulated NKILA expression in T cells in order to facilitate viral replication. Downregulated NKILA was mainly due to reduced acetylation of histone K27 on the promoter of NKILA by HIV-1 infection, which blocks NKILA expression. Knockdown of NKILA promoted the reactivation of latent HIV-1 upon phorbol myristate acetate (PMA) stimulation, while ectopic NKILA suppressed the reactivation in a well-established clinical model of withdrawal of azidothymidine (AZT) in vitro These findings improve our understanding of the functional suppression of HIV-1 replication and latency by NKILA through NF-κB signaling.IMPORTANCE The NF-κB pathway plays key roles in HIV-1 replication and reactivation of HIV-1 latency. A regulator inhibiting NF-κB activation may be a promising therapeutic strategy against HIV-1. Recently, NF-κB-interacting long noncoding RNA (NKILA) was identified to suppress the development of different human cancers by inhibiting IκB kinase (IKK)-induced IκB phosphorylation and NF-κB pathway activation, whereas the relationship between NKILA and HIV-1 replication is still unknown. Here, our results show that NKILA inhibits HIV-1 replication and reactivation by suppressing HIV-1 long terminal repeat (LTR)-driven transcription initiation. Moreover, NKILA inhibited the replication of HIV-1 clones with different coreceptor tropisms. This project may reveal a target for the development of novel anti-HIV drugs.
Assuntos
HIV-1/fisiologia , NF-kappa B/metabolismo , RNA Longo não Codificante/metabolismo , Latência Viral/fisiologia , Replicação Viral/fisiologia , Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/virologia , Imunoprecipitação da Cromatina , Regulação Viral da Expressão Gênica , Células HEK293 , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/fisiologia , HIV-1/genética , Humanos , Fosforilação , RNA Longo não Codificante/genética , RNA Longo não Codificante/farmacologia , Transdução de Sinais/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
A significant number of people living with human immunodeficiency virus type 1 (HIV-1) suffer from HIV-associated neurocognitive disorders (HAND). Many previous studies investigating HIV in astrocytes as a heterogenous population have established the relevance of astrocytes to HIV-associated neuropathogenesis. However, these studies were unable to differentiate the state of infection, i.e., active or latent, or to evaluate how this affects astrocyte biology. In this study, the pseudotyped doubly labeled fluorescent reporter red/green (R/G)-HIV-1 was used to identify and enrich restricted and active populations of HIV+ astrocytes based on the viral promoter activity. Here, we report that the majority of human astrocytes restricted R/G-HIV-1 gene expression early during infection and were resistant to reactivation by vorinostat and interleukin 1ß. However, actively infected astrocytes were inducible, leading to increased expression of viral proteins upon reactivation. R/G-HIV-1 infection also significantly decreased the cell proliferation and glutamate clearance ability of astrocytes, which may contribute to excitotoxicity. Moreover, transcriptome analyses to compare gene expression patterns of astrocyte harboring active versus restricted long terminal repeats (LTRs) revealed that the gene expression patterns were similar and that the active population demonstrated more widespread and robust changes. Our data suggest that harboring the HIV genome profoundly alters astrocyte biology and that strategies that keep the virus latent (e.g., block and lock) or those that reactivate the latent virus (e.g., shock and kill) would be detrimental to astrocyte function and possibly augment their contributions to HAND.IMPORTANCE More than 36 million people are living with HIV-1 worldwide, and despite antiretroviral therapy, 30 to 50% of the people living with HIV-1 suffer from mild to moderate neurocognitive disorders. HIV-1 reservoirs in the central nervous system (CNS) are challenging to address due to low penetration of antiretroviral drugs, lack of resident T cells, and permanent integration of provirus into neural cells such as microglia and astrocytes. Several studies have shown astrocyte dysfunction during HIV-1 infection. However, little is known about how HIV-1 latency affects their function. The significance of our research is in identifying that the majority of HIV+ astrocytes restrict HIV expression and were resistant to reactivation. Further, simply harboring the HIV genome profoundly altered astrocyte biology, resulting in a proinflammatory phenotype and functional changes. In this context, therapeutic strategies to reactivate or silence astrocyte HIV reservoirs, without excising proviral DNA, will likely lead to detrimental neuropathological outcomes during HIV CNS infection.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Regulação Viral da Expressão Gênica , Infecções por HIV/metabolismo , HIV-1/metabolismo , Transcriptoma , Astrócitos/virologia , Encéfalo/patologia , Linhagem Celular , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Infecções por HIV/genética , Infecções por HIV/terapia , HIV-1/genética , HumanosRESUMO
Unbiased shRNA library screens revealed that the estrogen receptor-1 (ESR-1) is a key factor regulating HIV-1 latency. In both Jurkat T cells and a Th17 primary cell model for HIV-1 latency, selective estrogen receptor modulators (SERMs, i.e., fulvestrant, raloxifene, and tamoxifen) are weak proviral activators and sensitize cells to latency-reversing agents (LRAs) including low doses of TNF-α (an NF-κB inducer), the histone deacetylase inhibitor vorinostat (soruberoylanilide hydroxamic acid, SAHA), and IL-15. To probe the physiologic relevance of these observations, leukapheresis samples from a cohort of 12 well-matched reproductive-age women and men on fully suppressive antiretroviral therapy were evaluated by an assay measuring the production of spliced envelope (env) mRNA (the EDITS assay) by next-generation sequencing. The cells were activated by T cell receptor (TCR) stimulation, IL-15, or SAHA in the presence of either ß-estradiol or an SERM. ß-Estradiol potently inhibited TCR activation of HIV-1 transcription, while SERMs enhanced the activity of most LRAs. Although both sexes responded to SERMs and ß-estradiol, females showed much higher levels of inhibition in response to the hormone and higher reactivity in response to ESR-1 modulators than males. Importantly, the total inducible RNA reservoir, as measured by the EDITS assay, was significantly smaller in the women than in the men. We conclude that concurrent exposure to estrogen is likely to limit the efficacy of viral emergence from latency and that ESR-1 is a pharmacologically attractive target that can be exploited in the design of therapeutic strategies for latency reversal.
Assuntos
Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/agonistas , HIV-1/fisiologia , Caracteres Sexuais , Transcrição Gênica/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Adulto , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células Jurkat , Masculino , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
The HIV-1 reservoir is a major obstacle to complete eradication of the virus. Although many proteins and RNAs have been characterized as regulators in HIV-1/AIDS pathogenesis and latency, only a few long noncoding RNAs (lncRNAs) have been shown to be closely associated with HIV-1 replication and latency. In this study, we demonstrated that lncRNA uc002yug.2 plays a key role in HIV-1 replication and latency. uc002yug.2 potentially enhances HIV-1 replication, long terminal repeat (LTR) activity, and the activation of latent HIV-1 in both cell lines and CD4+ T cells from patients. Further investigation revealed that uc002yug.2 activates latent HIV-1 through downregulating RUNX1b and -1c and upregulating Tat protein expression. The accumulated evidence supports our model that the Tat protein has the key role in the uc002yug.2-mediated regulatory effect on HIV-1 reactivation. Moreover, uc002yug.2 showed an ability to activate HIV-1 similar to that of suberoylanilide hydroxamic acid or phorbol 12-myristate 13-acetate using latently infected cell models. These findings improve our understanding of lncRNA regulation of HIV-1 replication and latency, providing new insights into potential targeted therapeutic interventions.IMPORTANCE The latent viral reservoir is the primary obstacle to curing HIV-1 disease. To date, only a few lncRNAs, which play major roles in various biological processes, including viral infection, have been identified as regulators in HIV-1 latency. In this study, we demonstrated that lncRNA uc002yug.2 is important for both HIV-1 replication and activation of latent viruses. Moreover, uc002yug.2 was shown to activate latent HIV-1 through regulating alternative splicing of RUNX1 and increasing the expression of Tat protein. These findings highlight the potential merit of targeting lncRNA uc002yug.2 as an activating agent for latent HIV-1.