Highly active antiretroviral therapy (HAART) suppresses human immunodeficiency virus (HIV) replication to undetectable levels but cannot fully eradicate the virus because a small reservoir of CD4+ T cells remains latently infected. Since HIV efficiently infects only activated CD4+ T cells and since latent HIV primarily resides in resting CD4+ T cells, it is generally assumed that latency is established when a productively infected cell recycles to a resting state, trapping the virus in a latent state. In this study, we use a dual reporter virus—HIV Duo-Fluo I, which identifies latently infected cells immediately after infection—to investigate how T cell activation affects the estab-lishment of HIV latency. We show that HIV latency can arise from the direct infection of both resting and activated CD4+ T cells. Importantly, returning productively infected cells to a resting state is not associated with a significant silencing of the integrated HIV. We further show that resting CD4+ T cells from human lymphoid tissue (tonsil, spleen) show increased latency after infection when compared to peripheral blood. Our findings raise significant questions regarding the most commonly accepted model for the establishment of latent HIV and suggest that infection of both resting and activated primary CD4+ T cells produce latency.
The study of HIV latency has been hindered because there are few latently infected cells in vivo, and we cannot distinguish latently infected cells from uninfected cells prior to reactivation of the latent provirus. In general, HIV latency is quantitatively studied by reactivating latently infected cells after latency has been established. However, this practice limits the investigation of how latency is established and how latent provirus can be reactivated. Our recently developed dual reporter virus, HIV Duo-Fluo I, can identify latently infected cells early after infection. In this study, we use HIV Duo-Fluo I to investigate how T cell activation affects the outcome of HIV infection.
Treatment with antiretroviral therapy dramatically increases the survival of HIV-infected individuals. However, treatment has to be continued for life because it does not lead to the full eradication of infection. HIV persists in resting CD4+ T cells, and possibly other cell types, and can reemerge from these cells when therapy is interrupted. Here, we review molecular mechanisms that have been proposed to contribute to HIV latency, as well as the relative roles of cis- and trans-acting mechanisms. We also discuss existing and future therapeutic opportunities regarding HIV latency that might lead to a future cure for HIV infection.
Transcriptional regulation; latency-reversing therapeutics; immunological mechanisms; cellular mechanisms; viral mechanisms
SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane.
Persistence of human immunodeficiency virus type 1 (HIV-1) in latently infected CD4+ T cells prevents eradication in HIV-infected treated patients. Latency is characterized by a reversible silencing of transcription of integrated HIV-1. Several molecular mechanisms have been described which contribute to latency, including the establishment and maintenance of repressive chromatin on the HIV-1 promoter. Histone deacetylation is a landmark modification associated with transcriptional repression of the HIV-1 promoter and inhibition of histone deacetylase enzymes (HDACs) reactivates latent HIV-1. Here we review the different HDAC inhibitors that have been studied in HIV-1 latency and their therapeutic potential in reactivating latent HIV-1.
HIV Latency; Histone Deacetylases; HDAC inhibitors; chromatin; therapeutics
Traditionally, the ketone body β-hydroxybutyrate (βOHB) has been looked upon as a carrier of energy from liver to peripheral tissues during fasting or exercise. However, βOHB also signals via extracellular receptors and acts as an endogenous inhibitor of histone deacetylases (HDACs). These recent findings support a model in which βOHB functions to link the environment, in this case the diet, and gene expression via chromatin modifications. Here, we review the regulation and functions of ketone bodies, the relationship between ketone bodies and calorie restriction, and the implications of HDAC inhibition by the ketone body βOHB in the modulation of metabolism, and diseases of aging.
acetylation; HDAC; calorie restriction; longevity; epigenetics
Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5−/− animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.
The biological and enzymatic function of SIRT4 is largely uncharacterized. We show that the C. elegans SIR-2.2 and SIR-2.3 orthologs of SIRT4 are ubiquitously expressed, also localize to mitochondria and function during oxidative stress. Further, we identified conserved interaction with mitochondrial biotin-dependent carboxylases (PC, PCC, MCCC), key enzymes in anaplerosis and ketone body formation. The carboxylases were found acetylated on multiple lysine residues and detailed analysis of mPC suggested that one of these residues, K748ac, might regulate enzymatic activity. Nevertheless, no changes in mPC acetylation levels and enzymatic activity could be detected upon overexpression or loss of functional SIRT4.
sirtuins; SIRT4; C. elegans; biotin-dependent carboxylase; pyruvate carboxylase; protein acetylation
Vascular endothelial growth factor (VEGF) promotes leukocyte adhesion to endothelial cells (ECs). Angiopoietin-1 (Ang-1) inhibits this response. Nuclear receptor-77 (Nur77) is a proangiogenic nuclear receptor. In the present study, we assessed the influence of Ang-1 and VEGF on Nur77 expression in ECs, and evaluated its role in Ang-1/VEGF-mediated leukocyte adhesion.
Methods and Results
Expression of Nur77 was evaluated with real-time polymerase chain reaction and immunoblotting. Adhesion of leukocytes to ECs was monitored with inverted microscopy. Nur77 expression or activity was inhibited using adenoviruses expressing dominant-negative form of Nur77, retroviruses expressing Nur77 in the antisense direction, and small interfering RNA oligos. Both Ang-1 and VEGF induce Nur77 expression, by >5- and 30-fold, respectively. When combined, Ang-1 potentiates VEGF-induced Nur77 expression. Ang-1 induces Nur77 through the phosphoinositide 3-kinase and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces Nur77 expression through the protein kinase D/histone deacetylase 7/myocyte enhancer factor 2 and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces nuclear factor-kappaB transcription factor, vascular cell adhesion molecule-1, and E-selectin expressions, and promotes leukocyte adhesion to ECs. Ang-1 inhibits these responses. This inhibitory effect of Ang-1 disappears when Nur77 expression is disrupted, restoring the inductive effects of VEGF on adhesion molecule expression, and increased leukocyte adhesion to ECs.
Nur77 promotes anti-inflammatory effects of Ang-1, and functions as a negative feedback inhibitor of VEGF-induced EC activation.
angiopoietin-1; endothelial cells; inflammation; leukocyte adhesion; nuclear receptor-77; vascular endothelial growth factor
Sirt3 is an NAD+-dependent deacetylase that regulates mitochondrial function by targeting metabolic enzymes and proteins. In fasting mice, Sirt3 expression is decreased in skeletal muscle resulting in increased mitochondrial protein acetylation. Deletion of Sirt3 led to impaired glucose oxidation in muscle, which was associated with decreased pyruvate dehydrogenase (PDH) activity, accumulation of pyruvate and lactate metabolites, and an inability of insulin to suppress fatty acid oxidation. Antibody-based acetyl-peptide enrichment and mass spectrometry of mitochondrial lysates from WT and Sirt3 KO skeletal muscle revealed that a major target of Sirt3 deacetylation is the E1α subunit of PDH (PDH E1α). Sirt3 knockout in vivo and Sirt3 knockdown in myoblasts in vitro induced hyperacetylation of the PDH E1α subunit, altering its phosphorylation leading to suppressed PDH enzymatic activity. The inhibition of PDH activity resulting from reduced levels of Sirt3 induces a switch of skeletal muscle substrate utilization from carbohydrate oxidation toward lactate production and fatty acid utilization even in the fed state, contributing to a loss of metabolic flexibility. Thus, Sirt3 plays an important role in skeletal muscle mitochondrial substrate choice and metabolic flexibility in part by regulating PDH function through deacetylation.
HIV latency is a major barrier to viral eradication from infected individuals. Lusic et al. show that HIV latency is established by spatially positioning the proviral chromatin in close proximity to promyelocytic leukemia (PML) nuclear bodies, a reversible process that recruits the methyltransferase enzyme G9a to the latent viral promoter.
Latent HIV persists in CD4+ T cells in infected patients under antiretroviral therapy (ART). Latency is associated with transcriptional silencing of the integrated provirus and driven, at least in part, by histone deacetylases (HDACs), a family of chromatin associated proteins that regulate histone acetylation and the accessibility of DNA to transcription factors. Remarkably, inhibition of HDACs is sufficient to reactivate a fraction of latent HIV in a variety of experimental systems. This basic observation led to the shock and kill idea that forcing the transcriptional activation of HIV might lead to virus expression, to virus-or host-induced cell death of the reactivated cells, and to the eradication of the pool of latently infected cells. Such intervention might possibly lead to a cure for HIV infected patients. Here, we review the basic biology of HDACs and their inhibitors, the role of HDACs in HIV latency and recent efforts to use HDAC inhibitors to reactivate latent HIV in vitro and in vivo.
HIV; Latency; HDAC; HDAC inhibitors
HIV latency constitutes the main barrier for clearing HIV infection from patients. Our inability to recognize and isolate latently infected cells hinders the study of latent HIV. We engineered two HIV-based viral reporters expressing different fluorescent markers: one HIV promoter-dependent marker for productive HIV infection, and a second marker under a constitutive promoter independent of HIV promoter activity. Infection of cells with these viruses allows the identification and separation of latently-infected cells from uninfected and productively infected cells. These reporters are sufficiently sensitive and robust for high-throughput screening to identify drugs that reactivate latent HIV. These reporters can be used in primary CD4 T lymphocytes and reveal a rare population of latently infected cells responsive to physiological stimuli. In summary, our HIV-1 reporters enable visualization and purification of latent cell populations and open up new perspectives for studies of latent HIV infection.
The therapeutic potential of pharmacologic inhibition of bromodomain and extraterminal (BET) proteins has recently emerged in hematological malignancies and chronic inflammation. We find that BET inhibitor compounds (JQ1, I-Bet, I-Bet151 and MS417) reactivate HIV from latency. This is evident in polyclonal Jurkat cell populations containing latent infectious HIV, as well as in a primary T-cell model of HIV latency. Importantly, we show that this activation is dependent on the positive transcription elongation factor p-TEFb but independent from the viral Tat protein, arguing against the possibility that removal of the BET protein BRD4, which functions as a cellular competitor for Tat, serves as a primary mechanism for BET inhibitor action. Instead, we find that the related BET protein, BRD2, enforces HIV latency in the absence of Tat, pointing to a new target for BET inhibitor treatment in HIV infection. In shRNA-mediated knockdown experiments, knockdown of BRD2 activates HIV transcription to the same extent as JQ1 treatment, while a lesser effect is observed with BRD4. In single-cell time-lapse fluorescence microscopy, quantitative analyses across ~2,000 viral integration sites confirm the Tat-independent effect of JQ1 and point to positive effects of JQ1 on transcription elongation, while delaying re-initiation of the polymerase complex at the viral promoter. Collectively, our results identify BRD2 as a new Tat-independent suppressor of HIV transcription in latently infected cells and underscore the therapeutic potential of BET inhibitors in the reversal of HIV latency.
HIV; latency; Tat; JQ1; MS417; I-BET; I-BET151; P-TEFb; BRD4; BRD2
T follicular helper (TFH) cells provide critical help to B cells during humoral immune responses. Here we report that mice with T cell-specific deletion of miR-17~92 family miRNAs (tKO mice) exhibited severely compromised TFH differentiation, germinal center formation, antibody responses, and failed to control chronic virus infection. Conversely, T cell-specific miR-17~92 transgenic mice spontaneously accumulated TFH cells and developed fatal immunopathology. Mechanistically, miR-17~92 family miRNAs control CD4+ T cell migration into B cell follicles by regulating ICOS-PI3K signaling intensity through suppressing the expression of the Akt phosphatase Phlpp2. These findings demonstrate that miR-17~92 family microRNAs play an essential role in TFH differentiation and establish Phlpp2 as an important mediator of their function in this process.
The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.
HIV establishes a state of latency in vivo and this latent reservoir, although small, is difficult to eradicate. To be able to better understand this state of latency, and to develop strategies to eliminate it, many groups have developed in vitro models of HIV latency. However, notable differences exist among cell model systems because compounds that reactivate latent HIV in a particular system often fail to do so uniformly across different models. To begin to understand the biological characteristics that are inherent to each HIV model of latency, we compared the response properties of five primary T cell, four J-Lat cell models and those obtained with patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models.
NAD+-dependent histone deacetylases (sirtuins)
play important roles in epigenetic regulation but also through nonhistone
substrates for other key cellular events and have been linked to the
pathogenesis of cancer, neurodegeneration, and metabolic diseases.
The subtype Sirt5 has been shown recently to act as a desuccinylating
and demalonylating enzyme. We have established an assay for biochemical
testing of Sirt5 using a small labeled succinylated lysine derivative.
We present a comparative study on the profiling of several established
sirtuin inhibitors on Sirt1–3 as well as Sirt5 and also present
initial results on a screening for new compounds that block Sirt5.
Thiobarbiturates were identified as new Sirt5 inhibitors in the low
micromolar range, which are selective over Sirt3 that can be found
in the same cell compartment as Sirt5.
sirtuin; Sirt5; desuccinylation; sirtinol; AMC assay
Regulating crosstalk between anoikis and survival signaling pathways is crucial to regulating tissue processes and mitigating diseases like cancer. Previously, we showed that anoikis activates a CD95/Fas-mediated signaling pathway regulated by receptor-interacting protein (RIP), a kinase that shuttles between Fas-mediated cell death and integrin/FAK-mediated survival pathways. Since sirtuin-3 (SIRT3), an NAD-dependent deacetylase, is known to regulate cell survival, metabolism, and tumorigenesis, we hypothesized that SIRT3 might engage in crosstalk with Fas/RIP/integrin/FAK survival-death pathways in cancer cell systems.
Using immunohistochemical staining, immunoblotting, human tissue microarrays, and overexpression and suppression approaches in vitro and in vivo we examined the roles of RIP and SIRT3 in oral squamous cell carcinoma (OSCC) anoikis resistance and tumorigenesis.
RIP and SIRT3 have an opposite expression profile in OSCC cells and tissues. Stable suppression of RIP enhances SIRT3 levels, whereas, stable suppression of SIRT3 does not impact RIP levels in OSCC cells. As OSCC cells become anoikis-resistant they form multicellular aggregates or oraspheres in suspension conditions, and their expression of SIRT3 increases as their RIP expression decreases. Also, anoikis-resistant OSCC cells with higher SIRT3 and low RIP expression induce an increased tumor burden and incidence in mice unlike their adherent OSCC cell counterparts. Furthermore, stable suppression of SIRT3 inhibits anoikis resistance and reduces tumor incidence.
RIP is a likely upstream negative regulator of SIRT3 in anoikis resistance, and an anoikis-resistant orasphere phenotype defined by higher SIRT3 and low RIP expression contributes to a more aggressive phenotype in OSCC development.
Efficient coupling of cellular energy production to metabolic demand is crucial to maintain organismal homeostasis. Here, we report that the mitochondrial Sirtuin Sirt4 regulates mitochondrial ATP homeostasis. We find that Sirt4 affects mitochondrial uncoupling via the adenine nucleotide translocator 2 (ANT2). Loss of Sirt4 expression leads to decreased cellular ATP levels in vitro and in vivo while Sirt4 overexpression is associated with increased ATP levels. Further, we provide evidence that lack of Sirt4 activates a retrograde signaling response from the mitochondria to the nucleus that includes AMPK, PGC1α, key regulators of β-oxidation such as Acetyl-CoA carboxylase, and components of the mitochondrial respiratory machinery. This study highlights the ability of Sirt4 to regulate ATP levels via ANT2 and a feedback loop involving AMPK.
Sirt4; ANT2; AMPK; ATP; mitochondrial signaling
Toll-like receptors (TLRs) are crucial for recognition of pathogen-associated molecular patterns by cells of the innate immune system. TLRs are present and functional in CD4+ T cells. Memory CD4+ T cells, predominantly central memory cells (TCM), constitute the main reservoir of latent HIV-1. However, how TLR ligands affect the quiescence of latent HIV within central memory CD4+ T cells has not been studied.
We evaluated the ability of a broad panel of TLR agonists to reactivate latent HIV-1. The TLR-1/2 agonist Pam3CSK4 leads to viral reactivation of quiescent HIV in a model of latency based on cultured TCM and in resting CD4+ T cells isolated from aviremic patients. In addition, we investigated the signaling pathway associated with Pam3CSK4 involved in HIV-1 reactivation. We show that the transcription factors NFκB, NFAT and AP-1 cooperate to induce viral reactivation downstream of TLR-1/2 stimulation. Furthermore, increasing levels of cyclin T1 is not required for TLR-mediated viral reactivation, but induction of viral expression requires activated pTEFb. Finally, Pam3CSK4 reactivates latent HIV-1 in the absence of T cell activation or proliferation, in contrast to antigen stimulation.
Our findings suggest that the signaling through TLR-1/2 pathway via Pam3CSK4 or other reagents should be explored as an anti-latency strategy either alone or in combination with other anti-latency drugs.
Age-related loss of muscle mass and function, sarcopenia, has a major impact on the quality of life in the elderly. Among the proposed causes of sarcopenia are mitochondrial dysfunction and accumulated oxidative damage during aging. Dietary restriction (DR), a robust dietary intervention that extends lifespan and modulates age-related pathology in a variety of species has been shown to protect from sarcopenia in rodents. Although the mechanism(s) by which DR modulates aging are still not defined, one potential mechanism is through modulation of oxidative stress and mitochondrial dysfunction. To directly test the protective effect of DR against oxidative stress induced muscle atrophy in vivo, we subjected mice lacking a key antioxidant enzyme, CuZnSOD (Sod1) to DR (40% of ad libitum fed diet). We have previously shown that the Sod1−/− mice exhibit an acceleration of sarcopenia associated with high oxidative stress, mitochondrial dysfunction, and severe neuromuscular innervation defects. Despite the dramatic atrophy phenotype in the Sod1−/− mice, DR led to a reversal or attenuation of reduced muscle function, loss of innervation and muscle atrophy in these mice. DR improves mitochondrial function as evidenced by enhanced Ca2+ regulation and reduction of mitochondrial reactive oxygen species (ROS). Furthermore, we show upregulation of SIRT3 and MnSOD in DR animals, consistent with reduced mitochondrial oxidative stress and reduced oxidative damage in muscle tissue measured as F2- isoprostanes. Collectively, our results demonstrate that DR is a powerful mediator of mitochondrial function, mitochondrial ROS production, and oxidative damage, providing a solid protection against oxidative stress induced neuromuscular defects and muscle atrophy in vivo even under conditions of high oxidative stress.
Concentrations of acetyl–coenzyme A and nicotinamide adenine dinucleotide (NAD+) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress.
Improving the control of energy homeostasis can lower cardiovascular risk in metabolically compromised individuals. To identify new regulators of whole-body energy control, we conducted a high-throughput screen in transgenic reporter zebrafish for small molecules that modulate the expression of the fasting-inducible gluconeogenic gene pck1. We show that this in vivo strategy identified several drugs that impact gluconeogenesis in humans, as well as metabolically uncharacterized compounds. Most notably, we find that the Translocator Protein (TSPO) ligands PK 11195 and Ro5-4864 are glucose lowering agents despite a strong inductive effect on pck1 expression. We show that these drugs are activators of a fasting-like energy state, and importantly that they protect high-fat diet induced obese mice from hepatosteatosis and glucose intolerance, two pathological manifestations of metabolic dysregulation. Thus, using a whole-organism screening strategy, this study has identified new small molecule activators of fasting metabolism.
Current antiretroviral therapy for HIV-1 infection effectively suppresses but does not eradicate HIV-1. Patients on Highly Active Anti-Retroviral Therapy (HAART) maintain a persistent low-level viremia requiring lifelong adherence to antiretroviral therapies. This viremia may arise from latently infected reservoirs such as resting memory CD4+ T-cells or sanctuary sites where drug penetration is suboptimal. Understanding the mechanisms of HIV latency will help efforts to eradicate the infection. This review examines the dynamics of persistent viremia, viral reservoirs, the mechanisms behind viral latency, and methods to purge the viral reservoirs. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development.
Heterochromatin; repression; latency; transcription; transcriptional interference; chromatin; primary latency models; virus; epigenetics; histone