N-acetylglucosamine supplementation fails to bypass the critical acetylation of glucosamine-6-phosphate required for Toxoplasma gondii replication and invasion.

The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite's ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies.

Brain HIV-1 latently-infected reservoirs targeted by the suicide gene strategy.

Reducing the pool of HIV-1 reservoirs in patients is a must to achieve functional cure. The most prominent HIV-1 cell reservoirs are resting CD4 + T cells and brain derived microglial cells. Infected microglial cells are believed to be the source of peripheral tissues reseedings and the emergence of drug resistance. Clearing infected cells from the brain is therefore crucial. However, many characteristics of microglial cells and the central nervous system make extremely difficult their eradication from brain reservoirs. Current methods, such as the "shock and kill", the "block and lock" and gene editing strategies cannot override these difficulties. Therefore, new strategies have to be designed when considering the elimination of brain reservoirs. We set up an original gene suicide strategy using latently infected microglial cells as model cells. In this paper we provide proof of concept of this strategy.

Toxoplasma gondii ROP16 kinase silences the cyclin B1 gene promoter by hijacking host cell UHRF1-dependent epigenetic pathways.

Toxoplasmosis, caused by the apicomplexan parasite Toxoplasma gondii, is one of the most common infections in the world due to the lifelong persistence of this parasite in a latent stage. This parasite hijacks host signaling pathways through epigenetic mechanisms which converge on key nuclear proteins. Here, we report a new parasite persistence strategy involving T. gondii rhoptry protein ROP16 secreted early during invasion, which targets the transcription factor UHRF1 (ubiquitin-like containing PHD and RING fingers domain 1), and leads to host cell cycle arrest. This is mediated by DNMT activity and chromatin remodeling at the cyclin B1 gene promoter through recruitment of phosphorylated UHRF1 associated with a repressive multienzymatic protein complex. This leads to deacetylation and methylation of histone H3 surrounding the cyclin B1 promoter to epigenetically silence its transcriptional activity. Moreover, T. gondii infection causes DNA hypermethylation in its host cell, by upregulation of DNMTs. ROP16 is already known to activate and phosphorylate protective immunity transcription factors such as STAT 3/6/5 and modulate host signaling pathways in a strain-dependent manner. Like in the case of STAT6, the strain-dependent effects of ROP16 on UHRF1 are dependent on a single amino-acid polymorphism in ROP16. This study demonstrates that Toxoplasma hijacks a new epigenetic initiator, UHRF1, through an early event initiated by the ROP16 parasite kinase.

An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression.

The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

HMGA1 recruits CTIP2-repressed P-TEFb to the HIV-1 and cellular target promoters.

Active positive transcription elongation factor b (P-TEFb) is essential for cellular and human immunodeficiency virus type 1 (HIV-1) transcription elongation. CTIP2 represses P-TEFb activity in a complex containing 7SK RNA and HEXIM1. Recently, the inactive 7SK/P-TEFb small nuclear RNP (snRNP) has been detected at the HIV-1 core promoter as well as at the promoters of cellular genes, but a recruiting mechanism still remains unknown to date. Here we show global synergy between CTIP2 and the 7SK-binding chromatin master-regulator HMGA1 in terms of P-TEFb-dependent endogenous and HIV-1 gene expression regulation. While CTIP2 and HMGA1 concordingly repress the expression of cellular 7SK-dependent P-TEFb targets, the simultaneous knock-down of CTIP2 and HMGA1 also results in a boost in Tat-dependent and independent HIV-1 promoter activity. Chromatin immunoprecipitation experiments reveal a significant loss of CTIP2/7SK/P-TEFb snRNP recruitment to cellular gene promoters and the HIV-1 promoter on HMGA1 knock-down. Our findings not only provide insights into a recruiting mechanism for the inactive 7SK/P-TEFb snRNP, but may also contribute to a better understanding of viral latency.

CTIP2 is a negative regulator of P-TEFb.

The positive transcription elongation factor b (P-TEFb) is involved in physiological and pathological events including inflammation, cancer, AIDS, and cardiac hypertrophy. The balance between its active and inactive form is tightly controlled to ensure cellular integrity. We report that the transcriptional repressor CTIP2 is a major modulator of P-TEFb activity. CTIP2 copurifies and interacts with an inactive P-TEFb complex containing the 7SK snRNA and HEXIM1. CTIP2 associates directly with HEXIM1 and, via the loop 2 of the 7SK snRNA, with P-TEFb. In this nucleoprotein complex, CTIP2 significantly represses the Cdk9 kinase activity of P-TEFb. Accordingly, we show that CTIP2 inhibits large sets of P-TEFb- and 7SK snRNA-sensitive genes. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Overexpression of the ß-myosin heavy chain protein contributes to the pathological cardiac wall thickening. The inactive P-TEFb complex associates with CTIP2 at the MYH7 gene promoter to repress its activity. Taken together, our results strongly suggest that CTIP2 controls P-TEFb function in physiological and pathological conditions.

The many lives of CTIP2: from AIDS to cancer and cardiac hypertrophy.

CTIP2 is a key transcriptional regulator involved in numerous physiological functions. Initial works have shown the importance of CTIP2 in the establishment and persistence of HIV latency in microglial cells, the main latent/quiescent viral reservoir in the brain. Recent studies have highlighted the importance of CTIP2 in several other pathologies, such as cardiac hypertrophy and various types of human malignancies. Targeting CTIP2 may therefore constitute a new approach in the treatment of these pathologies.

LSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing.

Microglial cells are the main HIV-1 targets in the central nervous system (CNS) and constitute an important reservoir of latently infected cells. Establishment and persistence of these reservoirs rely on the chromatin structure of the integrated proviruses. We have previously demonstrated that the cellular cofactor CTIP2 forces heterochromatin formation and HIV-1 gene silencing by recruiting HDAC and HMT activities at the integrated viral promoter. In the present work, we report that the histone demethylase LSD1 represses HIV-1 transcription and viral expression in a synergistic manner with CTIP2. We show that recruitment of LSD1 at the HIV-1 proximal promoter is associated with both H3K4me3 and H3K9me3 epigenetic marks. Finally, our data suggest that LSD1-induced H3K4 trimethylation is linked to hSET1 recruitment at the integrated provirus.

[CTIP2, a multifunctional protein: cellular physiopathology and therapeutic implications]. / CTIP2, une protéine multifonctionnelle - implication en physiopathologie cellulaire et en thérapeutique.

The transcription factor CTIP2 (BCL11B) is a multifunctional protein involved in numerous cell physiological processes. To date, many molecular mechanisms underlying this process have been discovered, which highlighted the importance of the epigenetic regulation of genes and the regulation of the elongation factor P-TEFb. Furthermore studies of the deregulation of CTIP2 showed the association of CTIP2 to numerous pathologies including cancer and cardiac hypertrophy. A better comprehension of the physiopathology of these diseases might lead to the design of therapeutical strategies intending to prevent CTIP2 deregulation. Moreover, CTIP2 and its associated proteins constitute potential targets in strategies aiming to reduce and/or purge HIV-1 cell reservoirs.

Pseudomonas DING proteins as human transcriptional regulators and HIV-1 antagonists.

BACKGROUND: Anti-HIV-1 therapy depends upon multiple agents that target different phases of the viral replication cycle. Recent reports indicate that plant and human DING proteins are unique in targeting viral gene transcription as the basis of their anti-HIV-1 therapy. METHODS: Two cloned DING genes from Pseudomonas were transiently expressed in human cells, and effects on NFκB-mediated transcription, HIV-1 transcription, and HIV-1 production were measured. RESULTS: Both DING proteins elevated NFκB-mediated transcription. In microglial cells, one protein, from P. aeruginosa PA14, suppressed HIV-1 transcription; the other protein, from P. fluorescens SBW25, was inactive. The PA14DING protein also reduces HIV-1 production in microglial cells. CONCLUSIONS: Structural differences between the two DING proteins highlight regions of the PA14DING protein essential to the anti-HIV-1 activity, and may guide the design of therapeutic agents.

Chromatin disruption in the promoter of bovine leukemia virus during transcriptional activation.

Bovine leukemia virus expression relies on its chromatin organization after integration into the host cell genome. Proviral latency, which results from transcriptional repression in vivo, represents a viral strategy to escape the host immune system and likely allows for tumor progression. Here, we discriminated two types of latency: an easily reactivable latent state of the YR2 provirus and a 'locked' latent state of the L267 provirus. The defective YR2 provirus was characterized by the presence of nuclease hypersensitive sites at the U3/R junction and in the R/U5 region of the 5'-long terminal repeat (5'-LTR), whereas the L267 provirus displayed a closed chromatin configuration at the U3/R junction. Reactivation of viral expression in YR2 cells by the phorbol 12-myristate 13-acetate (PMA) plus ionomycin combination was accompanied by a rapid but transient chromatin remodeling in the 5'-LTR, leading to an increased PU.1 and USF-1/USF-2 recruitment in vivo sustained by PMA/ionomycin-mediated USF phosphorylation. In contrast, viral expression was not reactivated by PMA/ionomycin in L267 cells, because the 5'-LTR U3/R region remained inaccessible to nucleases and hypermethylated at CpG dinucleotides. Remarkably, we elucidated the BLV 5'-LTR chromatin organization in PBMCs isolated from BLV-infected cows, thereby depicting the virus hiding in vivo in its natural host.

Targeting and eradicating latent CNS reservoirs of HIV-1: Original strategies and new models.

Nowadays, combination antiretroviral therapy (cART) is the standard treatment for all people with human immunodeficiency virus (HIV-1). Although cART is effective in treating productive infection, it does not eliminate latent reservoirs of the virus. This leads to lifelong treatment associated with the occurrence of side effects and the development of drug-resistant HIV-1. Suppression of viral latency is therefore the major hurdle to HIV-1 eradication. Multiple mechanisms exist to regulate viral gene expression and drive the transcriptional and post-transcriptional establishment of latency. Epigenetic processes are amongst the most studied mechanisms influencing both productive and latent infection states. The central nervous system (CNS) represents a key anatomical sanctuary for HIV and is the focal point of considerable research efforts. However, limited and difficult access to CNS compartments makes understanding the HIV-1 infection state in latent brain cells such as microglial cells, astrocytes, and perivascular macrophages challenging. This review examines the latest advances on epigenetic transformations involved in CNS viral latency and targeting of brain reservoirs. Evidence from clinical studies as well as in vivo and in vitro models of HIV-1 persistence in the CNS will be discussed, with a special focus on recent 3D in vitro models such as human brain organoids. Finally, the review will address therapeutic considerations for targeting latent CNS reservoirs.

Corrigendum: Editorial: The relevance of molecular mechanisms in HIV-1 latency and reactivation from latency.

[This corrects the article DOI: 10.3389/fcimb.2023.1190867.].

Engineering a versatile and retrievable cell macroencapsulation device for the delivery of therapeutic proteins.

Encapsulated cell therapy holds a great potential to deliver sustained levels of highly potent therapeutic proteins to patients and improve chronic disease management. A versatile encapsulation device that is biocompatible, scalable, and easy to administer, retrieve, or replace has yet to be validated for clinical applications. Here, we report on a cargo-agnostic, macroencapsulation device with optimized features for protein delivery. It is compatible with adherent and suspension cells, and can be administered and retrieved without burdensome surgical procedures. We characterized its biocompatibility and showed that different cell lines producing different therapeutic proteins can be combined in the device. We demonstrated the ability of cytokine-secreting cells encapsulated in our device and implanted in human skin to mobilize and activate antigen-presenting cells, which could potentially serve as an effective adjuvant strategy in cancer immunization therapies. We believe that our device may contribute to cell therapies for cancer, metabolic disorders, and protein-deficient diseases.

Erratum: Immortalized human myoblast cell lines for the delivery of therapeutic proteins using encapsulated cell technology.

[This corrects the article DOI: 10.1016/j.omtm.2022.07.017.].

Achieving a cure for HIV infection: do we have reasons to be optimistic?

The introduction of highly active antiretroviral therapy (HAART) in 1996 has transformed a lethal disease to a chronic pathology with a dramatic decrease in mortality and morbidity of AIDS-related symptoms in infected patients. However, HAART has not allowed the cure of HIV infection, the main obstacle to HIV eradication being the existence of quiescent reservoirs. Several other problems have been encountered with HAART (such as side effects, adherence to medication, emergence of resistance and cost of treatment), and these motivate the search for new ways to treat these patients. Recent advances hold promise for the ultimate cure of HIV infection, which is the topic of this review. Besides these new strategies aiming to eliminate the virus, efforts must be made to improve current HAART. We believe that the cure of HIV infection will not be attained in the short term and that a strategy based on purging the reservoirs has to be associated with an aggressive HAART strategy.

[DING proteins, their biochemical and structural properties, and their ability to inhibit HIV-1 replication]. / Les protéines DING: propriétés biochimiques, structurales, et capacité a inhiber la réplication du virus VIH-1.

DING proteins comprise an intriguing phosphate-binding protein family present in all animal phyla. Five different DING representatives have been described in humans. Eukaryotic DING proteins are mostly involved in cellular processes such as cell cycle regulation, and also in pathological process such as rheumatoid arthritis and kidney stone formation. Although these proteins are ubiquitous in eukaryotes, no relevant locus or ORF has yet been found in sequenced genomes. This lack of sequence information has considerably hampered functional and structural studies of these proteins, and has required the use of novel and original techniques such as ab initio protein sequencing based on a combination of X-ray crystallography and mass spectrometry. Sub-Angstrom structural resolution has elucidated the molecular binding mechanism of phosphate ions by these high-affinity proteins. Immunohistochemical studies show that these proteins are present in a wide variety of mouse tissues. Some DING proteins, particularly human phosphate binding protein (HPBP), can inhibit HIV replication. This inhibition takes place at the transcriptional step, which is not targeted by any current antiretroviral drug. Initial studies suggest that HPBP warrants animal testing. This recent discovery opens new possibilities for the treatment of HIV infection.