Immediate-early Transcription Research Articles

Evaluation of a Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)-Based Microneutralization Assay for Assessing Clinical Human Cytomegalovirus-Neutralizing Antibody Activity.

Development of a vaccine for human cytomegalovirus (hCMV) is critical because of the severe consequences of infection in congenitally infected newborns and immunocompromised patients. The assessment of hCMV-neutralizing antibody activity is crucial for vaccine development. This study evaluated a RT-qPCR assay targeting the immediate-early gene transcript of hCMV for determining microneutralizing antibody activity. The assay was evaluated for sensitivity, specificity, and precision using endotheliotropic clinical isolate VR1814 that infects fibroblasts, epithelial, and endothelial cells. The RT-qPCR-based neutralization assay was compared with an immunostaining-based neutralization assay using virions present in hCMV-positive urine, saliva, and breast-milk samples. Our results showed that hCMV replication was detectable at 20 h post-infection with a limit of detection of 1 infectious units (IU)/reaction. The RT-qPCR assay had a dynamic range of 1 to 1.0 × 104 IU/reaction, with coefficients of variation ranging from 0.94% to 15.08%. The RT-qPCR results were in high agreement with the immunostaining assay for hCMV-antibody neutralization assessment. Overall, the RT-qPCR neutralization assay is a reliable, rapid, efficient, and sensitive alternative method for evaluating hCMV-neutralizing activity in vitro.

Upregulation of IQGAP2 by EBV transactivator Rta and its influence on EBV life cycle.

Epstein-Barr virus (EBV) is a human oncogenic γ-herpesvirus that establishes persistent infection in more than 90% of the world's population. EBV has two life cycles, latency and lytic replication. Reactivation of EBV from latency to the lytic cycle is initiated and controlled by two viral immediate-early transcription factors, Zta and Rta, encoded by BZLF1 and BRLF1, respectively. In this study, we found that IQGAP2 expression was elevated in EBV-infected B cells and identified Rta as a viral gene responsible for the IQGAP2 upregulation in both B cells and nasopharyngeal carcinoma cell lines. Mechanistically, we showed that Rta increases IQGAP2 expression through direct binding to the Rta-responsive element in the IQGAP2 promoter. We also demonstrated the direct interaction between Rta and IQGAP2 as well as their colocalization in the nucleus. Functionally, we showed that the induced IQGAP2 is required for the Rta-mediated Rta promoter activation in the EBV lytic cycle progression and may influence lymphoblastoid cell line clumping morphology through regulating E-cadherin expression. IMPORTANCE Elevated levels of antibodies against EBV lytic proteins and increased EBV DNA copy numbers in the sera have been reported in patients suffering from Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma, indicating that EBV lytic cycle progression may play an important role in the pathogenesis of EBV-associated diseases and highlighting the need for a more complete mechanistic understanding of the EBV lytic cycle. Rta acts as an essential transcriptional activator to induce lytic gene expression and thus trigger EBV reactivation. In this study, scaffolding protein IQGAP2 was found to be upregulated prominently following EBV infection via the direct binding of Rta to the RRE in the IQGAP2 promoter but not in response to other biological stimuli. Importantly, IQGAP2 was demonstrated to interact with Rta and promote the EBV lytic cycle progression. Suppression of IQGAP2 was also found to decrease E-cadherin expression and affect the clumping morphology of lymphoblastoid cell lines.

Histone H3 phospho-regulation by KimH3 in both interphase and mitosis.

Histone H3 is phosphorylated at Ser10 by multiple kinases, and many of them are anti-cancer targets. Here, we report the first kinase that can phosphorylate H3Ser10 in both interphase and mitosis, which we named KimH3 (kinase of interphase and mitotic Histone H3). Meta-analysis indicates that KimH3 is upregulated in a broad spectrum of human cancers and its high expression is correlated with reduced the median survival time of cancer patients. In mitosis, CDK1 phosphorylates KimH3, which then phosphorylates H3Ser10 to regulate cell cycle procession. In interphase, EGF induces KimH3 activation and H3Ser10 phosphorylation, which is involved in MAPK-ERK1/2 signaling pathway to activate immediate-early genes transcription. Consequently, a small molecule inhibitor of KimH3 significantly inhibited tumor growth in mice. This is not only consistent with the dual roles of KimH3 in both interphase and mitotic Histone H3 phosphorylation, but also reveals it as an important potential anti-cancer target.

PMON56 LH synthesis is regulated by RNA-binding protein ZFP36 bound with AU-rich elements in gonadotropes

Abstract Gonadotropin secretion, which is stimulated by gonadotropin-releasing hormone (GnRH), is a critical feature of reproductive control and maintenance of fertility. GnRH stimulation increases LH secretion 50-100-fold over baseline per pulse, whereas Lhb gene expression increases a modest 1.4-fold. This observation shows that significant post-transcriptional regulation of gonadotropin synthesis and secretion must occur. We have previously shown that GnRH-induced Dusp1 expression, a factor central for feedback control of GnRH signaling in gonadotropes, depends on interaction of the 3'UTR of its mRNA with the RNA binding protein (RBP) ELAVL1, which binds AU-rich elements (ARE) in target mRNAs (1). Recently, using RIP-Chip analysis, we have shown that GnRH receptor (Gnrhr) expression depends on interaction of its mRNA with ELAVL1. Gnrhr mRNA is among the most highly represented ELAVL1-associated mRNAs (2). Further, many mRNAs encoding proteins essential to gonadotropin gene expression, including the immediate-early transcription factor genes Egr1, 2, and 4, Fos, Fosb, Jun, and Junb, as well as Dusp1 and 2, show increased binding to ELAVL1 after GnRH treatment, although gonadotropin Lhb mRNA is not, due to the absence of ARE site to be bound. Typically, association with ELAVL1 stabilize expression of target mRNAs, promoting their translation. Also, of interest is the role of the RBP ZFP36, which competes with ELAVL1 for binding to ARE's but has the opposing effect of directing mRNA to the degradation pathway. Significantly, 5 minutes pulsatile GnRH stimulated ZFP36 protein production in 30-60 minutes in LbetaT2 cells. And, knockdown of ELAVL1 results in the deterioration of Gnrhr expression and of the immediate-early gene Egr1 a transcription factor central to Lhb expression. We have examined the direct role of ZFP36 protein in GnRH-induced gene expression. Interestingly, Gnrhr and Lhb mRNA expression levels were decreased by ZFP36 overexpression in LbetaT2 cells compared with control cells. Also, GnRH-stimulated EGR1 production by was decreased by ZFP36 overexpression. These observations are consistent with previous results with ELAVL1 knockdown showing decreased mRNA stability. We cloned Egr1, Gnrhr and Zfp36 3'UTRs which contain AREs bound by ELAVL1 into luciferase reporter genes in pmirGLO vector to evaluate whether the 3'UTR is directly affected by ZFP36. These Egr1, Gnrhr and Zfp36 3'UTR contained mirGLOs were transfected in both ZFP36 overexpression and control cells and luciferase activity was monitored. The results showed that luciferase expression from reporters bearing the target 3'UTR were significantly reduced in ZFP36 overexpressed cells. Collectively, our studies show that ELAVL1, ZFP36 and their target mRNAs are regulated by GnRH and this indirectly regulates Lhb gene expression in gonadotropes. ) Do MT, T Kim et al. Mol Cell Endocrinology 382 (1): 1346-1357, 2014 ) Terasaka T et al. Endocrinology 160(8): 1999-2014, 2019 Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function.

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor–binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.

Abstract P2032: Cardiac Nuclear Receptor Subfamily 4 Group A Member 2 Activates Cell Cycle Progression And Induces Heart Failure

Transcription factors play a fundamental role in cardiovascular adaptation to stress. Nuclear receptor subfamily 4 group A member 2 (NR4A2; NURR1) is an immediate-early gene and transcription factor with a versatile role throughout many organs. In the adult mammalian heart and particularly in cardiac myocytes, NR4A2 is strongly up-regulated in response to beta-adrenergic stimulation. The physiological implications of this increase remain unknown. In this study, we aimed to interrogate the consequences of cardiac NR4A2 up-regulation under normal conditions and in response to pressure overload. In mice, tamoxifen-dependent, cardiomyocyte-restricted overexpression of NR4A2 led to left ventricular hypertrophy and dilation, heart failure, and death within 40 days. Chronic NR4A2 induction also precipitated cardiac decompensation during transverse aortic constriction (TAC)-induced pressure overload. Mechanistically, NR4A2 caused adult cardiac myocytes to return to a fetal-like phenotype, with a switch to glycolytic metabolism and disassembly of sarcomeric structures. NR4A2 also re-activated cell cycle progression and stimulated DNA replication and karyokinesis but failed to induce cytokinesis, thereby promoting polyploidization and multinucleation of cardiac myocytes. Activation of cell cycle checkpoints led to induction of an apoptotic response which ultimately resulted in excessive loss of cardiac myocytes and impaired left ventricular contractile function. In summary, myocyte-specific overexpression of NR4A2 in the postnatal mammalian heart results in increased cell cycle re-entry and DNA replication but does not result in cardiac myocyte division. Our findings expose a novel function for the nuclear receptor as a critical regulator in the self-renewal of the cardiac myocyte and heart regeneration.

Prolonged cardiac NR4A2 activation causes dilated cardiomyopathy in mice

Transcription factors play a fundamental role in cardiovascular adaptation to stress. Nuclear receptor subfamily 4 group A member 2 (NR4A2; NURR1) is an immediate-early gene and transcription factor with a versatile role throughout many organs. In the adult mammalian heart, and particularly in cardiac myocytes, NR4A2 is strongly up-regulated in response to beta-adrenergic stimulation. The physiologic implications of this increase remain unknown. In this study, we aimed to interrogate the consequences of cardiac NR4A2 up-regulation under normal conditions and in response to pressure overload. In mice, tamoxifen-dependent, cardiomyocyte-restricted overexpression of NR4A2 led to cardiomyocyte hypertrophy, left ventricular dilation, heart failure, and death within 40 days. Chronic NR4A2 induction also precipitated cardiac decompensation during transverse aortic constriction (TAC)-induced pressure overload. Mechanistically, NR4A2 caused adult cardiac myocytes to return to a fetal-like phenotype, with a switch to glycolytic metabolism and disassembly of sarcomeric structures. NR4A2 also re-activated cell cycle progression and stimulated DNA replication and karyokinesis but failed to induce cytokinesis, thereby promoting multinucleation of cardiac myocytes. Activation of cell cycle checkpoints led to induction of an apoptotic response which ultimately resulted in excessive loss of cardiac myocytes and impaired left ventricular contractile function. In summary, myocyte-specific overexpression of NR4A2 in the postnatal mammalian heart results in increased cell cycle re-entry and DNA replication but does not result in cardiac myocyte division. Our findings expose a novel function for the nuclear receptor as a critical regulator in the self-renewal of the cardiac myocyte and heart regeneration.

Single-cell deletion analyses show control of pro-T cell developmental speed and pathways by Tcf7, Spi1, Gata3, Bcl11a, Erg, and Bcl11b.

As early T cell precursors transition from multipotentiality to T lineage commitment, they change expression of multiple transcription factors. It is unclear whether individual transcription factors directly control choices between T cell identity and some alternative fate or whether these factors mostly affect proliferation or survival during the normal commitment process. Here, we unraveled the impacts of deleting individual transcription factors at two stages in early T cell development, using synchronized in vitro differentiation systems, single-cell RNA-seq with batch indexing, and controlled gene-disruption strategies. First, using a customized method for single-cell CRISPR disruption, we defined how the early-acting transcription factors Bcl11a, Erg, Spi1 (PU.1), Gata3, and Tcf7 (TCF1) function before commitment. The results revealed a kinetic tug of war within individual cells between T cell factors Tcf7 and Gata3 and progenitor factors Spi1 and Bcl11a, with an unexpected guidance role for Erg. Second, we tested how activation of transcription factor Bcl11b during commitment altered ongoing cellular programs. In knockout cells where Bcl11b expression was prevented, the cells did not undergo developmental arrest, instead following an alternative path as T lineage commitment was blocked. A stepwise, time-dependent regulatory cascade began with immediate-early transcription factor activation and E protein inhibition, finally leading Bcl11b knockout cells toward exit from the T cell pathway. Last, gene regulatory networks of transcription factor cross-regulation were extracted from the single-cell transcriptome results, characterizing the specification network operating before T lineage commitment and revealing its links to both the Bcl11b knockout alternative network and the network consolidating T cell identity during commitment.

P. aeruginosa type III and type VI secretion systems modulate early response gene expression in type II pneumocytes in vitro

BackgroundLung airway epithelial cells are part of innate immunity and the frontline of defense against bacterial infections. During infection, airway epithelial cells secrete proinflammatory mediators that participate in the recruitment of immune cells. Virulence factors expressed by bacterial pathogens can alter epithelial cell gene expression and modulate this response. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, expresses numerous virulence factors to facilitate establishment of infection and evade the host immune response. This study focused on identifying the role of two major P. aeruginosa virulence factors, type III (T3SS) and type VI (T6SS) secretion systems, on the early transcriptome response of airway epithelial cells in vitro.ResultsWe performed RNA-seq analysis of the transcriptome response of type II pneumocytes during infection with P. aeruginosa in vitro. We observed that P. aeruginosa differentially upregulates immediate-early response genes and transcription factors that induce proinflammatory responses in type II pneumocytes. P. aeruginosa infection of type II pneumocytes was characterized by up-regulation of proinflammatory networks, including MAPK, TNF, and IL-17 signaling pathways. We also identified early response genes and proinflammatory signaling pathways whose expression change in response to infection with P. aeruginosa T3SS and T6SS mutants in type II pneumocytes. We determined that T3SS and T6SS modulate the expression of EGR1, FOS, and numerous genes that are involved in proinflammatory responses in epithelial cells during infection. T3SS and T6SS were associated with two distinct transcriptomic signatures related to the activation of transcription factors such as AP1, STAT1, and SP1, and the secretion of pro-inflammatory cytokines such as IL-6 and IL-8.ConclusionsTaken together, transcriptomic analysis of epithelial cells indicates that the expression of immediate-early response genes quickly changes upon infection with P. aeruginosa and this response varies depending on bacterial viability and injectosomes. These data shed light on how P. aeruginosa modulates host epithelial transcriptome response during infection using T3SS and T6SS.

Bivalent-histone-marked immediate-early gene regulation is vital for VEGF-responsive angiogenesis.

Endothelial cells (ECs) are phenotypically heterogeneous, mainly due to their dynamic response to the tissue microenvironment. Vascular endothelial cell growth factor (VEGF), the best-known angiogenic factor, activates calcium-nuclear factor of activated Tcells (NFAT) signaling following acute angiogenic gene transcription. Here, we evaluate the global mapping of VEGF-mediated dynamic transcriptional events, focusing on major histone-code profiles using chromatin immunoprecipitation sequencing (ChIP-seq). Remarkably, the gene loci of immediate-early angiogenic transcription factors (TFs) exclusively acquire bivalent H3K4me3-H3K27me3 double-positive histone marks after the VEGF stimulus. Moreover, NFAT-associated Pax transactivation domain-interacting protein (PTIP) directs bivalently marked TF genes transcription through a limited polymerase II running. The non-canonical polycomb1 variant PRC1.3 specifically binds to and allows the transactivation of PRC2-enriched bivalent angiogenic TFs until conventional PRC1-mediated gene silencing is achieved. Knockdown of these genes abrogates post-natal aberrant neovessel formation via the selective inhibition of indispensable bivalent angiogenic TF gene transcription. Collectively, the reported dynamic histone mark landscape may uncover the importance of immediate-early genes and the development of advanced anti-angiogenic strategies.

Epstein-Barr Virus miR-BHRF1-3 Targets the BZLF1 3'UTR and Regulates the Lytic Cycle.

Suppression of lytic viral gene expression is a key aspect of the Epstein-Barr virus (EBV) life cycle to facilitate the establishment of latent infection. Molecular mechanisms regulating transitions between EBV lytic replication and latency are not fully understood. Here, we investigated the impact of viral microRNAs on the EBV lytic cycle. Through functional assays, we found that miR-BHRF1-3 attenuates EBV lytic gene expression following reactivation. To understand the miRNA targets contributing to this activity, we performed Ago PAR-CLIP analysis on EBV-positive, reactivated Burkitt's lymphoma cells and identified multiple miR-BHRF1-3 interactions with viral transcripts. Using luciferase reporter assays, we confirmed a miRNA interaction site within the 3'UTR of BZLF1 which encodes the essential immediate early (IE) transactivator Zta. Comparison of >850 published EBV genomes identified sequence polymorphisms within the miR-BHRF1-3 locus that deleteriously affect miRNA expression and function. Molecular interactions between the homologous viral miRNA, miR-rL1-17, and IE transcripts encoded by rhesus lymphocryptovirus were further identified. Our data demonstrate that regulation of IE gene expression by a BHRF1 miRNA is conserved among lymphocryptoviruses, and further reveal virally-encoded genetic elements that orchestrate viral antigen expression during the lytic cycle. IMPORTANCE Epstein-Barr virus infection is predominantly latent in healthy individuals, while periodic cycles of reactivation are thought to facilitate persistent lifelong infection. Lytic infection has been linked to development of certain EBV-associated diseases. Here, we demonstrate that EBV miR-BHRF1-3 can suppress lytic replication by directly inhibiting Zta expression. Moreover, we identify nucleotide variants that impact the function of miR-BHRF1-3, which may contribute to specific EBV pathologies.

Does the Zinc Finger Antiviral Protein (ZAP) Shape the Evolution of Herpesvirus Genomes?

An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses, reducing their fitness, and ultimately minimising pathogenic effects. In turn, viruses have evolved sophisticated counter-measures that mediate evasion of host defence mechanisms. A key aspect of host defences is the ability to differentiate between self and non-self. Previous studies have demonstrated significant suppression of CpG and UpA dinucleotide frequencies in the coding regions of RNA and small DNA viruses. Artificially increasing these dinucleotide frequencies results in a substantial attenuation of virus replication, suggesting dinucleotide bias could facilitate recognition of non-self RNA. The interferon-inducible gene, zinc finger antiviral protein (ZAP) is the host factor responsible for sensing CpG dinucleotides in viral RNA and restricting RNA viruses through direct binding and degradation of the target RNA. Herpesviruses are large DNA viruses that comprise three subfamilies, alpha, beta and gamma, which display divergent CpG dinucleotide patterns within their genomes. ZAP has recently been shown to act as a host restriction factor against human cytomegalovirus (HCMV), a beta-herpesvirus, which in turn evades ZAP detection by suppressing CpG levels in the major immediate-early transcript IE1, one of the first genes expressed by the virus. While suppression of CpG dinucleotides allows evasion of ZAP targeting, synonymous changes in nucleotide composition that cause genome biases, such as low GC content, can cause inefficient gene expression, especially in unspliced transcripts. To maintain compact genomes, the majority of herpesvirus transcripts are unspliced. Here we discuss how the conflicting pressures of ZAP evasion, the need to maintain compact genomes through the use of unspliced transcripts and maintaining efficient gene expression may have shaped the evolution of herpesvirus genomes, leading to characteristic CpG dinucleotide patterns.

Persistent increases of PKMζ in memory-activated neurons trace LTP maintenance during spatial long-term memory storage.

PKMζ is an autonomously active PKC isoform crucial for the maintenance of synaptic long-term potentiation (LTP) and long-term memory. Unlike other kinases that are transiently stimulated by second messengers, PKMζ is persistently activated through sustained increases in protein expression of the kinase. Therefore, visualizing increases in PKMζ expression during long-term memory storage might reveal the sites of its persistent action and thus the location of memory-associated LTP maintenance in the brain. Using quantitative immunohistochemistry validated by the lack of staining in PKMζ-null mice, we examined the amount and distribution of PKMζ in subregions of the hippocampal formation of wild-type mice during LTP maintenance and spatial long-term memory storage. During LTP maintenance in hippocampal slices, PKMζ increases in the pyramidal cell body and stimulated dendritic layers of CA1 for at least 2hr. During spatial memory storage, PKMζ increases in CA1 pyramidal cells for at least 1month, paralleling the persistence of the memory. During the initial expression of the memory, we tagged principal cells with immediate-early gene Arc promoter-driven transcription of fluorescent proteins. The subset of memory-tagged CA1 cells selectively increases expression of PKMζ during memory storage, and the increase persists in dendritic compartments within stratum radiatum for 1month, indicating long-term storage of information in the CA3-to-CA1 pathway. We conclude that persistent increases in PKMζ trace the molecular mechanism of LTP maintenance and thus the sites of information storage within brain circuitry during long-term memory.

A long noncoding RNA antisense to ICAM-1 is involved in allergic asthma associated hyperreactive response of airway epithelial cells.

Epithelial cells of the conducting airways are a pivotal first line of defense against airborne pathogens and allergens that orchestrate inflammatory responses and mucociliary clearance. Nonetheless, the molecular mechanisms responsible for epithelial hyperreactivity associated with allergic asthma are not completely understood. Transcriptomic analysis of human airway epithelial cells (HAECs), differentiated in-vitro at air-liquid interface (ALI), showed 725 differentially expressed immediate-early transcripts, including putative long noncoding RNAs (lncRNAs). A novel lncRNA on the antisense strand of ICAM-1 or LASI was identified, which was induced in LPS-primed HAECs along with mucin MUC5AC and its transcriptional regulator SPDEF. LPS-primed expression of LASI, MUC5AC, and SPDEF transcripts were higher in ex-vivo cultured asthmatic HAECs that were further augmented by LPS treatment. Airway sections from asthmatics with increased mucus load showed higher LASI expression in MUC5AC+ goblet cells following multi-fluorescent in-situ hybridization and immunostaining. LPS- or IL-13-induced LASI transcripts were mostly enriched in the nuclear/perinuclear region and were associated with increased ICAM-1, IL-6, and CXCL-8 expression. Blocking LASI expression reduced the LPS or IL-13-induced epithelial inflammatory factors andMUC5AC expression, suggesting that the novel lncRNA LASI could play a key role in LPS-primed trained airway epithelial responses that are dysregulated in allergic asthma.

Human cytomegalovirus major immediate early transcripts arise predominantly from the canonical major immediate early promoter in reactivating progenitor-derived dendritic cells.

Human cytomegalovirus latency and reactivation is a major source of morbidity in immune-suppressed patient populations. Lifelong latent infections are established in CD34+progenitor cells in the bone marrow, which are hallmarked by a lack of major lytic gene expression, genome replication and virus production. A number of studies have shown that inhibition of the major immediate early promoter (MIEP) – the promoter that regulates immediate early (IE) gene expression – is important for the establishment of latency and that, by extension, reactivation requires reversal of this repression of the MIEP. The identification of novel promoters (termed ip1 and ip2) downstream of the MIEP that can drive IE gene expression has led to speculation over the precise role of the MIEP in reactivation. In this study we show that IE transcripts arise from both the MIEP and ip2 promoter in the THP1 cell macrophage cell line and also CD14+monocytes stimulated with phorbol ester. In contrast, we show that in in vitro generated dendritic cells or macrophages that support HCMV reactivation IE transcripts arise predominantly from the MIEP and not the intronic promoters. Furthermore, inhibition of histone modifying enzyme activity confirms the view that the MIEP is predominantly regulated by the activity of cellular chromatin. Finally, we observe that ip2-derived IE transcription is cycloheximide-sensitive in reactivating DCs, behaviour consistent with an early gene designation. Taken together, these data argue that MIEP activity is still important for HCMV reactivation but ip2 activity could play cell-type-specific roles in reactivation.

Evaluation on the antiviral activity of genipin against white spot syndrome virus in crayfish

White spot syndrome virus (WSSV) is a serious epidemic pathogen of crustaceans and cause severe economic losses to aquaculture. However, no commercial drugs presently available to control WSSV infection. Genipin (GN) is a bioactive compound extracted from the fruit of Gardenia jasminoides and exhibits potential antiviral activity. In the study, the antiviral activity of GN against WSSV was investigated in crayfish Procambarus clarkii and in shrimp Litopenaeus vannamei. In vitro antiviral test showed that GN could inhibit WSSV replication in crayfish and in shrimp, and the highest inhibition on WSSV was over 99% when treatment with 50 mg/kg of GN for 24 h. In vivo antiviral test proved that GN could be used to treat and prevent WSSV infection. GN could also effectively protect crayfish from WSSV infection by reducing the mortality rate of WSSV-infected crayfish. Moreover, GN attenuated the WSSV-induced oxidative stress and inflammatory by upregulation the expression of antioxidant-related genes and downregulation the expression of inflammatory-related genes, respectively. Mechanically, GN inhibited WSSV replication at least via decreasing STAT (signal transducer and activator of transcription) gene expression to block WSSV immediate-early gene ie1 transcription. Additionally, the inhibition of BI-1 (Bax inhibitor-1) gene expression also played an important role in the suppression of WSSV infection. In conclusion, GN represented a potential therapeutic and preventive agent to block WSSV infection.

Transcripts expressed in cytomegalovirus latency coding for an antigenic IE/E phase peptide that drives "memory inflation".

Roizman's definition of herpesviral latency, which applies also to cytomegaloviruses (CMVs), demands maintenance of reactivation-competent viral genomes after clearance of productive infection. It is more recent understanding that failure to complete the productive viral cycle for virus assembly and release does not imply viral gene silencing at all genetic loci and all the time. It rather appears that CMV latency is transcriptionally "noisy" in that silenced viral genes get desilenced from time to time in a stochastic manner, leading to "transcripts expressed in latency" (TELs). If a TEL happens to code for a protein that contains a CD8 T cell epitope, protein processing can lead to the presentation of the antigenic peptide and restimulation of cognate CD8 T cells during latency. This mechanism is discussed as a potential driver of epitope-selective accumulation of CD8 T cells over time, a phenomenon linked to CMV latency and known as "memory inflation" (MI). So far, expression of an epitope-encoding TEL was shown only for the major immediate-early (MIE) gene m123/ie1 of murine cytomegalovirus (mCMV), which codes for the prototypic MI-driving antigenic peptide YPHFMPTNL that is presented by the MHC class-I molecule Ld. The only known second MI-driving antigenic peptide of mCMV in the murine MHC haplotype H-2d is AGPPRYSRI presented by the MHC-I molecule Dd. This peptide is very special in that it is encoded by the early (E) phase gene m164 and by an overlapping immediate-early (IE) transcript governed by a promoter upstream of m164. If MI is driven by presentation of TEL-derived antigenic peptides, as the hypothesis says, one should find corresponding TELs. We show here that E-phase and IE-phase transcripts that code for the MI-driving antigenic peptide AGPPRYSRI are independently and stochastically expressed in latently infected lungs.

Gardenia jasminoides Ellis inhibit white spot syndrome virus replication in red swamp crayfish Procambarus clarkii

As one of the most dangerous pathogens in the aquaculture of crustaceans, white spot syndrome virus (WSSV) induces a high mortality rate and huge economic losses in crustacean industries. Up to now, there are no commercially available drugs to control the virus. Thus, it is important to find an antiviral drug to combat highly lethal WSSV outbreaks. In the present study, the anti-WSSV activities of 23 medicinal plant extracts were investigated in crayfish Procambarus clarkii. In order to better evaluate the antiviral activities of plant extracts, it was important to build a WSSV infection model in crayfish first. The results of WSSV infection showed that WSSV could infect most tissues of crayfish, and the heaviest infected tissue is the gills. The time course curve of WSSV amount change in crayfish assumed a very similar shape with the typical viral one-step growth curve. On the basis of virus infection model, the anti-WSSV activities of 23 plant extracts were investigated. Among these plants, Gardenia jasminoides showed the highest inhibition on WSSV replication, with 92.31%. A further study showed that G. jasminoides improved survival rate of WSSV-challenged crayfish. Mechanistically, G. jasminoides reduced Hsp70 expression and virus immediate-early gene ie1, early gene DNApol, late gene VP28 transcript, indicating that G. jasminoides inhibited WSSV replication via blocking the immediate-early stage gene ie1 transcript. Furthermore, the expression of antioxidants (cMnSOD, mMnSOD, CAT and GST) and apoptosis-related factors (Bax and Bax-inhibitor-1) were significantly increased in G. jasminoides-treated crayfish, suggesting that G. jasminoides could modulate apoptosis-related factors and possessed anti-oxidative activity in crayfish. Taken together, G. jasminoides could inhibit WSSV replication and improve survival rate of WSSV-challenged crayfish. This outcome offers a scientific basis for the use of extracts of G. jasminoides against WSSV, and it is worth focusing on the isolation and identification of effective active compounds from G. jasminoides.

The Cellular Coactivator HCF-1 Is Required for Glucocorticoid Receptor-Mediated Transcription of Bovine Herpesvirus 1 Immediate Early Genes.

Following productive infection, bovine herpesvirus 1 (BoHV-1) establishes latency in sensory neurons. As in other alphaherpesviruses, expression of BoHV-1 immediate early (IE) genes is regulated by an enhancer complex containing the viral IE activator VP16, the cellular transcription factor Oct-1, and transcriptional coactivator HCF-1, which is assembled on an IE enhancer core element (TAATGARAT). Expression of the IE transcription unit that encodes the viral IE activators bICP0 and bICP4 may also be induced by the activated glucocorticoid receptor (GR) via two glucocorticoid response elements (GREs) located upstream of the enhancer core. Strikingly, lytic infection and reactivation from latency are consistently enhanced by glucocorticoid treatment in vivo As the coactivator HCF-1 is essential for IE gene expression of alphaherpesviruses and recruited by multiple transcription factors, we tested whether HCF-1 is required for glucocorticoid-induced IE gene expression. Depletion of HCF-1 reduced GR-mediated activation of the IE promoter in mouse neuroblastoma cells (Neuro-2A). More importantly, HCF-1-mediated GR activation of the promoter was dependent on the presence of GRE sites but independent of the TAATGARAT enhancer core element. HCF-1 was also recruited to the GRE region of a promoter lacking the enhancer core, consistent with a direct role of the coactivator in mediating GR-induced transcription. Similarly, during productive lytic infection, HCF-1 and GR occupied the IE region containing the GREs. These studies indicate HCF-1 is critical for GR activation of the viral IE genes and suggests that glucocorticoid induction of viral reactivation proceeds via an HCF-1-GR mechanism in the absence of the viral IE activator VP16.IMPORTANCE BoHV-1 transcription is rapidly activated during stress-induced reactivation from latency. The immediate early transcription unit 1 (IEtu1) promoter is regulated by the GR via two GREs. The IEtu1 promoter regulates expression of two viral transcriptional regulatory proteins, infected cell proteins 0 and 4 (bICP0 and bICP4), and thus must be stimulated during reactivation. This study demonstrates that activation of the IEtu1 promoter by the synthetic corticosteroid dexamethasone requires HCF-1. Interestingly, the GRE sites, but not the IE enhancer core element (TAATGARAT), were required for HCF-1-mediated GR promoter activation. The GR and HCF-1 were recruited to the IEtu1 promoter in transfected and infected cells. Collectively, these studies indicate that HCF-1 is critical for GR activation of the viral IE genes and suggest that an HCF-1-GR complex can stimulate the IEtu1 promoter in the absence of the viral IE activator VP16.

NPAS4 recruits CCK basket cell synapses and enhances cannabinoid-sensitive inhibition in the mouse hippocampus.

Experience-dependent expression of immediate-early gene transcription factors (IEG-TFs) can transiently change the transcriptome of active neurons and initiate persistent changes in cellular function. However, the impact of IEG-TFs on circuit connectivity and function is poorly understood. We investigate the specificity with which the IEG-TF NPAS4 governs experience-dependent changes in inhibitory synaptic input onto CA1 pyramidal neurons (PNs). We show that novel sensory experience selectively enhances somatic inhibition mediated by cholecystokinin-expressing basket cells (CCKBCs) in an NPAS4-dependent manner. NPAS4 specifically increases the number of synapses made onto PNs by individual CCKBCs without altering synaptic properties. Additionally, we find that sensory experience-driven NPAS4 expression enhances depolarization-induced suppression of inhibition (DSI), a short-term form of cannabinoid-mediated plasticity expressed at CCKBC synapses. Our results indicate that CCKBC inputs are a major target of the NPAS4-dependent transcriptional program in PNs and that NPAS4 is an important regulator of plasticity mediated by endogenous cannabinoids.