RESUMO
Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.
Assuntos
Epigênese Genética , Infecções por Vírus Epstein-Barr , Regulação Viral da Expressão Gênica , Centro Germinativo , Herpesvirus Humano 4 , Latência Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Centro Germinativo/imunologia , Centro Germinativo/virologia , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/imunologia , Citocinas/metabolismo , Linfócitos B/virologia , Linfócitos B/metabolismoRESUMO
Ethylene induces anthocyanin biosynthesis in most fruits, including apple (Malus domestica) and plum (Prunus spp.). By contrast, ethylene inhibits anthocyanin biosynthesis in pear (Pyrus spp.), but the underlying molecular mechanism remains unclear. In this study, we identified and characterized an ethylene-induced ETHYLENE RESPONSE FACTOR (ERF) transcription factor, PpETHYLENE RESPONSE FACTOR9 (PpERF9), which functions as a transcriptional repressor. Our analyses indicated PpERF9 can directly inhibit expression of the MYB transcription factor gene PpMYB114 by binding to its promoter. Additionally, PpERF9 inhibits the expression of the transcription factor gene PpRELATED TO APETALA2.4 (PpRAP2.4), which activates PpMYB114 expression, by binding to its promoter, thus forming a PpERF9-PpRAP2.4-PpMYB114 regulatory circuit. Furthermore, PpERF9 interacts with the co-repressor PpTOPLESS1 (PpTPL1) via EAR motifs to form a complex that removes the acetyl group on histone H3 and maintains low levels of acetylated H3 in the PpMYB114 and PpRAP2.4 promoter regions. The resulting suppressed expression of these 2 genes leads to decreased anthocyanin biosynthesis in pear. Collectively, these results indicate that ethylene inhibits anthocyanin biosynthesis by a mechanism that involves PpERF9-PpTPL1 complex-mediated histone deacetylation of PpMYB114 and PpRAP2.4. The data presented herein will be useful for clarifying the relationship between chromatin status and hormone signaling, with implications for plant biology research.
Assuntos
Malus , Pyrus , Pyrus/genética , Pyrus/metabolismo , Fatores de Transcrição/metabolismo , Antocianinas/metabolismo , Histonas/metabolismo , Regulação da Expressão Gênica de Plantas , Etilenos/metabolismo , Frutas/metabolismo , Malus/genética , Malus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
IMPORTANCE: Marek's disease virus (MDV) is a highly infectious and oncogenic virus that can induce severe T cell lymphomas in chickens. MDV encodes more than 100 genes, most of which have unknown functions. This work indicated that the LORF9 gene is necessary for MDV early cytolytic replication in B lymphocytes. In addition, we have found that the LORF9 deletion mutant has a comparative immunological protective effect with CVI988/Rispens vaccine strain against very virulent MDV challenge. This is a significant discovery that LORF9 can be exploited as a possible target for the development of an MDV gene deletion vaccine.
Assuntos
Herpesvirus Galináceo 2 , Vacinas contra Doença de Marek , Doença de Marek , Doenças das Aves Domésticas , Animais , Linfócitos B , Galinhas , Deleção de Genes , Herpesvirus Galináceo 2/genética , Doença de Marek/prevenção & controle , Doença de Marek/genética , Vacinas contra Doença de Marek/genética , Replicação ViralRESUMO
BACKGROUND: Lamellibrachia luymesi dominates cold sulfide-hydrocarbon seeps and is known for its ability to consume bacteria for energy. The symbiotic relationship between tubeworms and bacteria with particular adaptations to chemosynthetic environments has received attention. However, metabolic studies have primarily focused on the mechanisms and pathways of the bacterial symbionts, while studies on the animal hosts are limited. RESULTS: Here, we sequenced the transcriptome of L. luymesi and generated a transcriptomic database containing 79,464 transcript sequences. Based on GO and KEGG annotations, we identified transcripts related to sulfur metabolism, sterol biosynthesis, trehalose synthesis, and hydrolysis. Our in-depth analysis identified sulfation pathways in L. luymesi, and sulfate activation might be an important detoxification pathway for promoting sulfur cycling, reducing byproducts of sulfide metabolism, and converting sulfur compounds to sulfur-containing organics, which are essential for symbiotic survival. Moreover, sulfide can serve directly as a sulfur source for cysteine synthesis in L. luymesi. The existence of two pathways for cysteine synthesis might ensure its participation in the formation of proteins, heavy metal detoxification, and the sulfide-binding function of haemoglobin. Furthermore, our data suggested that cold-seep tubeworm is capable of de novo sterol biosynthesis, as well as incorporation and transformation of cycloartenol and lanosterol into unconventional sterols, and the critical enzyme involved in this process might have properties similar to those in the enzymes from plants or fungi. Finally, trehalose synthesis in L. luymesi occurs via the trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) pathways. The TPP gene has not been identified, whereas the TPS gene encodes a protein harbouring conserved TPS/OtsA and TPP/OtsB domains. The presence of multiple trehalases that catalyse trehalose hydrolysis could indicate the different roles of trehalase in cold-seep tubeworms. CONCLUSIONS: We elucidated several molecular pathways of sulfate activation, cysteine and cholesterol synthesis, and trehalose metabolism. Contrary to the previous analysis, two pathways for cysteine synthesis and the cycloartenol-C-24-methyltransferase gene were identified in animals for the first time. The present study provides new insights into particular adaptations to chemosynthetic environments in L. luymesi and can serve as the basis for future molecular studies on host-symbiont interactions and biological evolution.
Assuntos
Poliquetos , Trealose , Animais , Esteróis , Cisteína , Hidrocarbonetos , Enxofre , Sulfetos/metabolismo , Sulfatos/metabolismoRESUMO
Epstein-Barr virus (EBV) persists in human cells as episomes. EBV episomes are chromatinized and their 3D conformation varies greatly in cells expressing different latency genes. We used HiChIP, an assay which combines genome-wide chromatin conformation capture followed by deep sequencing (Hi-C) and chromatin immunoprecipitation (ChIP), to interrogate the EBV episome 3D conformation in different cancer cell lines. In an EBV-transformed lymphoblastoid cell line (LCL) GM12878 expressing type III EBV latency genes, abundant genomic interactions were identified by H3K27ac HiChIP. A strong enhancer was located near the BILF2 gene and looped to multiple genes around BALFs loci. Perturbation of the BILF2 enhancer by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) altered the expression of BILF2 enhancer-linked genes, including BARF0 and BALF2, suggesting that this enhancer regulates the expression of linked genes. H3K27ac ChIP followed by deep sequencing (ChIP-seq) identified several strong EBV enhancers in T/NK (natural killer) lymphoma cells that express type II EBV latency genes. Extensive intragenomic interactions were also found which linked enhancers to target genes. A strong enhancer at BILF2 also looped to the BALF loci. CRISPRi also validated the functional connection between BILF2 enhancer and BARF1 gene. In contrast, H3K27ac HiChIP found significantly fewer intragenomic interactions in type I EBV latency gene-expressing primary effusion lymphoma (PEL) cell lines. These data provided new insight into the regulation of EBV latency gene expression in different EBV-associated tumors. IMPORTANCE EBV is the first human DNA tumor virus identified, discovered over 50 years ago. EBV causes ~200,000 cases of various cancers each year. EBV-encoded oncogenes, noncoding RNAs, and microRNAs (miRNAs) can promote cell growth and survival and suppress senescence. Regulation of EBV gene expression is very complex. The viral C promoter regulates the expression of all EBV nuclear antigens (EBNAs), some of which are very far away from the C promoter. Another way by which the virus activates remote gene expression is through DNA looping. In this study, we describe the viral genome looping patterns in various EBV-associated cancer cell lines and identify important EBV enhancers in these cells. This study also identified novel opportunities to perturb and eventually control EBV gene expression in these cancer cells.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Plasmídeos , Latência Viral , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos/genética , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , MicroRNAs/metabolismo , Neoplasias/virologia , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Virais/genética , Latência Viral/genéticaRESUMO
Marek's disease virus (MDV) is a potent oncogenic alphaherpesvirus that elicits a rapid onset of malignant T-cell lymphomas in chickens. Three MDV types, including GaHV-2 (MDV-1), GaHV-3 (MDV-2) and MeHV-1 (HVT), have been identified and all encode a US3 protein kinase. MDV-1 US3 is important for efficient virus growth in vitro. To study the role of US3 in MDV replication and pathogenicity, we generated an MDV-1 US3-null virus and chimeric viruses by replacing MDV-1 US3 with MDV-2 or HVT US3. Using MD as a natural virus-host model, we showed that both MDV-2 and HVT US3 partially rescued the growth deficiency of MDV-1 US3-null virus. In addition, deletion of MDV-1 US3 attenuated the virus resulting in higher survival rate and lower MDV specific tumor incidence, which could be partially compensated by MDV-2 and HVT US3. We also identified chicken histone deacetylase 1 (chHDAC1) as a common US3 substrate for all three MDV types while only US3 of MDV-1 and MDV-2 phosphorylate chHDAC2. We further determined that US3 of MDV-1 and HVT phosphorylate chHDAC1 at serine 406 (S406), while MDV-2 US3 phosphorylates S406, S410, and S415. In addition, MDV-1 US3 phosphorylates chHDAC2 at S407, while MDV-2 US3 targets S407 and S411. Furthermore, biochemical studies show that MDV US3 mediated phosphorylation of chHDAC1 and 2 affect their stability, transcriptional regulation activity, and interaction network. Using a class I HDAC specific inhibitor, we showed that MDV US3 mediated phosphorylation of chHDAC1 and 2 is involved in regulation of virus replication. Overall, we identified novel substrates for MDV US3 and characterized the role of MDV US3 in MDV pathogenesis.
Assuntos
Herpesvirus Galináceo 2/patogenicidade , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Doença de Marek/virologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Animais , Galinhas , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Doença de Marek/metabolismo , Doença de Marek/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Virais/genéticaRESUMO
Dynamic alteration of the epitranscriptome exerts regulatory effects on the lifecycle of oncogenic viruses in vitro. However, little is known about these effects in vivo because of the general lack of suitable animal infection models of these viruses. Using a model of rapid-onset Marek's disease lymphoma in chickens, we investigated changes in viral and host messenger RNA (mRNA) N6-methyladenosine (m6 A) modification during Marek's disease virus (MDV) infection in vivo. We found that the expression of major epitranscriptomic proteins varies among viral infection phases, reprogramming both the viral and the host epitranscriptomes. Specifically, the methyltransferase-like 3 (METTL3)/14 complex was suppressed during the lytic and reactivation phases of the MDV lifecycle, whereas its expression was increased during the latent phase and in MDV-induced tumors. METTL3/14 overexpression inhibits, whereas METTL3/14 knockdown enhances, MDV gene expression and replication. These findings reveal the dynamic features of the mRNA m6 A modification program during viral replication in vivo, especially in relation to key pathways involved in tumorigenesis.
Assuntos
Doença de Marek , Animais , Doença de Marek/genética , Vírus Oncogênicos/genética , Galinhas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Marek's disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes T cell lymphoma in chickens. MDV-encoded Meq and vIL8 proteins play important roles in transformation and early cytolytic infection, respectively. Previous studies identified a spliced transcript, meq-vIL8, formed by alternative splicing of meq and vIL8 genes in MDV lymphoblastoid tumour cells. To determine the role of Meq-vIL8 in MDV pathogenesis, we generated a recombinant MDV (MDV-meqΔSD) by mutating the splice donor site in the meq gene to abrogate the expression of Meq-vIL8. As expected, our results show that MDV-meqΔSD virus grows similarly to the parental and revertant viruses in cell culture, suggesting that Meq-vIL8 is dispensable for MDV growth in vitro. We further characterized the pathogenic properties of MDV-meqΔSD virus in chickens. Our results show that lack of Meq-vIL8 did not affect virus replication during the early cytolytic phase, as determined by immunohistochemistry analysis and/or viral genome copy number, but significantly enhanced viral DNA load in the late phase of infection in the spleen and brain of infected chickens. In addition, we observed that abrogation of Meq-vIL8 expression reduced the mean death time and increased the prevalence of persistent neurological disease, common features of highly virulent strains of MDV, in inoculated chickens. In conclusion, our study shows that Meq-vIL8 is an important virulence factor of MDV.
Assuntos
Herpesvirus Galináceo 2/genética , Herpesvirus Galináceo 2/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , DNA Viral/genética , Técnica Indireta de Fluorescência para Anticorpo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Fatores de Virulência , Replicação ViralRESUMO
Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the subfamily Alphaherpesvirinae Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, US3. The functions of US3 have been extensively studied in other alphaherpesviruses; however, the biological functions of MDV US3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV US3 and identified chicken CREB (chCREB) as a substrate of MDV US3. We show that wild-type MDV US3, but not kinase-dead US3 (US3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of the CREB-responsive gene and activation of CREB target gene expression. Using US3 deletion and US3 kinase-dead recombinant MDV, we identified US3-responsive MDV genes during infection and found that the majority of US3-responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation sequencing (ChIP-seq) studies determined that some US3-regulated genes colocalized with Meq (an MDV-encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the ICP4/LAT region, which is also regulated by US3. Furthermore, biochemical studies demonstrated that MDV US3 interacts with Meq in transfected cells and MDV-infected chicken embryonic fibroblasts in a phosphorylation-dependent manner. Finally, in vitro kinase studies revealed that Meq is a US3 substrate. MDV US3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of the CREB/Meq heterodimer, which targets cellular and viral gene expression.IMPORTANCE MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. US3 encoded by alphaherpesviruses is a multifunctional kinase involved in the regulation of various cellular pathways. Using an MDV genome quantitative reverse transcriptase PCR (qRT-PCR) array and chromatin immunoprecipitation, we elucidated the role of MDV US3 in viral and cellular gene regulation. Our results provide insights into how viral kinase regulates host cell signaling pathways to activate both viral and host gene expression. This is an important step toward understanding host-pathogen interaction through activation of signaling cascades.
Assuntos
Herpesvirus Galináceo 2/enzimologia , Herpesvirus Galináceo 2/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Alphaherpesvirinae/genética , Animais , Linhagem Celular , Transformação Celular Viral/genética , Galinhas/virologia , Imunoprecipitação da Cromatina , Dosagem de Genes , Regulação Viral da Expressão Gênica , Células HEK293 , Humanos , Doença de Marek/virologia , Fosforilação , Aves Domésticas , Regiões Promotoras Genéticas , Transdução de Sinais , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Hypoxia occurs in many human solid tumors and activates multiple cellular adaptive-response pathways, including the unfolded protein response (UPR) in the endoplasmic reticulum (ER). Wnt/ß-catenin signaling plays a critical role in tumorigenesis, and ß-catenin has been shown to enhance hypoxia-inducible factor 1α (HIF1α)-activated gene expression, thereby supporting cell survival during hypoxia. However, the molecular interplay between hypoxic ER stress, Wnt/ß-catenin signaling, and HIF1α-mediated gene regulation during hypoxia remains incompletely understood. Here, we report that hypoxic ER stress reduces ß-catenin stability, which, in turn, enhances the activity of spliced X-box-binding protein 1 (XBP1s), a transcription factor and signal transducer of the UPR, in HIF1α-mediated hypoxic responses. We observed that in the RKO colon cancer cell line, which possesses a Wnt-stimulated ß-catenin signaling cascade, increased ER stress during hypoxia is accompanied by a reduction in low-density lipoprotein receptor-related protein 6 (LRP6), and this reduction in LRP6 decreased ß-catenin accumulation and impaired Wnt/ß-catenin signaling. Of note, ß-catenin interacted with both XBP1s and HIF1α, suppressing XBP1s-mediated augmentation of HIF1α target gene expression. Furthermore, Wnt stimulation or ß-catenin overexpression blunted XBP1s-mediated cell survival under hypoxia. Together, these results reveal an unanticipated role for the Wnt/ß-catenin pathway in hindering hypoxic UPR-mediated responses that increase cell survival. Our findings suggest that the molecular cross-talks between hypoxic ER stress, LRP6/ß-catenin signaling, and the HIF1α pathway may represent an unappreciated mechanism that enables some tumor subtypes to survive and grow in hypoxic conditions.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Sobrevivência Celular/fisiologia , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , Via de Sinalização Wnt , Proteína 1 de Ligação a X-Box/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Epstein-Barr virus (EBV) uses a biphasic lifecycle of latency and lytic reactivation to infect >95% of adults worldwide. Despite its central role in EBV persistence and oncogenesis, much remains unknown about how EBV latency is maintained. We used a human genome-wide CRISPR/Cas9 screen to identify that the nuclear protein SFPQ was critical for latency. SFPQ supported expression of linker histone H1, which stabilizes nucleosomes and regulates nuclear architecture, but has not been previously implicated in EBV gene regulation. H1 occupied latent EBV genomes, including the immediate early gene BZLF1 promoter. Upon reactivation, SFPQ was sequestered into sub-nuclear puncta, and EBV genomic H1 occupancy diminished. Enforced H1 expression blocked EBV reactivation upon SFPQ knockout, confirming it as necessary downstream of SFPQ. SFPQ knockout triggered reactivation of EBV in B and epithelial cells, as well as of Kaposi's sarcoma-associated herpesvirus in B cells, suggesting a conserved gamma-herpesvirus role. These findings highlight SFPQ as a major regulator of H1 expression and EBV latency.
Assuntos
Herpesvirus Humano 4 , Histonas , Fator de Processamento Associado a PTB , Ativação Viral , Latência Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Histonas/metabolismo , Ativação Viral/genética , Latência Viral/genética , Fator de Processamento Associado a PTB/metabolismo , Fator de Processamento Associado a PTB/genética , Regulação Viral da Expressão Gênica , Linfócitos B/virologia , Linfócitos B/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Sistemas CRISPR-Cas , Regiões Promotoras Genéticas/genética , Transativadores/metabolismo , Transativadores/genética , Genoma ViralRESUMO
High pressure is both an environmental challenge to which deep sea biology has to adapt, and a highly sensitive thermodynamic tool that can be used to trigger structural changes in biological molecules and assemblies. Lipid membranes are amongst the most pressure sensitive biological assemblies and pressure can have a large influence on their structure and properties. In this chapter, we will explore the use of high pressure small angle X-ray diffraction and high pressure microscopy to measure and quantify changes in the lateral structure of lipid membranes under both equilibrium high pressure conditions and in response to pressure jumps.
Assuntos
Pressão Hidrostática , Bicamadas Lipídicas , Difração de Raios X , Difração de Raios X/métodos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Espalhamento a Baixo Ângulo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , TermodinâmicaRESUMO
Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor signal transducer and activator of transcription 3 (STAT3). To better understand the role of STAT3 during gammaherpesvirus latency and the B cell response to infection, we used the model pathogen murine gammaherpesvirus 68 (MHV68). Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak MHV68 latency approximately sevenfold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to wild-type (WT) littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeric mice consisting of WT and STAT3 knockout B cells. We discovered a dramatic reduction in latency in STAT3 knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that MHV68 infection shifts the gene signature toward proliferation and away from type I and type II IFN responses. Loss of STAT3 largely reversed the virus-driven transcriptional shift without impacting the viral gene expression program. STAT3 promoted B cell processes of the germinal center, including IL-21-stimulated downregulation of surface CD23 on B cells infected with MHV68 or EBV. Together, our data provide mechanistic insights into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.IMPORTANCEThere are no directed therapies to the latency program of the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor signal transducer and activator of transcription 3 (STAT3) is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes that are key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.
Assuntos
Infecções por Vírus Epstein-Barr , Gammaherpesvirinae , Infecções por Herpesviridae , Herpesvirus Humano 8 , Rhadinovirus , Sarcoma de Kaposi , Animais , Humanos , Camundongos , Gammaherpesvirinae/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 8/metabolismo , Camundongos Endogâmicos C57BL , Rhadinovirus/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Latência Viral/genéticaRESUMO
Radiotherapy could regulate systemic antitumor immunity, while the immune state in the tumor microenvironment (TME) also affects the efficacy of radiotherapy. We have found that higher CD8+ T cell infiltration is associated with longer overall survival of lung adenocarcinoma and melanoma patients receiving radiotherapy. 8-Gray radiation increased the transcriptional levels of chemokines in tumor cells in vitro. However, it was not sufficient to induce significant lymphocyte infiltration in vivo. Dipeptidyl peptidase 4 (DPP4) has been reported to inactivate chemokines via post-translational truncation. Single-cell sequencing revealed that dendritic cells (DCs) had a higher DPP4 expression among other cells in the TME and upregulated DPP4 expression after radiation. Combining a DPP4 inhibitor with radiotherapy could promote chemokines expression and T cell infiltration in the TME, enhancing the antitumor effect of radiotherapy. Moreover, this therapy further enhanced the therapeutic efficacy of anti-PD-1. In this study, we demonstrated the underlying mechanism of why radiotherapy failed to induce sufficient T cell infiltration and proposed an effective strategy to promote T cell infiltration and sensitize radiotherapy. These findings demonstrate the translational value of DPP4 inhibition as a complementary approach to enhance the efficacy of radiotherapy and the combination of radiotherapy with immunotherapy.
Assuntos
Inibidores da Dipeptidil Peptidase IV , Neoplasias , Humanos , Dipeptidil Peptidase 4/genética , Inibidores da Dipeptidil Peptidase IV/farmacologia , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Quimiocinas/metabolismo , Neoplasias/tratamento farmacológico , Linfócitos T CD8-Positivos , Microambiente TumoralRESUMO
Previous studies focused on the effects of light on fruit appearance, especially the peel color. However, the effect of light on fruit internal quality and the underlying mechanisms are unclear. In this study, we analyzed the effects of blue light on the appearance and internal quality of mango fruit (Mangifera indica L.). Blue light simultaneously induced peel anthocyanin and flesh sucrose/carotenoid biosynthesis in mango fruit. Analyses of co-expression networks and gene expression trends in mango fruit peel and flesh identified candidate genes, including transcription factor genes, involved in blue light-regulated anthocyanin, carotenoid, and sucrose biosynthesis pathways. Key blue light signaling-related genes (MiCRY and MiHY5) and blue light-triggered phytohormones were involved in these pathways. Additionally, there were common and tissue-specific pathways for the blue light-promoted accumulation of anthocyanins, carotenoids, and sucrose. Our results provide new insights into the regulatory effects of light on the appearance and internal quality of mango fruit.
Assuntos
Antocianinas , Mangifera , Antocianinas/metabolismo , Mangifera/genética , Frutas/genética , Frutas/metabolismo , Transcriptoma , Carotenoides/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
The local coordination environment around the active centers has a major impact on tuning the intrinsic activity of M-N-C catalysts. Herein, a porous graphene with Fe-N5 active sites modified with Fe clusters is successfully fabricated by using Fe3+-SCN- and NaHCO3 as the metal precursor and pore-forming agent, respectively. The unique Fe-N5 configuration accompanying Fe clusters and the improved ORR activity are confirmed by various characterization techniques and theoretical calculations. Benefiting from the pores, mass and electron transfer channels are successfully constructed, making more active sites accessible and facilitating the ORR process. As a consequence, the as-prepared catalyst has an excellent ORR activity with a half-wave potential of 0.89 V, comparable selectivity, and superior stability. In addition, a homemade primary zinc-air battery using this material as the cathode catalyst has a maximum power density of 0.205 W/cm2, revealing the potential of the as-constructed CSA-Fe-N-C catalyst to replace precious Pt catalysts.
RESUMO
Marek's disease virus (MDV) induces immunosuppression and neoplastic disease in chickens. The virus is controllable via an attenuated meq deletion mutant virus, which has the disadvantage of retaining the ability to induce lymphoid organ atrophy. To overcome this deficiency and produce more vaccine candidates, a recombinant MDV was generated from the highly virulent Md5BAC strain, in which both meq and a cytolytic replication-related gene, pp38, were deleted. Replication of the double deletion virus, Md5BAC ΔmeqΔpp38, was comparable with that of the parental virus in vitro. The double deletion virus was shown to be fully attenuated and to reduce lymphoid organ atrophy in vivo. Crucially, Md5BAC ΔmeqΔpp38 confers superior protection against highly virulent virus compared with a commercial vaccine strain, CVI988/Rispens. Transcriptomic profiling indicated that Md5BAC ΔmeqΔpp38 induced a different host immune response from CVI988/Rispens. In summary, a novel, effective, and safe vaccine candidate for prevention and control of MD caused by highly virulent MDV is reported. IMPORTANCE MDV is a highly contagious immunosuppressive and neoplastic pathogen. The virus can be controlled through vaccination via an attenuated meq deletion mutant virus that retains the ability to induce lymphoid organ atrophy. In this study, we overcame the deficiency by generating meq and pp38 double deletion mutant virus. Indeed, the successfully generated meq and pp38 double deletion mutant virus had significantly reduced replication capacity in vivo but not in vitro. It was fully attenuated and conferred superior protection efficacy against very virulent MDV challenge. In addition, the possible immunological protective mechanism of the double deletion mutant virus was shown to be different from that of the gold standard MDV vaccine, CVI988/Rispens. Overall, we successfully generated an attenuated meq deletion mutant virus and widened the range of potential vaccine candidates. Importantly, this study provides for the first time the theoretical basis of vaccination induced by fully attenuated gene-deletion mutant virus.
Assuntos
Herpesvirus Galináceo 2 , Vacinas contra Doença de Marek , Doença de Marek , Proteínas Oncogênicas Virais , Doenças das Aves Domésticas , Animais , Doença de Marek/prevenção & controle , Doença de Marek/genética , Deleção de Genes , Proteínas Oncogênicas Virais/genética , Galinhas , Herpesvirus Galináceo 2/genética , Vacinas contra Doença de Marek/genética , AtrofiaRESUMO
Marek's disease (MD) is a neoplastic disease of chickens caused by an avian alphaherpesvirus, Marek's disease virus (MDV, also known as Gallid alphaherpesvirus 2 [GaHV2]). A total of 14 microRNA (miRNA) precursors and 26 mature miRNAs have been identified in MDV genome, which were grouped in three distinct clusters. In recent years, our studies revealed the role of MDV encoded cluster 3 miRNAs (or miR-M8-M10) and the specific function of its three members, miR-M6, miR-M7 and miR-M10, in regulating MDV replication and pathogenesis. In this study, we characterized the unique function of the other two members, miR-M8 and miR-M13, in cluster 3 miRNAs. Our results show that miR-M8 and miR-M13 are not important for MDV plaque formation and genome replication in vitro. Animal experiment results show that deletion of miR-M8-5p and miR-M13-5p eliminates the bursa atrophy, but not thymus atrophy, of MDV inoculated chickens. In addition, we found that the survival curve and MD incidences were not affected by disruption of miR-M8 and miR-M13. Taken together, this study uncovers the unique role of miR-M8 and miR-M13 in MDV replication and pathogenesis, which filled the gap in the research of MDV encoded miRNAs.
Assuntos
Herpesvirus Galináceo 2 , Doença de Marek , MicroRNAs , Animais , Atrofia/veterinária , Galinhas , Herpesvirus Galináceo 2/genética , MicroRNAs/genéticaRESUMO
Chicken infectious anemia (CIA) is an immunosuppressive disease caused by the chicken infectious anemia virus (CIAV) resulting in heavy economic losses once an outbreak is established. This study conducted a systematic analysis of the epidemiology and pathology of CIA in Henan province, China. A total of 437 clinical tissue samples and 120 poultry disease-related live attenuated vaccines were collected during 2017-2020; of which 45 were positive for CIAV nucleic acid, with a positive rate of 8.08%. Our results showed that genome sequence similarity among a total of 12 CIAV isolates was high, and ranged from 97.1 to 99.3%, and their similarity to the vaccine strains Cux-1 and Del-Ros ranged from 97.8 to 98.6%. However, There were mutations in the locus of the major capsid proteins VP1, VP2, and VP3 among all isolates. The subsequent sequence analysis indicated that the isolates of HN-4 and HN-8 showed genetic recombination and follow up animal experiments revealed that HN-4 might be a pathogenic strain. Our results reveal that both field infection and non-CIAV vaccines contamination promote the epidemiology of CIAV in China and some dominant epidemic viruses have undergone recombination and evolution. This study provides important information to help with the prevention and control of CIAV in the poultry industry.
RESUMO
Marek's disease virus (MDV) is an oncogenic avian alphaherpesvirus whose genome consists of unique long (UL) and short (US) regions that are flanked by inverted repeat regions. More than 100 open reading frames (ORFs) have been annotated in the MDV genome, and are involved in various aspects of MDV biology and pathogenesis. Within UL and US regions of MDV, there are several unique ORFs, some of which have recently been shown to be important for MDV replication and pathogenesis. In this review, we will summarize the current knowledge on these ORFs and compare their location in different MDV strains.