RESUMEN
In the version of this article initially published, the label (CASP4-C285A-HA) above the second and fifth lanes in the right blot in Fig. 1e is incorrect; the correct label is CASP4-C258A-HA. Also, the two labels at right above the plot in Fig. 6c were switched; the far right label should be 'Co-housed Serpinb1a-/-' (in red font) and the label just to its left (above the fourth column) should be 'Co-housed WT' (in black font). Finally, the bottom two symbols in the key to Fig. 7d were switched; the red circle should identify 1CARD-SUMO (TEV) and the blue triangle should identify 1CARD-SUMO + SERPINB1 (TEV). The errors have been corrected in the HTML and PDF versions of the article.
RESUMEN
Inflammatory caspases (caspase-1, caspase-4, caspase-5 and caspase-11 (caspase-1/-4/-5/-11)) mediate host defense against microbial infections, processing pro-inflammatory cytokines and triggering pyroptosis. However, precise checkpoints are required to prevent their unsolicited activation. Here we report that serpin family B member 1 (SERPINB1) limited the activity of those caspases by suppressing their caspase-recruitment domain (CARD) oligomerization and enzymatic activation. While the reactive center loop of SERPINB1 inhibits neutrophil serine proteases, its carboxy-terminal CARD-binding motif restrained the activation of pro-caspase-1/-4/-5/-11. Consequently, knockdown or deletion of SERPINB1 prompted spontaneous activation of caspase-1/-4/-5/-11, release of the cytokine IL-1ß and pyroptosis, inducing elevated inflammation after non-hygienic co-housing with pet-store mice and enhanced sensitivity to lipopolysaccharide- or Acinetobacter baumannii-induced endotoxemia. Our results reveal that SERPINB1 acts as a vital gatekeeper of inflammation by restraining neutrophil serine proteases and inflammatory caspases in a genetically and functionally separable manner.
Asunto(s)
Caspasas/inmunología , Mediadores de Inflamación/inmunología , Inflamación/inmunología , Serpinas/inmunología , Animales , Caspasas/genética , Caspasas/metabolismo , Línea Celular , Células Cultivadas , Activación Enzimática/inmunología , Células HEK293 , Humanos , Inflamación/genética , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Piroptosis/efectos de los fármacos , Piroptosis/inmunología , Células RAW 264.7 , Interferencia de ARN , Serina Proteasas/inmunología , Serina Proteasas/metabolismo , Serpinas/genética , Serpinas/metabolismo , Células THP-1 , Células U937RESUMEN
Recurrent spillovers of α- and ß-coronaviruses (CoV) such as severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome-CoV, SARS-CoV-2, and possibly human CoV have caused serious morbidity and mortality worldwide. In this study, six receptor-binding domains (RBDs) derived from α- and ß-CoV that are considered to have originated from animals and cross-infected humans were linked to a heterotrimeric scaffold, proliferating cell nuclear antigen (PCNA) subunits, PCNA1, PCNA2, and PCNA3. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens, like jewels in a crown. Prime-boost immunizations with 6RBD-np in mice induced significantly high Ab titers against RBD antigens derived from α- and ß-CoV and increased interferon (IFN-γ) production, with full protection against the SARS-CoV-2 wild type and Delta challenges. The mosaic 6RBD-np has the potential to induce intergenus cross-reactivity and to be developed as a pan-CoV vaccine against future CoV spillovers.
Asunto(s)
COVID-19 , Nanopartículas , Humanos , Animales , Ratones , SARS-CoV-2 , Anticuerpos Antivirales , COVID-19/prevención & control , Anticuerpos Neutralizantes , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Primary effusion lymphoma (PEL) is an aggressive B cell lymphoma that is etiologically linked to Kaposi's sarcoma-associated herpesvirus (KSHV). Despite standard multi-chemotherapy treatment, PEL continues to cause high mortality. Thus, new strategies to control PEL are needed urgently. Here, we show that a phosphodegron motif within the KSHV protein, latency-associated nuclear antigen (LANA), specifically interacts with E3 ubiquitin ligase FBW7, thereby competitively inhibiting the binding of the anti-apoptotic protein MCL-1 to FBW7. Consequently, LANA-FBW7 interaction enhances the stability of MCL-1 by preventing its proteasome-mediated degradation, which inhibits caspase-3-mediated apoptosis in PEL cells. Importantly, MCL-1 inhibitors markedly suppress colony formation on soft agar and tumor growth of KSHV+PEL/BCBL-1 in a xenograft mouse model. These results strongly support the conclusion that high levels of MCL-1 expression enable the oncogenesis of PEL cells and thus, MCL-1 could be a potential drug target for KSHV-associated PEL. This work also unravels a mechanism by which an oncogenic virus perturbs a key component of the ubiquitination pathway to induce tumorigenesis.
Asunto(s)
Antígenos Virales/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Herpesvirus Humano 8/fisiología , Linfoma de Efusión Primaria/virología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Nucleares/metabolismo , Sarcoma de Kaposi/virología , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Apoptosis , Proliferación Celular , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Femenino , Humanos , Linfoma de Efusión Primaria/genética , Linfoma de Efusión Primaria/metabolismo , Linfoma de Efusión Primaria/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas Nucleares/genética , Fosforilación , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patología , Células Tumorales Cultivadas , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Enhancers play indispensable roles in cell proliferation and survival through spatiotemporally regulating gene transcription. Active enhancers and superenhancers often produce noncoding enhancer RNAs (eRNAs) that precisely control RNA polymerase II activity. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic gamma-2 herpesvirus that causes Kaposi's sarcoma and primary effusion lymphoma (PEL). It is well characterized that KSHV utilizes host epigenetic machineries to control the switch between two lifecycles, latency and lytic replication. However, how KSHV impacts host epigenome at different stages of viral lifecycle is not well understood. Using global run-on sequencing (GRO-seq) and chromatin-immunoprecipitation sequencing (ChIP-seq), we profiled the dynamics of host transcriptional regulatory elements during latency and lytic replication of KSHV-infected PEL cells. This revealed that a number of critical host genes for KSHV latency, including MYC proto-oncogene, were under the control of superenhancers whose activities were globally repressed upon viral reactivation. The eRNA-expressing MYC superenhancers were located downstream of the MYC gene in KSHV-infected PELs and played a key role in MYC expression. RNAi-mediated depletion or dCas9-KRAB CRISPR inhibition of eRNA expression significantly reduced MYC mRNA level in PELs, as did the treatment of an epigenomic drug that globally blocks superenhancer function. Finally, while cellular IRF4 acted upon eRNA expression and superenhancer function for MYC expression during latency, KSHV viral IRF4 repressed cellular IRF4 expression, decreasing MYC expression and thereby, facilitating lytic replication. These results indicate that KSHV acts as an epigenomic driver that modifies host epigenomic status upon reactivation by effectively regulating host enhancer function.
Asunto(s)
Regulación Viral de la Expresión Génica/genética , Herpesvirus Humano 8/genética , Linfoma de Efusión Primaria/genética , Línea Celular , Epigenómica/métodos , Genes myc/genética , Herpesvirus Humano 8/patogenicidad , Humanos , Proteínas Inmediatas-Precoces/genética , Linfoma de Efusión Primaria/metabolismo , Linfoma de Efusión Primaria/virología , Proteínas Nucleares/metabolismo , Proto-Oncogenes Mas , ARN/metabolismo , Sarcoma de Kaposi/virología , Transactivadores/metabolismo , Transcripción Genética/genética , Proteínas Virales/metabolismo , Activación Viral/genética , Latencia del Virus/genética , Replicación Viral/genéticaRESUMEN
Three-dimensional (3D) cell culture is well documented to regain intrinsic metabolic properties and to better mimic the in vivo situation than two-dimensional (2D) cell culture. Particularly, proline metabolism is critical for tumorigenesis since pyrroline-5-carboxylate (P5C) reductase (PYCR/P5CR) is highly expressed in various tumors and its enzymatic activity is essential for in vitro 3D tumor cell growth and in vivo tumorigenesis. PYCR converts the P5C intermediate to proline as a biosynthesis pathway, whereas proline dehydrogenase (PRODH) breaks down proline to P5C as a degradation pathway. Intriguingly, expressions of proline biosynthesis PYCR gene and proline degradation PRODH gene are up-regulated directly by c-Myc oncoprotein and p53 tumor suppressor, respectively, suggesting that the proline-P5C metabolic axis is a key checkpoint for tumor cell growth. Here, we report a metabolic reprogramming of 3D tumor cell growth by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Metabolomic analyses revealed that KSHV infection increased nonessential amino acid metabolites, specifically proline, in 3D culture, not in 2D culture. Strikingly, the KSHV K1 oncoprotein interacted with and activated PYCR enzyme, increasing intracellular proline concentration. Consequently, the K1-PYCR interaction promoted tumor cell growth in 3D spheroid culture and tumorigenesis in nude mice. In contrast, depletion of PYCR expression markedly abrogated K1-induced tumor cell growth in 3D culture, not in 2D culture. This study demonstrates that an increase of proline biosynthesis induced by K1-PYCR interaction is critical for KSHV-mediated transformation in in vitro 3D culture condition and in vivo tumorigenesis.
Asunto(s)
Transformación Celular Neoplásica/patología , Herpesvirus Humano 8/metabolismo , Prolina/metabolismo , Pirrolina Carboxilato Reductasas/metabolismo , Sarcoma de Kaposi/patología , Proteínas Virales/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Proliferación Celular , Humanos , Metabolómica , Ratones , Prolina Oxidasa/metabolismo , Sarcoma de Kaposi/virología , Esferoides Celulares , Ensayos Antitumor por Modelo de Xenoinjerto , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
The ubiquitin proteasome system (UPS) is a protein degradation machinery that is crucial for cellular homeostasis in eukaryotes. Therefore, it is not surprising that the UPS coordinates almost all host cellular processes, including host-pathogen interactions. This protein degradation machinery acts predominantly by tagging substrate proteins designated for degradation with a ubiquitin molecule. These ubiquitin tags have been involved at various steps of the innate immune response. Hence, herpesviruses have evolved ways to antagonize the host defense mechanisms by targeting UPS components such as ubiquitin E3 ligases and deubiquitinases (DUBs) that establish a productive infection. This review delineates how herpesviruses usurp the critical roles of ubiquitin E3 ligases and DUBs in innate immune response to escape host-antiviral immune response, with particular focus on retinoic acid-inducible gene I (RIG-I)-like receptors (RLR), cyclic-GMP-AMP (cGAMP) synthase (cGAS), stimulator of interferon (IFN) genes (STING) pathways, and inflammasome signaling.
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Herpesviridae/inmunología , Inmunidad Innata , Transducción de Señal , Ubiquitina/metabolismo , Animales , Humanos , Factores Inmunológicos/metabolismo , Inflamación/patologíaRESUMEN
UV-induced cell pigmentation represents an important mechanism against skin cancers. Sun-exposed skin secretes α-MSH, which induces the lineage-specific transcriptional factor MITF and activates melanogenesis in melanocytes. Here, we show that the autophagic tumor suppressor UVRAG plays an integral role in melanogenesis by interaction with the biogenesis of lysosome-related organelles complex 1 (BLOC-1). This interaction is required for BLOC-1 stability and for BLOC-1-mediated cargo sorting and delivery to melanosomes. Absence of UVRAG dispersed BLOC-1 distribution and activity, resulting in impaired melanogenesis in vitro and defective melanocyte development in zebrafish in vivo. Furthermore, our results establish UVRAG as an important effector for melanocytes' response to α-MSH signaling as a direct target of MITF and reveal the molecular basis underlying the association between oncogenic BRAF and compromised UV protection in melanoma.
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Melaninas/biosíntesis , Melanosomas/metabolismo , Pigmentación de la Piel/efectos de la radiación , Proteínas Supresoras de Tumor/metabolismo , Rayos Ultravioleta , Animales , Células HEK293 , Humanos , Melaninas/genética , Melanoma/genética , Melanoma/metabolismo , Melanosomas/genética , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Supresoras de Tumor/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The emergence of multidrug-resistant (MDR) bacteria through the abuse and long-term use of antibiotics is a serious health problem worldwide. Therefore, novel antimicrobial agents that can cure an infection from MDR bacteria, especially gram-negative bacteria, are urgently needed. Antimicrobial peptides, part of the innate immunity system, have been studied to find bactericidal agents potent against MDR bacteria. However, they have many problems, such as restrained systemic activity and cytotoxicity. In a previous study, we suggested that the K58-R78 domain of Romo1, a mitochondrial protein encoded by the nucleus, was a promising treatment candidate for sepsis caused by MDR bacteria. Here, we performed sequence optimization to enhance the antimicrobial activity of this peptide and named it as AMPR-22 (antimicrobial peptide derived from Romo1). It showed broad-spectrum antimicrobial activity against 17 sepsis-causing bacteria, including MDR strains, by inducing membrane permeabilization. Moreover, treatment with AMPR-22 enabled a remarkable survival rate in mice injected with MDR bacteria in a murine model of sepsis. Based on these results, we suggest that AMPR-22 could be prescribed as a first-line therapy (prior to bacterial identification) for patients diagnosed with sepsis.
Asunto(s)
Proteínas de la Membrana/química , Proteínas Mitocondriales/química , Fragmentos de Péptidos/uso terapéutico , Proteínas Citotóxicas Formadoras de Poros/uso terapéutico , Sepsis/tratamiento farmacológico , Animales , Células Cultivadas , Farmacorresistencia Bacteriana , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/farmacología , Dominios Proteicos , Sepsis/microbiologíaRESUMEN
Epigenetic histone modifications play critical roles in the control of gene transcription. Recently, an increasing number of histone H2A deubiquitinases have been identified and characterized. However, the physiological functions for this entire group of histone H2A deubiquitinases remain unknown. In this study, we revealed that the histone H2A deubiquitinase MYSM1 plays an essential and intrinsic role in early B cell development. MYSM1 deficiency results in a block in early B cell commitment and a defect of B cell progenitors in expression of EBF1 and other B lymphoid genes. We further demonstrated that MYSM1 derepresses EBF1 transcription in B cell progenitors by orchestrating histone modifications and transcription factor recruitment to the EBF1 locus. Thus, this study not only uncovers the essential role for MYSM1 in gene transcription during early B cell development but also underscores the biological significance of reversible epigenetic histone H2A ubiquitination.
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Linfocitos B/citología , Linfocitos B/enzimología , Diferenciación Celular , Endopeptidasas/metabolismo , Histonas/metabolismo , Factores de Transcripción/metabolismo , Animales , Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linaje de la Célula/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas , Unión Proteica , Transactivadores/genética , Factores de Transcripción/genética , Transcripción Genética , Proteasas Ubiquitina-EspecíficasRESUMEN
Hepatitis C virus (HCV) p7 is known to be a nonselective cation channel for HCV maturation. Because the interaction of HCV proteins with host lipids in the endoplasmic reticulum membrane is crucial for the budding process, the identification of p7-lipid interactions could be important for understanding the HCV life cycle. Here, we report that p7 interacts with phosphatidylserine (PS) to induce membrane permeabilization. The interaction of p7 with PS was not inhibited by Gd3+ ions, which have been known to interact with negatively charged lipids, but channel activity and p7-induced mitochondrial depolarization were inhibited by Gd3+ ions. From the present results, we suggest that the p7-PS interaction plays an essential role in regulating its ion channel function and could be a potential molecular target for anti-HCV therapy.
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Hepacivirus/fisiología , Hepatitis C/virología , Canales Iónicos/antagonistas & inhibidores , Fosfatidilserinas/metabolismo , Proteínas Virales/metabolismo , Permeabilidad de la Membrana Celular , Retículo Endoplásmico/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virología , Mitocondrias/metabolismoRESUMEN
Primary effusion lymphoma (PEL), strongly linked with latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), constitutively expresses cellular interferon regulatory factor 4 (IRF4) while suppressing the expression of B cell lymphoma 6 (BCL6). Recently, it was shown that IRF4, a key transcriptional repressor of BCL6, might be a pivotal regulator of KSHV for balancing between latency and its reactivation in PEL cells. However, the action of the BCL6-IRF4 transcription factor axis during KSHV's life cycle is not clear. Herein we found that the KSHV lytic protein viral interferon regulatory factor 4 (vIRF4) dramatically enhanced the transcriptional activity of the BCL6 through the inhibition of its negative regulator IRF4. Using a chromatin immunoprecipitation (ChIP) assay, we further showed that vIRF4 bound to the specific promoter region of IRF4, contributing to a dramatic suppression of IRF4 gene expression. Correspondingly, we also found BCL6 expression to be positively and inversely correlated with vIRF4 and IRF4 expression, respectively, during KSHV reactivation. Finally, we observed that these processes require efficient KSHV lytic replication. Thus, our findings suggest a crucial role of the BCL6-IRF4 axis in triggering the transition between KSHV latency and lytic reactivation.
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Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Humano 8/metabolismo , Factores Reguladores del Interferón/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Activación Transcripcional/fisiología , Proteínas Virales/metabolismo , Activación Viral/fisiología , Latencia del Virus/fisiología , Regulación hacia Abajo , Replicación Viral/fisiologíaRESUMEN
Viroporin p7 of the hepatitis C virus (HCV) acts as an ion channel for pH equilibration to stabilize HCV particles; most studies of p7 have focused on this role. However, pH equilibration by p7 via its ion channel activity does not fully explain the importance of p7 in HCV particle production. Indeed, several researchers have suggested p7 to have an unidentified ion channel-independent function. Here, we show that p7 has a novel role as a lipid raft adhesion factor, which is independent of its ion channel activity. We found that p7 targets not only the liquid-disordered (Ld) phase, but also the negatively-charged liquid-ordered (Lo) phase that can be represented as a lipid raft. p7 clusters at the phase boundary of the neutral Ld phase and the negatively-charged Lo phase. Interestingly, p7 targeting the Lo phase facilitates membrane-to-membrane adhesion, and this activity is not inhibited by p7 ion channel inhibitors. Our results demonstrated that HCV p7 has dual roles as a viroporin and as a lipid raft adhesion factor. This ion channel-independent function of p7 might be an attractive target for development of anti-HCV compounds.
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Hepacivirus/genética , Hepatitis C/genética , Proteínas Virales/genética , Secuencia de Aminoácidos/genética , Adhesión Celular/genética , Línea Celular , Regulación Viral de la Expresión Génica , Hepacivirus/metabolismo , Hepacivirus/patogenicidad , Hepatitis C/patología , Hepatitis C/virología , Humanos , Concentración de Iones de Hidrógeno , Microdominios de Membrana/genética , Microdominios de Membrana/metabolismo , Proteínas Virales/metabolismo , Replicación Viral/genéticaRESUMEN
Before an infection can be completely established, the host immediately turns on the innate immune system through activating the interferon (IFN)-mediated antiviral pathway. Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes a unique antagonistic mechanism of type I IFN-mediated host antiviral immunity by incorporating four viral interferon regulatory factors (vIRF1-4). Herein, we characterized novel immune evasion strategies of vIRF4 to inhibit the IRF7-mediated IFN-α production. KSHV vIRF4 specifically interacts with IRF7, resulting in inhibition of IRF7 dimerization and ultimately suppresses IRF7-mediated activation of type I IFN. These results suggest that each of the KSHV vIRFs, including vIRF4, subvert IFN-mediated anti-viral response via different mechanisms. Therefore, it is indicated that KSHV vIRFs are indeed a crucial immunomodulatory component of their life cycles.
Asunto(s)
Herpesvirus Humano 8/inmunología , Evasión Inmune , Factor 7 Regulador del Interferón/inmunología , Factores Reguladores del Interferón/inmunología , Interferón-alfa/inmunología , Proteínas Virales/inmunología , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , Herpesvirus Humano 8/química , Humanos , Inmunidad Innata , Factor 7 Regulador del Interferón/genética , Factores Reguladores del Interferón/genética , Interferón-alfa/antagonistas & inhibidores , Interferón-alfa/genética , Luciferasas/genética , Luciferasas/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Virus Sendai/genética , Virus Sendai/inmunología , Transducción de Señal , Transfección , Proteínas Virales/genéticaRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the ß-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G1-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G1-S transition, possibly providing an intracellular milieu favorable for its replication.
Asunto(s)
Puntos de Control del Ciclo Celular , Genes bcl-1 , Herpesvirus Humano 8/fisiología , Interacciones Huésped-Patógeno , Factores Reguladores del Interferón/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Sialoglicoproteínas/antagonistas & inhibidores , Proteínas Virales/metabolismo , beta Catenina/antagonistas & inhibidores , Regulación hacia Abajo , Replicación ViralRESUMEN
Transcription of herpesvirus late genes depends on several virus-encoded proteins whose function is not completely understood. Here, we identify a viral trimeric complex of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 31 (ORF31), ORF24, and ORF34 that is required for late gene expression but not viral DNA replication. We found that (i) ORF34 bridges the interaction between ORF31 and ORF24, (ii) the amino-terminal cysteine-rich and carboxyl-terminal basic domains of ORF31 mediate the ORF31-ORF34 interaction required for late gene expression, and (iii) a complex consisting of ORF24, ORF31, and ORF34 specifically binds to the K8.1 late promoter. Together, our results support the model that a subset of lytic viral proteins assembles into a transcriptional activator complex to induce expression of late genes.
Asunto(s)
Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/genética , Multimerización de Proteína , Proteínas Virales/metabolismo , Humanos , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
Besides an essential transcriptional factor for B cell development and function, cellular interferon regulatory factor 4 (c-IRF4) directly regulates expression of the c-Myc gene, which is not only associated with various B cell lymphomas but also required for herpesvirus latency and pathogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma and primary effusion lymphoma, has developed a unique mechanism to deregulate host antiviral innate immunity and growth control by incorporating four viral homologs (vIRF1 to -4) of cellular IRFs into its genome. Previous studies have shown that several KSHV latent proteins, including vIRF3, vFLIP, and LANA, target the expression, function, and stability of c-Myc to establish and maintain viral latency. Here we report that the KSHV vIRF4 lytic protein robustly suppresses expression of c-IRF4 and c-Myc, reshaping host gene expression profiles to facilitate viral lytic replication. Genomewide gene expression analysis revealed that KSHV vIRF4 grossly affects host gene expression by upregulating and downregulating 118 genes and 166 genes, respectively, by at least 2-fold. Remarkably, vIRF4 suppressed c-Myc expression by 11-fold, which was directed primarily by the deregulation of c-IRF4 expression. Real-time quantitative PCR (RT-qPCR), single-molecule in situ hybridization, and chromatin immunoprecipitation assays showed that vIRF4 not only reduces c-IRF4 expression but also competes with c-IRF4 for binding to the specific promoter region of the c-Myc gene, resulting in drastic suppression of c-Myc expression. Consequently, the loss of vIRF4 function in the suppression of c-IRF4 and c-Myc expression ultimately led to a reduction of KSHV lytic replication capacity. These results indicate that the KSHV vIRF4 lytic protein comprehensively targets the expression and function of c-IRF4 to downregulate c-Myc expression, generating a favorable environment for viral lytic replication. Finally, this study further reinforces the important role of the c-Myc gene in KSHV lytic replication and latency.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , Factores Reguladores del Interferón/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Virales/metabolismo , Replicación Viral/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Regulación Viral de la Expresión Génica/genética , Humanos , Immunoblotting , Hibridación Fluorescente in Situ , Análisis por Micromatrices , Plásmidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The establishment of latency is an essential step for the life-long persistent infection and pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). While the KSHV genome is chromatin-free in the virions, the viral DNA in latently infected cells has a chromatin structure with activating and repressive histone modifications that promote latent gene expression but suppress lytic gene expression. Here, we report a comprehensive epigenetic study of the recruitment of chromatin regulatory factors onto the KSHV genome during the pre-latency phase of KSHV infection. This demonstrates that the KSHV genome undergoes a biphasic chromatinization following de novo infection. Initially, a transcriptionally active chromatin (euchromatin), characterized by high levels of the H3K4me3 and acetylated H3K27 (H3K27ac) activating histone marks, was deposited on the viral episome and accompanied by the transient induction of a limited number of lytic genes. Interestingly, temporary expression of the RTA protein facilitated the increase of H3K4me3 and H3K27ac occupancy on the KSHV episome during de novo infection. Between 24-72 hours post-infection, as the levels of these activating histone marks declined on the KSHV genome, the levels of the repressive H3K27me3 and H2AK119ub histone marks increased concomitantly with the decline of lytic gene expression. Importantly, this transition to heterochromatin was dependent on both Polycomb Repressive Complex 1 and 2. In contrast, upon infection of human gingiva-derived epithelial cells, the KSHV genome underwent a transcription-active euchromatinization, resulting in efficient lytic gene expression. Our data demonstrate that the KSHV genome undergoes a temporally-ordered biphasic euchromatin-to-heterochromatin transition in endothelial cells, leading to latent infection, whereas KSHV preferentially adopts a transcriptionally active euchromatin in oral epithelial cells, resulting in lytic gene expression. Our results suggest that the differential epigenetic modification of the KSHV genome in distinct cell types is a potential determining factor for latent infection versus lytic replication of KSHV.
Asunto(s)
Eucromatina/genética , Genoma Viral , Infecciones por Herpesviridae/genética , Herpesvirus Humano 8/genética , Heterocromatina/genética , Latencia del Virus/genética , Células Cultivadas , Ensamble y Desensamble de Cromatina/fisiología , Regulación Viral de la Expresión Génica , Células HEK293 , Infecciones por Herpesviridae/virología , Histonas/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Genome-wide chromatin immunoprecipitation assays indicate that the promoter-proximal pausing of RNA polymerase II (RNAPII) is an important postinitiation step for gene regulation. During latent infection, the majority of Kaposi's sarcoma-associated herpesvirus (KSHV) genes is silenced via repressive histone marks on their promoters. Despite the absence of their expression during latency, however, several lytic promoters are enriched with activating histone marks, suggesting that mechanisms other than heterochromatin-mediated suppression contribute to preventing lytic gene expression. Here, we show that the RNAPII-mediated transcription of the KSHV OriLytL, K5, K6, and K7 (OriLytL-K7) lytic genes is paused at the elongation step during latency. Specifically, the RNAPII-mediated transcription is stalled by the host's negative elongation factor (NELF) at the promoter regions of OriLytL-K7 lytic genes during latency, leading to the hyperphosphorylation of the serine 5 residue and the hypophosphorylation of the serine 2 of the C-terminal domain of the RNAPII large subunit, a hallmark of stalled RNAPII. Consequently, depletion of NELF expression induced transition of stalled RNAPII into a productive transcription elongation at the promoter-proximal regions of OriLytL-K7 lytic genes, leading to their RTA-independent expression. Using an RTA-deficient recombinant KSHV, we also showed that expression of the K5, K6, and K7 lytic genes was highly inducible upon external stimuli compared to other lytic genes that lack RNAPII on their promoters during latency. These results indicate that the transcription elongation of KSHV OriLytL-K7 lytic genes is inhibited by NELF during latency, but can also be promptly reactivated in an RTA-independent manner upon external stimuli.
Asunto(s)
Herpesvirus Humano 8/fisiología , ARN Polimerasa II/fisiología , Factores de Transcripción/fisiología , Secuencia de Bases , Línea Celular , Regulación Viral de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Virales , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/patogenicidad , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/fisiología , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Transactivadores/fisiología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Latencia del Virus/genética , Latencia del Virus/fisiologíaRESUMEN
Efficient genetic modification of herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) has come to rely on bacterial artificial chromosome (BAC) technology. In order to facilitate this approach, we generated a new KSHV BAC clone, called BAC16, derived from the rKSHV.219 virus, which stems from KSHV and Epstein-Barr virus-coinfected JSC1 primary effusion lymphoma (PEL) cells. Restriction enzyme and complete sequencing data demonstrate that the KSHV of JSC1 PEL cells showed a minimal level of sequence variation across the entire viral genome compared to the complete genomic sequence of other KSHV strains. BAC16 not only stably propagated in both Escherichia coli and mammalian cells without apparent genetic rearrangements, but also was capable of robustly producing infectious virions (â¼5 × 10(7)/ml). We also demonstrated the utility of BAC16 by generating deletion mutants of either the K3 or K5 genes, whose products are E3 ligases of the membrane-associated RING-CH (MARCH) family. While previous studies have shown that individual expression of either K3 or K5 results in efficient downregulation of the surface expression of major histocompatibility complex class I (MHC-I) molecules, we found that K5, but not K3, was the primary factor critical for the downregulation of MHC-I surface expression during KSHV lytic reactivation or following de novo infection. The data presented here demonstrate the utility of BAC16 for the generation and characterization of KSHV knockout and mutant recombinants and further emphasize the importance of functional analysis of viral genes in the context of the KSHV genome besides the study of individual gene expression.