RESUMEN
Epstein-Barr virus (EBV) infection can engender severe B cell lymphoproliferative diseases1,2. The primary infection is often asymptomatic or causes infectious mononucleosis (IM), a self-limiting lymphoproliferative disorder3. Selective vulnerability to EBV has been reported in association with inherited mutations impairing T cell immunity to EBV4. Here we report biallelic loss-of-function variants in IL27RA that underlie an acute and severe primary EBV infection with a nevertheless favourable outcome requiring a minimal treatment. One mutant allele (rs201107107) was enriched in the Finnish population (minor allele frequency = 0.0068) and carried a high risk of severe infectious mononucleosis when homozygous. IL27RA encodes the IL-27 receptor alpha subunit5,6. In the absence of IL-27RA, phosphorylation of STAT1 and STAT3 by IL-27 is abolished in T cells. In in vitro studies, IL-27 exerts a synergistic effect on T-cell-receptor-dependent T cell proliferation7 that is deficient in cells from the patients, leading to impaired expansion of potent anti-EBV effector cytotoxic CD8+ T cells. IL-27 is produced by EBV-infected B lymphocytes and an IL-27RA-IL-27 autocrine loop is required for the maintenance of EBV-transformed B cells. This potentially explains the eventual favourable outcome of the EBV-induced viral disease in patients with IL-27RA deficiency. Furthermore, we identified neutralizing anti-IL-27 autoantibodies in most individuals who developed sporadic infectious mononucleosis and chronic EBV infection. These results demonstrate the critical role of IL-27RA-IL-27 in immunity to EBV, but also the hijacking of this defence by EBV to promote the expansion of infected transformed B cells.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Interleucina-27 , Receptores de Interleucina , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Adulto Joven , Alelos , Linfocitos B/patología , Linfocitos B/virología , Linfocitos T CD8-positivos/patología , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/terapia , Finlandia , Frecuencia de los Genes , Herpesvirus Humano 4 , Homocigoto , Mononucleosis Infecciosa/complicaciones , Mononucleosis Infecciosa/genética , Mononucleosis Infecciosa/terapia , Interleucina-27/inmunología , Interleucina-27/metabolismo , Mutación con Pérdida de Función , Receptores de Interleucina/deficiencia , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Epigénesis Genética , Infecciones por Virus de Epstein-Barr , Regulación Viral de la Expresión Génica , Centro Germinal , Herpesvirus Humano 4 , Latencia del Virus , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Humanos , Centro Germinal/inmunología , Centro Germinal/virología , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/inmunología , Citocinas/metabolismo , Linfocitos B/virología , Linfocitos B/metabolismoRESUMEN
Epstein-Barr Virus (EBV) is associated with numerous cancers including B cell lymphomas. In vitro, EBV transforms primary B cells into immortalized Lymphoblastoid Cell Lines (LCLs) which serves as a model to study the role of viral proteins in EBV malignancies. EBV induced cellular transformation is driven by viral proteins including EBV-Nuclear Antigens (EBNAs). EBNA-LP is important for the transformation of naïve but not memory B cells. While EBNA-LP was thought to promote gene activation by EBNA2, EBNA-LP Knockout (LPKO) virus-infected cells express EBNA2-activated cellular genes efficiently. Therefore, a gap in knowledge exists as to what roles EBNA-LP plays in naïve B cell transformation. We developed a trans-complementation assay wherein transfection with wild-type EBNA-LP rescues the transformation of peripheral blood- and cord blood-derived naïve B cells by LPKO virus. Despite EBNA-LP phosphorylation sites being important in EBNA2 co-activation; neither phospho-mutant nor phospho-mimetic EBNA-LP was defective in rescuing naïve B cell outgrowth. However, we identified conserved leucine-rich motifs in EBNA-LP that were required for transformation of adult naïve and cord blood B cells. Because cellular PPAR-g coactivator (PGC) proteins use leucine-rich motifs to engage transcription factors including YY1, a key regulator of DNA looping and metabolism, we examined the role of EBNA-LP in engaging transcription factors. We found a significant overlap between EBNA-LP and YY1 in ChIP-Seq data. By Cut&Run, YY1 peaks unique to WT compared to LPKO LCLs occur at more highly expressed genes. Moreover, Cas9 knockout of YY1 in primary B cells prior to EBV infection indicated YY1 to be important for EBV-mediated transformation. We confirmed EBNA-LP and YY1 biochemical association in LCLs by endogenous co-immunoprecipitation and found that the EBNA-LP leucine-rich motifs were required for YY1 interaction in LCLs. We propose that EBNA-LP engages YY1 through conserved leucine-rich motifs to promote EBV transformation of naïve B cells.
Asunto(s)
Linfocitos B , Transformación Celular Viral , Herpesvirus Humano 4 , Proteínas Virales , Factor de Transcripción YY1 , Humanos , Linfocitos B/virología , Linfocitos B/metabolismo , Linfocitos B/inmunología , Factor de Transcripción YY1/metabolismo , Proteínas Virales/metabolismo , Proteínas Virales/genética , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Secuencias de Aminoácidos , Leucina/metabolismoRESUMEN
Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.
Asunto(s)
Linfocitos B , Linfoma de Burkitt , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Proteínas Proto-Oncogénicas c-myc , Latencia del Virus , Animales , Linfoma de Burkitt/virología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Linfoma de Burkitt/genética , Humanos , Ratones , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Linfocitos B/virología , Linfocitos B/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Apoptosis , Proteínas Virales/metabolismo , Proteínas Virales/genéticaRESUMEN
Epstein-Barr virus (EBV) is a human tumor virus associated with a variety of malignancies, including nasopharyngeal carcinoma, gastric cancers, and B-cell lymphomas. N6-methyladenosine (m6A) modifications modulate a wide range of cellular processes and participate in the regulation of virus-host cell interactions. Here, we discovered that EBV infection downregulates toll-like receptor 9 (TLR9) m6A modification levels and thus inhibits TLR9 expression. TLR9 has multiple m6A modification sites. Knockdown of METTL3, an m6A "writer", decreases TLR9 protein expression by inhibiting its mRNA stability. Mechanistically, Epstein-Barr nuclear antigen 1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway. Additionally, YTHDF1 was identified as an m6A "reader" of TLR9, enhancing TLR9 expression by promoting mRNA translation in an m6A -dependent manner, which suggests that EBV inhibits TLR9 translation by "hijacking" host m6A modification mechanism. Using the METTL3 inhibitor STM2457 inhibits TLR9-induced B cell proliferation and immunoglobulin secretion, and opposes TLR9-induced immune responses to assist tumor cell immune escape. In clinical lymphoma samples, the expression of METTL3, YTHDF1, and TLR9 was highly correlated with immune cells infiltration. This study reveals a novel mechanism that EBV represses the important innate immunity molecule TLR9 through modulating the host m6A modification system.
Asunto(s)
Adenosina , Herpesvirus Humano 4 , Metiltransferasas , Proteínas de Unión al ARN , Receptor Toll-Like 9 , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Linfocitos B/metabolismo , Linfocitos B/inmunología , Linfocitos B/virología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Evasión Inmune , Metiltransferasas/metabolismo , Metiltransferasas/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/genética , Línea Celular TumoralRESUMEN
Equine herpesvirus type 1 (EHV-1) is a contagious respiratory pathogen that infects the mucosa of the upper respiratory tract (URT). Mucosal immune responses at the URT provide the first line of defense against EHV-1 and are crucial for orchestrating immunity. To define host-pathogen interactions, we characterized B-cell responses, antibody isotype functions, and EHV-1 replication of susceptible (non-immune) and clinically protected (immune) horses after experimental EHV-1 infection. Nasal secretion and nasal wash samples were collected and used for the isolation of DNA, RNA, and mucosal antibodies. Shedding of infectious virus, EHV-1 copy numbers, viral RNA expression, and host B-cell activation in the URT were compared based on host immune status. Mucosal EHV-1-specific antibody responses were associated with EHV-1 shedding and viral RNA transcription. Finally, mucosal immunoglobulin G (IgG) and IgA isotypes were purified and tested for neutralizing capabilities. IgG1 and IgG4/7 neutralized EHV-1, while IgG3/5, IgG6, and IgA did not. Immune horses secreted high amounts of mucosal EHV-1-specific IgG4/7 antibodies and quickly upregulated B-cell pathway genes, while EHV-1 was undetected by virus isolation and PCR. RNA transcription analysis reinforced incomplete viral replication in immune horses. In contrast, complete viral replication with high viral copy numbers and shedding of infectious viruses was characteristic for non-immune horses, together with low or absent EHV-1-specific neutralizing antibodies during viral replication. These data confirm that pre-existing mucosal IgG1 and IgG4/7 and rapid B-cell activation upon EHV-1 infection are essential for virus neutralization, regulation of viral replication, and mucosal immunity against EHV-1.IMPORTANCEEquine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion storms, and neurologic outbreaks known as equine herpes myeloencephalopathy (EHM). EHV-1 is transmitted with respiratory secretions by nose-to-nose contact or via fomites. The virus initially infects the epithelium of the upper respiratory tract (URT). Host-pathogen interactions and mucosal immunity at the viral entry site provide the first line of defense against the EHV-1. Robust mucosal immunity can be essential in protecting against EHV-1 and to reduce EHM outbreaks. It has previously been shown that immune horses do not establish cell-associated viremia, the prerequisite for EHM. Here, we demonstrate how mucosal antibodies can prevent the replication of EHV-1 at the epithelium of the URT and, thereby, the progression of the virus to the peripheral blood. The findings improve the mechanistic understanding of mucosal immunity against EHV-1 and can support the development of enhanced diagnostic tools, vaccines against EHM, and the management of EHV-1 outbreaks.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Infecciones por Herpesviridae , Herpesvirus Équido 1 , Enfermedades de los Caballos , Inmunoglobulina G , Replicación Viral , Animales , Herpesvirus Équido 1/inmunología , Caballos , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/veterinaria , Infecciones por Herpesviridae/virología , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Enfermedades de los Caballos/virología , Enfermedades de los Caballos/inmunología , Inmunoglobulina G/inmunología , Inmunidad Mucosa , Esparcimiento de Virus/inmunología , Linfocitos B/inmunología , Linfocitos B/virología , Interacciones Huésped-Patógeno/inmunologíaRESUMEN
Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt's lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50-100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF)FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers. IMPORTANCE: The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr , Proteínas F-Box , Herpesvirus Humano 4 , Proteínas Proto-Oncogénicas c-bcl-6 , Humanos , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/genética , Línea Celular Tumoral , Linfocitos B/metabolismo , Linfocitos B/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Proteolisis , Proliferación Celular , Ubiquitinación , Linfoma de Burkitt/virología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , Antígenos Virales/metabolismo , Antígenos Virales/genética , Centro Germinal/metabolismo , Centro Germinal/virología , Proteína-Arginina N-MetiltransferasasRESUMEN
Epstein-Barr virus (EBV) is a highly successful pathogen that infects ~95% of the adult population and is associated with diverse cancers and autoimmune diseases. The most abundant viral factor in latently infected cells is not a protein but a noncoding RNA called EBV-encoded RNA 1 (EBER1). Even though EBER1 is highly abundant and was discovered over forty years ago, the function of EBER1 has remained elusive. EBER1 interacts with the ribosomal protein L22, which normally suppresses the expression of its paralog L22-like 1 (L22L1). Here we show that when L22 binds EBER1, it cannot suppress L22L1, resulting in L22L1 being expressed and incorporated into ribosomes. We further show that L22L1-containing ribosomes preferentially translate mRNAs involved in the oxidative phosphorylation pathway. Moreover, upregulation of L22L1 is indispensable for growth transformation and immortalization of resting B cells upon EBV infection. Taken together, our results suggest that the function of EBER1 is to modulate host gene expression at the translational level, thus bypassing the need for dysregulating host gene transcription.
Asunto(s)
Herpesvirus Humano 4 , Fosforilación Oxidativa , ARN Viral , Proteínas Ribosómicas , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Humanos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , ARN Viral/genética , ARN Viral/metabolismo , Linfocitos B/virología , Interacciones Huésped-Patógeno/genética , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Ribosomas/metabolismo , Ribosomas/genética , Proteínas de Unión al ARNRESUMEN
Epstein-Barr virus (EBV) is a ubiquitous and predominantly B cell tropic virus. One of the most common viruses to infect humans, EBV, is best known as the causative agent of infectious mononucleosis (IM). Although most people experience asymptomatic infection, EBV is a potent immune stimulus and as such it elicits robust proliferation and activation of the B-lymphocytes it infects as well as the immune cells that respond to infection. In certain individuals, such as those with inherited or acquired defects affecting the immune system, failure to properly control EBV leads to the accumulation of EBV-infected B cells and EBV-reactive immune cells, which together contribute to the development of often life-threatening cytokine storm syndromes (CSS). Here, we review the normal immune response to EBV and discuss several CSS associated with EBV, such as chronic active EBV infection, hemophagocytic lymphohistiocytosis, and post-transplant lymphoproliferative disorder. Given the critical role for cytokines in driving inflammation and contributing to disease pathogenesis, we also discuss how targeting specific cytokines provides a rational and potentially less toxic treatment for EBV-driven CSS.
Asunto(s)
Síndrome de Liberación de Citoquinas , Citocinas , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Humanos , Síndrome de Liberación de Citoquinas/inmunología , Síndrome de Liberación de Citoquinas/virología , Herpesvirus Humano 4/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/complicaciones , Citocinas/inmunología , Citocinas/metabolismo , Linfohistiocitosis Hemofagocítica/inmunología , Linfohistiocitosis Hemofagocítica/virología , Linfocitos B/inmunología , Linfocitos B/virología , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/virología , AnimalesRESUMEN
The present study analyzed B-cell clonality and bovine leukemia virus (BLV) provirus integration sites in cattle with enzootic bovine leukosis (EBL) having BLV proviral copy numbers less or greater than the number of bovine nucleated cells. EBL cattle with BLV copy numbers less than the number of bovine nucleated cells showed monoclonal and biclonal proliferation of B-cells with one BLV provirus integration site. On the other hand, EBL cattle with BLV copy numbers greater than the number of bovine nucleated cells showed monoclonal proliferation of B-cells with two BLV provirus integration sites. These results suggest that superinfection of BLV can occur in EBL cattle.
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Linfocitos B , ADN Viral , Leucosis Bovina Enzoótica , Virus de la Leucemia Bovina , Provirus , Animales , Virus de la Leucemia Bovina/genética , Leucosis Bovina Enzoótica/virología , Bovinos , Provirus/genética , ADN Viral/genética , Linfocitos B/virología , Integración Viral , Proliferación CelularRESUMEN
When Epstein-Barr virus (EBV) infection is suspected, identification of infected cells is important to understand the pathogenesis, determinine the treatment strategy, and predict the prognosis. We used the PrimeFlow™ RNA Assay Kit with a probe to detect EBV-encoded small RNAs (EBERs) and multiple surface markers, to identify EBV-infected cells by flow cytometry. We analyzed a total of 24 patients [11 with chronic active EBV disease (CAEBV), 3 with hydroa vacciniforme lymphoproliferative disorder, 2 with X-linked lymphoproliferative disease type 1 (XLP1), 2 with EBV-associated hemophagocytic lymphohistiocytosis, and 6 with posttransplant lymphoproliferative disorder (PTLD)]. We compared infected cells using conventional quantitative PCR methods and confirmed that infected cell types were identical in most patients. Patients with CAEBV had widespread infection in T and NK cells, but a small amount of B cells were also infected, and infection in patients with XLP1 and PTLD was not limited to B cells. EBV-associated diseases are believed to be complex pathologies caused by EBV infecting a variety of cells other than B cells. We also demonstrated that infected cells were positive for HLA-DR in patients with CAEBV. EBER flow FISH can identify EBV-infected cells with high sensitivity and is useful for elucidating the pathogenesis.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Humanos , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/genética , Niño , Masculino , Femenino , Preescolar , Hibridación Fluorescente in Situ , Adolescente , Trastornos Linfoproliferativos/virología , Trastornos Linfoproliferativos/diagnóstico , Trastornos Linfoproliferativos/etiología , ARN Viral/análisis , Citometría de Flujo/métodos , Linfocitos B/virología , Adulto , Sensibilidad y Especificidad , Lactante , Células Asesinas Naturales/virologíaRESUMEN
The human immunodeficiency virus (HIV-1) matrix protein p17 (p17) is released from infected cells as a protein capable of deregulating the biological activity of different cells. P17 variants (vp17s), more frequently detected in the plasma of HIV-1+ patients with rather than without lymphoma and characterized by amino acids insertions in their C-terminal region, were found to trigger B cell growth and clonogenicity. Vp17s endowed with B-cell-growth-promoting activity are drastically destabilized, whereas, in a properly folded state, reference p17 (refp17) does not exert any biological activity on B cell growth and clonogenicity. However, misfolding of refp17 is necessary to expose a masked functional epitope, interacting with the protease-activated receptor 1 (PAR-1), endowed with B cell clonogenicity. Indeed, it is worth noting that changes in the secondary structure can strongly impact the function of a protein. Here, we performed computational studies to show that the gain of function of vp17s is linked to dramatic conformational changes due to structural modification in the secondary-structure elements and in the rearrangement of the hydrogen bond (H-bond) network. In particular, all clonogenic vp17s showed the disengagement of two critical residues, namely Trp16 and Tyr29, from their hydrophobic core. Biological data showed that the mutation of Trp16 and Tyr29 to Ala in the refp17 backbone, alone or in combination, resulted in a protein endowed with B cell clonogenic activity. These data show the pivotal role of the hydrophobic component in maintaining refp17 stability and identify a novel potential therapeutic target to counteract vp17-driven lymphomagenesis in HIV-1+ patients.
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Linfocitos B , Antígenos VIH , VIH-1 , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Humanos , Antígenos VIH/genética , Antígenos VIH/metabolismo , Antígenos VIH/química , VIH-1/genética , VIH-1/fisiología , Linfocitos B/virología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Infecciones por VIH/virología , Proliferación Celular , Pliegue de ProteínaRESUMEN
Hematopoiesis is a tightly regulated process that gets skewed toward myelopoiesis. This restrains lymphopoiesis, but the role of lymphocytes in this process is not well defined. To unravel the intricacies of neutrophil responses in COVID-19, we performed bulk RNAseq on neutrophils from healthy controls and COVID-19 patients. Principal component analysis revealed distinguishing neutrophil gene expression alterations in COVID-19 patients. ICU and ward patients displayed substantial transcriptional changes, with ICU patients exhibiting a more pronounced response. Intriguingly, neutrophils from COVID-19 patients, notably ICU patients, exhibited an enrichment of immunoglobulin (Ig) and B cell lineage-associated genes, suggesting potential lineage plasticity. We validated our RNAseq findings in a larger cohort. Moreover, by reanalyzing single-cell RNA sequencing (scRNAseq) data on human bone marrow (BM) granulocytes, we identified the cluster of granulocyte-monocyte progenitors (GMP) enriched with Ig and B cell lineage-associated genes. These cells with lineage plasticity may serve as a resource depending on the host's needs during severe systemic infection. This distinct B cell subset may play a pivotal role in promoting myelopoiesis in response to infection. The scRNAseq analysis of BM neutrophils in infected mice further supported our observations in humans. Finally, our studies using an animal model of acute infection implicate IL-7/GM-CSF in influencing neutrophil and B cell dynamics. Elevated GM-CSF and reduced IL-7 receptor expression in COVID-19 patients imply altered hematopoiesis favoring myeloid cells over B cells. Our findings provide novel insights into the relationship between the B-neutrophil lineages during severe infection, hinting at potential implications for disease pathogenesis. IMPORTANCE: This study investigates the dynamics of hematopoiesis in COVID-19, focusing on neutrophil responses. Through RNA sequencing of neutrophils from healthy controls and COVID-19 patients, distinct gene expression alterations are identified, particularly in ICU patients. Notably, neutrophils from COVID-19 patients, especially in the ICU, exhibit enrichment of immunoglobulin and B cell lineage-associated genes, suggesting potential lineage plasticity. Validation in a larger patient cohort and single-cell analysis of bone marrow granulocytes support the presence of granulocyte-monocyte progenitors with B cell lineage-associated genes. The findings propose a link between B-neutrophil lineages during severe infection, implicating a potential role for these cells in altered hematopoiesis favoring myeloid cells over B cells. Elevated GM-CSF and reduced IL-7 receptor expression in stress hematopoiesis suggest cytokine involvement in these dynamics, providing novel insights into disease pathogenesis.
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COVID-19 , Hematopoyesis , Neutrófilos , Humanos , Animales , COVID-19/inmunología , COVID-19/virología , Ratones , Neutrófilos/inmunología , Neutrófilos/virología , Linfocitos B/inmunología , Linfocitos B/virología , SARS-CoV-2/genética , Médula Ósea/virología , Linaje de la Célula , Masculino , Femenino , Persona de Mediana Edad , Análisis de la Célula Individual , Mielopoyesis/genética , Análisis de Secuencia de ARNRESUMEN
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.
Asunto(s)
Herpesvirus Humano 4 , Histonas , Factor de Empalme Asociado a PTB , Activación Viral , Latencia del Virus , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Humanos , Histonas/metabolismo , Activación Viral/genética , Latencia del Virus/genética , Factor de Empalme Asociado a PTB/metabolismo , Factor de Empalme Asociado a PTB/genética , Regulación Viral de la Expresión Génica , Linfocitos B/virología , Linfocitos B/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Sistemas CRISPR-Cas , Regiones Promotoras Genéticas/genética , Transactivadores/metabolismo , Transactivadores/genética , Genoma ViralRESUMEN
CD150, also termed signaling lymphocyte activation molecule family member 1, is a cell surface receptor expressed on T cells, B cells, dendritic cells (DCs) and some tumors. Stimulation of CD150 on immune cells induces cell proliferation and cytokine production. However, the function of CD150 in EpsteinBarr virus (EBV)infected B cells is still not fully understood. In the present study, CD150 expression on B cells increased rapidly following EBV infection, and various CD150 antibodies, measles viral proteins and recombinant CD150 proteins induced the secretion of multiple cytokines in both CD150+ EBVtransformed B cells and EBV+ lymphoma cells. Notably, the IL1α protein level showed the greatest increase among all cytokines measured. The culture supernatant containing these cytokines induced the rapid differentiation of monocytes to DCs after only 2 days in vitro, which was faster than the established DC maturation time. Furthermore, knockdown of CD150 expression led to a reduction in the secretion of multiple cytokines, and monocyte differentiation was partially inhibited by antiIL1α and antigranulocytemacrophage colonystimulating factor neutralizing antibodies. Collectively, the results of the present study suggest that CD150 activation triggers cytokine production in EBVtransformed B cells, and that measles virus coinfection might affect immune responses through the production of various cytokines in EBV+ lymphoma cells.
Asunto(s)
Linfocitos B , Diferenciación Celular , Citocinas , Herpesvirus Humano 4 , Monocitos , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Humanos , Herpesvirus Humano 4/inmunología , Linfocitos B/metabolismo , Linfocitos B/inmunología , Linfocitos B/virología , Citocinas/metabolismo , Monocitos/metabolismo , Monocitos/inmunología , Monocitos/virología , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/virología , Activación de Linfocitos/inmunologíaRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus that is linked directly to the development of Kaposi's sarcoma. KSHV establishes a latent infection in B cells, which can be reactivated to initiate lytic replication, producing infectious virions. Using pharmacological and genetic silencing approaches, we showed that the voltage-gated K+ channel Kv1.3 in B cells enhanced KSHV lytic replication. The KSHV replication and transcription activator (RTA) protein increased the abundance of Kv1.3 and led to enhanced K+ channel activity and hyperpolarization of the B cell membrane. Enhanced Kv1.3 activity promoted intracellular Ca2+ influx, leading to the Ca2+-driven nuclear localization of KSHV RTA and host nuclear factor of activated T cells (NFAT) proteins and subsequently increased the expression of NFAT1 target genes. KSHV lytic replication and infectious virion production were inhibited by Kv1.3 blockers or silencing. These findings highlight Kv1.3 as a druggable host factor that is key to the successful completion of KSHV lytic replication.
Asunto(s)
Herpesvirus Humano 8 , Canal de Potasio Kv1.3 , Factores de Transcripción NFATC , Replicación Viral , Herpesvirus Humano 8/fisiología , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/antagonistas & inhibidores , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Inmediatas-Precoces/genética , Transactivadores/metabolismo , Transactivadores/genética , Linfocitos B/virología , Linfocitos B/metabolismo , Calcio/metabolismo , Sarcoma de Kaposi/virología , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/genéticaRESUMEN
Epstein-Barr virus (EBV) is an aetiologic risk factor for the development of multiple sclerosis (MS). However, the role of EBV-infected B cells in the immunopathology of MS is not well understood. Here we characterized spontaneous lymphoblastoid cell lines (SLCLs) isolated from MS patients and healthy controls (HC) ex vivo to study EBV and host gene expression in the context of an individual's endogenous EBV. SLCLs derived from MS patient B cells during active disease had higher EBV lytic gene expression than SLCLs from MS patients with stable disease or HCs. Host gene expression analysis revealed activation of pathways associated with hypercytokinemia and interferon signalling in MS SLCLs and upregulation of forkhead box protein 1 (FOXP1), which contributes to EBV lytic gene expression. We demonstrate that antiviral approaches targeting EBV replication decreased cytokine production and autologous CD4+ T cell responses in this ex vivo model. These data suggest that dysregulation of intrinsic B cell control of EBV gene expression drives a pro-inflammatory, pathogenic B cell phenotype that can be attenuated by suppressing EBV lytic gene expression.
Asunto(s)
Linfocitos B , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Esclerosis Múltiple , Humanos , Herpesvirus Humano 4/genética , Esclerosis Múltiple/virología , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/virología , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/complicaciones , Citocinas/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD4-Positivos/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Transcriptoma , Replicación Viral , Regulación Viral de la Expresión Génica , Línea Celular , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Perfilación de la Expresión Génica , Adulto , Femenino , MasculinoRESUMEN
Epstein-Barr virus (EBV), the first virus found to induce cancer in humans, has been frequently detected in various types of B cell lymphomas. During its latent phase, EBV expresses a limited set of proteins crucial for its persistence. Induction of the lytic phase of EBV has shown promise in the treatment of EBV-associated malignancies. The present study assessed the ability of phomaherbarine A, a novel compound derived from the endophytic fungus Phoma herbarum DBE-M1, to stimulate lytic replication of EBV in B95-8 cells. Phomaherbarine A was found to efficiently initiate the expression of both early and late EBV lytic genes in B95-8 cells, with this initiation being further heightened by the addition of phorbol myristate acetate and sodium butyrate. Moreover, phomaherbarine A demonstrated notable cytotoxicity against the EBV-associated B cell lymphoma cell lines B95-8 and Raji. Mechanistically, phomaherbarine A induces apoptosis in these cells through the activation of caspase-3/7. When combined with ganciclovir, phomaherbarine A does not interfere with the reduction of viral replication by ganciclovir and sustains its apoptosis induction. In conclusion, these findings indicate that phomaherbarine A may be a promising candidate for therapeutic intervention in patients with EBV-associated B cell lymphomas.
Asunto(s)
Apoptosis , Linfocitos B , Herpesvirus Humano 4 , Activación Viral , Humanos , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/fisiología , Activación Viral/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos B/virología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Replicación Viral/efectos de los fármacos , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Antivirales/farmacología , Ascomicetos/efectos de los fármacos , Linfoma de Células B/virología , Linfoma de Células B/tratamiento farmacológico , Latencia del Virus/efectos de los fármacosRESUMEN
Antecedentes. El tratamiento de la infección por VIH-1 con terapia antirretroviral de alta eficiencia (TAAE) promueve la supervisión viral, el incremento de las cifras de las células T CD4+ y el mejoramiento de numerosas funciones inmunes. Objetivos. Comparar los niveles séricos de IgG, IgA e IgM en individuos con infección VIH/SIDA tratados con TAAE en relación con los niveles detectados en individuos sin TAAE y establecer relaciones entre niveles de inmunoglobulinas (Ig) y el estado inmunológico y virológico. Población. En este estudio transversal participaron 69 individuos, que fueron clasificados en 2 grupos según recibieran o no TAAE y subclasificados según el nivel de linfocitos T CD4+ (<200 CD4/mL o ò500 CD4/mL). El grupo de control estuvo integrado por 20 sujetos que concurrieron para análisis prenupciales. Resultados. En pacientes sin TAAE hubo aumento de IgG, IgA eIgM respecto de los controles, correlación negativa entre niveles de Ig y cifras de linfocitos T CD4+ y correlación positiva entre carga viral y niveles séricos de IgA, mientras que en pacientes con tratamiento solo hubo incremento de IgG e IgM. En pacientes sin TAAE con <200 CD4/mL hubo un aumento de IgA e IgM respecto de los pacientes con TAAE y similares niveles de linfocitos T CD4+. En pacientes sin tratamiento con ò500 CD4/mL hubo aumento de IgG e IgM respecto de los pacientes con tratamiento y ò500 CD4/mL. Conclusión. Estos hallazgos sugieren una modulación de la activación policlonal de las células B por la TAAE en el contexto de la recuperación inmunológica y la reducción de la replicación viral (AU)
Asunto(s)
Estudio Comparativo , Humanos , Masculino , Adulto , Persona de Mediana Edad , Síndrome de Inmunodeficiencia Adquirida/inmunología , Inmunoglobulinas/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Inmunoglobulinas/sangre , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Inhibidores de la Proteasa del VIH/uso terapéutico , Linfocitos B/virología , Carga Viral , Linfocitos T CD4-Positivos/efectos de los fármacos , Fármacos Anti-VIH , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , Resultado del Tratamiento , Sistema Inmunológico/efectos de los fármacosRESUMEN
Un paciente de 43 años de edad consultó por una historia de dos años con episodios recurrentes de urticaria en miembros y tronco, y edema de labios y párpados. Se le realizaron varios estudios de diagnóstico, incluyendo mediciones del C1 inhibidor y análisis de marcadores para infecciones virales, entre otras cosas. Los resultados fueron dentro de los parámetros normales salvo los títulos de IgG anti-EBV que resultaron positivos en dilucione de 1/80. Se efectuó la técnica de transcripción reversa y PCR (RT-PCR) para determinar la presencia de EBV en forma replicativa, y se determinó la presencia de ARN con secuencias del gen BLLF1. El paciente recibió un tratamiento durante 40 días con acyclovir 200 mg 4 veces al día. Al final de dicho período, los síntomas (urticaria y angioedema) desaparecieron por completo, y la técnica de RT-PCR resultó negativa para el gen BLLF1 del virus de Epstein-Barr. Sobre las bases del seguimiento clínico y los resultados de la técnica de RT-PCR, concluimos que la infección activa por el EBV puede jugar un rol en la expresión de urticaria y angioedema en pacientes susceptibles