RESUMO
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.
Assuntos
Linfócitos B , Transformação Celular Viral , Herpesvirus Humano 4 , Proteínas Virais , Fator de Transcrição YY1 , Humanos , Linfócitos B/virologia , Linfócitos B/metabolismo , Linfócitos B/imunologia , Fator de Transcrição YY1/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Motivos de Aminoácidos , Leucina/metabolismoRESUMO
Science is humanity's best insurance against threats from nature, but it is a fragile enterprise that must be nourished and protected. The preponderance of scientific evidence indicates a natural origin for SARS-CoV-2. Yet, the theory that SARS-CoV-2 was engineered in and escaped from a lab dominates media attention, even in the absence of strong evidence. We discuss how the resulting anti-science movement puts the research community, scientific research, and pandemic preparedness at risk.
RESUMO
In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.
Assuntos
Pesquisa Biomédica , Contenção de Riscos Biológicos , Virologia , Humanos , COVID-19 , Estados Unidos , Vírus , Pesquisa Biomédica/normasRESUMO
Multiple coronaviruses (CoVs) can cause respiratory diseases in humans. While prophylactic vaccines designed to prevent infection are available for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), incomplete vaccine efficacy, vaccine hesitancy, and the threat of other pathogenic CoVs for which vaccines do not exist have highlighted the need for effective antiviral therapies. While antiviral compounds targeting the viral polymerase and protease are already in clinical use, their sensitivity to potential resistance mutations as well as their breadth against the full range of human and preemergent CoVs remain incompletely defined. To begin to fill that gap in knowledge, we report here the development of an improved, noninfectious, cell-based fluorescent assay with high sensitivity and low background that reports on the activity of viral proteases, which are key drug targets. We demonstrate that the assay is compatible with not only the SARS-CoV-2 Mpro protein but also orthologues from a range of human and nonhuman CoVs as well as clinically reported SARS-CoV-2 drug-resistant Mpro variants. We then use this assay to define the breadth of activity of two clinically used protease inhibitors, nirmatrelvir and ensitrelvir. Continued use of this assay will help define the strengths and limitations of current therapies and may also facilitate the development of next-generation protease inhibitors that are broadly active against both currently circulating and preemergent CoVs. IMPORTANCE Coronaviruses (CoVs) are important human pathogens with the ability to cause global pandemics. Working in concert with vaccines, antivirals specifically limit viral disease in people who are actively infected. Antiviral compounds that target CoV proteases are already in clinical use; their efficacy against variant proteases and preemergent zoonotic CoVs, however, remains incompletely defined. Here, we report an improved, noninfectious, and highly sensitive fluorescent method of defining the sensitivity of CoV proteases to small molecule inhibitors. We use this approach to assay the activity of current antiviral therapies against clinically reported SARS-CoV-2 protease mutants and a panel of highly diverse CoV proteases. Additionally, we show this system is adaptable to other structurally nonrelated viral proteases. In the future, this assay can be used to not only better define the strengths and limitations of current therapies but also help develop new, broadly acting inhibitors that more broadly target viral families.
Assuntos
Antivirais , Inibidores de Proteases , Proteases Virais , Humanos , Antivirais/farmacologia , COVID-19 , Inibidores de Proteases/farmacologia , SARS-CoV-2RESUMO
Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that typically causes asymptomatic infection but can promote B lymphoid tumors in the immune suppressed. In vitro, EBV infection of primary B cells stimulates glycolysis during immortalization into lymphoblastoid cell lines (LCLs). Lactate export during glycolysis is crucial for continued proliferation of many cancer cells-part of a phenomenon known as the "Warburg effect"- and is mediated by monocarboxylate transporters (MCTs). However, the role of MCTs has yet to be studied in EBV-associated malignancies, which display Warburg-like metabolism in vitro. Here, we show that EBV infection of B lymphocytes directly promotes temporal induction of MCT1 and MCT4 through the viral proteins EBNA2 and LMP1, respectively. Functionally, MCT1 was required for early B cell proliferation, and MCT4 up-regulation promoted acquired resistance to MCT1 antagonism in LCLs. However, dual MCT1/4 inhibition led to LCL growth arrest and lactate buildup. Metabolic profiling in LCLs revealed significantly reduced oxygen consumption rates (OCRs) and NAD+/NADH ratios, contrary to previous observations of increased OCR and unaltered NAD+/NADH ratios in MCT1/4-inhibited cancer cells. Furthermore, U-13C6-glucose labeling of MCT1/4-inhibited LCLs revealed depleted glutathione pools that correlated with elevated reactive oxygen species. Finally, we found that dual MCT1/4 inhibition also sensitized LCLs to killing by the electron transport chain complex I inhibitors phenformin and metformin. These findings were extended to viral lymphomas associated with EBV and the related gammaherpesvirus KSHV, pointing at a therapeutic approach for targeting both viral lymphomas.
Assuntos
Linfoma/metabolismo , Linfoma/virologia , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Linfócitos B/virologia , Linhagem Celular Tumoral , Proliferação de Células , Infecções por Vírus Epstein-Barr/virologia , Glucose/metabolismo , Glutationa/metabolismo , Herpesvirus Humano 4/fisiologia , Herpesvirus Humano 8/fisiologia , Humanos , Ácido Láctico/metabolismo , Linfoma/patologia , Metformina/farmacologia , NAD/metabolismo , Consumo de Oxigênio , Fenformin/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Epstein-Barr virus (EBV) reactivation is commonly observed in lung transplant recipients (LTRs). However, cellular immune responses to EBV in adult LTRs have not been well described. We aimed to study CD4/CD8 ratio, EBV-specific T cells polyfunctional responses and phenotypic changes in natural killer (NK) cells in adult LTRs presenting with EBV-associated diseases. The CD4/CD8 ratio was significantly decreased in LTRs with EBV DNAemia compared with LTRs without EBV DNAemia and healthy controls (HCs). Stimulation with EBV lytic antigen BZLF1 peptide pools induced significant individual and polyfunctional responses from CD8+ CD69+ T cells. Frequencies of CD8+ CD69+ T cells expressing CD107a were significantly higher in LTRs without EBV DNAemia than in LTRs with DNAemia. Frequencies of CD8+ CD69+ T cells concurrently expressing CD107a, IFN-γ, and TNF-α were significantly greater in LTRs with and without EBV DNAemia than in HCs. Finally, BZLF1 induced significantly higher frequencies of CD8+ CD69+ T cells expressing CD107a and IFN-γ in LTRs without EBV DNAemia when compared with EBNA3B. Frequency of more differentiated CD56dim CD16pos NK cells was significantly decreased in LTRs with EBV DNAemia and PTLD compared with HCs. In conclusion, we noted the presence of significant changes in circulating cellular immune responses to EBV in adult LTRs.
Assuntos
Infecções por Vírus Epstein-Barr , Transplante de Pulmão , Humanos , Adulto , Herpesvirus Humano 4 , Linfócitos T CD8-Positivos , Interferon gama , Transplante de Pulmão/efeitos adversosRESUMO
Endemic Burkitt lymphoma (eBL), the most prevalent pediatric cancer in sub-Saharan Africa, is distinguished by its inclusion of Epstein-Barr virus (EBV). In order to better understand the impact of EBV variation in eBL tumorigenesis, we improved viral DNA enrichment methods and generated a total of 98 new EBV genomes from both eBL cases (n = 58) and healthy controls (n = 40) residing in the same geographic region in Kenya. Using our unbiased methods, we found that EBV type 1 was significantly more prevalent in eBL patients (74.5%) than in healthy children (47.5%) (odds ratio = 3.24, 95% confidence interval = 1.36 to 7.71, P = 0.007), as opposed to similar proportions in both groups. Controlling for EBV type, we also performed a genome-wide association study identifying six nonsynonymous variants in the genes EBNA1, EBNA2, BcLF1, and BARF1 that were enriched in eBL patients. In addition, viruses isolated from plasma of eBL patients were identical to their tumor counterparts consistent with circulating viral DNA originating from the tumor. We also detected three intertypic recombinants carrying type 1 EBNA2 and type 2 EBNA3 regions, as well as one novel genome with a 20-kb deletion, resulting in the loss of multiple lytic and virion genes. Comparing EBV types, viral genes displayed differential variation rates as type 1 appeared to be more divergent, while type 2 demonstrated novel substructures. Overall, our findings highlight the complexities of the EBV population structure and provide new insight into viral variation, potentially deepening our understanding of eBL oncogenesis.IMPORTANCE Improved viral enrichment methods conclusively demonstrate EBV type 1 to be more prevalent in eBL patients than in geographically matched healthy controls, which previously underrepresented the prevalence of EBV type 2. Genome-wide association analysis between cases and controls identifies six eBL-associated nonsynonymous variants in EBNA1, EBNA2, BcLF1, and BARF1 genes. Analysis of population structure reveals that EBV type 2 exists as two genomic subgroups and was more commonly found in female than in male eBL patients.
Assuntos
Linfoma de Burkitt/genética , Linfoma de Burkitt/virologia , Infecções por Vírus Epstein-Barr/virologia , Genoma Viral , Herpesvirus Humano 4/genética , Adolescente , Criança , Pré-Escolar , DNA Viral , Infecções por Vírus Epstein-Barr/epidemiologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Feminino , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Lactente , Quênia/epidemiologia , Masculino , Razão de Chances , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Análise de Sequência de DNA , Proteínas Virais/genéticaRESUMO
Epstein-Barr virus (EBV)-driven posttransplant lymphoproliferative disorder (PTLD) is a serious complication following lung transplant. The extent to which the presence of EBV in PTLD tissue is associated with survival is uncertain. Moreover, whether the heterogeneity in expression of EBV latency programs is related to the timing of PTLD onset remains unexplored. We retrospectively performed a comprehensive histological evaluation of EBV markers at the tissue level in 34 adult lung transplant recipients with early- and late-onset PTLD. Early-onset PTLD, occurring within the first 12 months posttransplant, had higher odds to express EBV markers. The presence of EBV in PTLD was not associated with a difference in survival relative to EBV-negative tumors. However, we found evidence of heterogeneous expression of EBV latency programs, including type III, IIb, IIa, and 0/I. Our study suggests that the heterogeneous expression of EBV latency programs may represent a mechanism for immune evasion in patients with PLTD after lung transplants. The recognition of multiple EBV latency programs can be used in personalized medicine in patients who are nonresponsive to traditional types of chemotherapy and can be potentially evaluated in other types of solid organ transplants.
Assuntos
Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/genética , Pulmão/virologia , Transtornos Linfoproliferativos/virologia , Transplante de Órgãos/efeitos adversos , Adulto , Infecções por Vírus Epstein-Barr/etiologia , Infecções por Vírus Epstein-Barr/mortalidade , Feminino , Expressão Gênica , Humanos , Pulmão/metabolismo , Pulmão/cirurgia , Transtornos Linfoproliferativos/etiologia , Transtornos Linfoproliferativos/mortalidade , Masculino , Pessoa de Meia-Idade , Transplante de Órgãos/mortalidade , Estudos Retrospectivos , Transplantados , Proteínas Virais/genética , Proteínas Virais/metabolismo , Latência Viral/genéticaRESUMO
Burkitt lymphoma (BL) is an aggressive, MYC-driven lymphoma comprising 3 distinct clinical subtypes: sporadic BLs that occur worldwide, endemic BLs that occur predominantly in sub-Saharan Africa, and immunodeficiency-associated BLs that occur primarily in the setting of HIV. In this study, we comprehensively delineated the genomic basis of BL through whole-genome sequencing (WGS) of 101 tumors representing all 3 subtypes of BL to identify 72 driver genes. These data were additionally informed by CRISPR screens in BL cell lines to functionally annotate the role of oncogenic drivers. Nearly every driver gene was found to have both coding and non-coding mutations, highlighting the importance of WGS for identifying driver events. Our data implicate coding and non-coding mutations in IGLL5, BACH2, SIN3A, and DNMT1. Epstein-Barr virus (EBV) infection was associated with higher mutation load, with type 1 EBV showing a higher mutational burden than type 2 EBV. Although sporadic and immunodeficiency-associated BLs had similar genetic profiles, endemic BLs manifested more frequent mutations in BCL7A and BCL6 and fewer genetic alterations in DNMT1, SNTB2, and CTCF. Silencing mutations in ID3 were a common feature of all 3 subtypes of BL. In vitro, mass spectrometry-based proteomics demonstrated that the ID3 protein binds primarily to TCF3 and TCF4. In vivo knockout of ID3 potentiated the effects of MYC, leading to rapid tumorigenesis and tumor phenotypes consistent with those observed in the human disease.
Assuntos
Linfoma de Burkitt/genética , Sequenciamento Completo do Genoma/métodos , Animais , Humanos , CamundongosRESUMO
Apoptosis is critical to B cell maturation, but studies of apoptotic regulation in primary human B cells is lacking. In this study, we sought to better understand the mechanisms of apoptotic regulation in normal and activated B cells. Using intracellular BH3 profiling, we defined the Bcl2 dependency of B cell subsets from human peripheral blood and tonsillar lymphoid tissue as well as mitogen-activated B cells. We found that naive and memory B cells were BCL-2-dependent, whereas germinal center B cells were MCL-1-dependent and plasma cells were BCL-XL-dependent. B cells stimulated to proliferate ex vivo by CpG or CD40L/IL-4 became more dependent on MCL-1 and BCL-XL As B cell lymphomas often rely on survival mechanisms derived from normal and activated B cells, these findings offer new insight into potential therapeutic strategies for lymphomas.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proliferação de Células/fisiologia , Mitógenos/farmacologia , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Humanos , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteína bcl-X/metabolismoRESUMO
Recent evidence has shown that the Epstein-Barr virus (EBV) oncogene LMP1 is not expressed at high levels early after EBV infection of primary B cells, despite its being essential for the long-term outgrowth of immortalized lymphoblastoid cell lines (LCLs). In this study, we found that expression of LMP1 increased 50-fold between 7 days postinfection and the LCL state. Metabolic labeling of nascent transcribed mRNA indicated that this was primarily a transcription-mediated event. EBNA2, the key viral transcription factor regulating LMP1, and CTCF, an important chromatin insulator, were recruited to the LMP1 locus similarly early and late after infection. However, the activating histone H3K9Ac mark was enriched at the LMP1 promoter in LCLs relative to that in infected B cells early after infection. We found that high c-Myc activity in EBV-infected lymphoma cells as well as overexpression of c-Myc in an LCL model system repressed LMP1 transcription. Finally, we found that chemical inhibition of c-Myc both in LCLs and early after primary B cell infection increased LMP1 expression. These data support a model in which high levels of endogenous c-Myc activity induced early after primary B cell infection directly repress LMP1 transcription.IMPORTANCE EBV is a highly successful pathogen that latently infects more than 90% of adults worldwide and is also causally associated with a number of B cell malignancies. During the latent life cycle, EBV expresses a set of viral oncoproteins and noncoding RNAs with the potential to promote cancer. Critical among these is the viral latent membrane protein LMP1. Prior work suggests that LMP1 is essential for EBV to immortalize B cells, but our recent work indicates that LMP1 is not produced at high levels during the first few weeks after infection. Here we show that transcription of the LMP1 gene can be negatively regulated by a host transcription factor, c-Myc. Ultimately, understanding the regulation of EBV oncogenes will allow us to better treat cancers that rely on these viral products for survival.
Assuntos
Linfócitos B/metabolismo , Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/virologia , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas da Matriz Viral/genética , Linhagem Celular , Histonas , Humanos , Regiões Promotoras Genéticas , Transcrição Gênica , Latência ViralRESUMO
Epstein-Barr virus (EBV) is an oncogenic herpesvirus that has been causally linked to the development of B-cell and epithelial malignancies. Early after infection, EBV induces a transient period of hyperproliferation that is suppressed by the activation of the DNA damage response and a G1/S-phase growth arrest. This growth arrest prevents long-term outgrowth of the majority of infected cells. We developed a method to isolate and characterize infected cells that arrest after this early burst of proliferation and integrated gene expression and metabolic profiling to gain a better understanding of the pathways that attenuate immortalization. We found that the arrested cells have a reduced level of mitochondrial respiration and a decrease in the expression of genes involved in the TCA cycle and oxidative phosphorylation. Indeed, the growth arrest in early infected cells could be rescued by supplementing the TCA cycle. Arrested cells were characterized by an increase in the expression of p53 pathway gene targets, including sestrins leading to activation of AMPK, a reduction in mTOR signaling, and, consequently, elevated autophagy that was important for cell survival. Autophagy was also critical to maintain early hyperproliferation during metabolic stress. Finally, in assessing the metabolic changes from early infection to long-term outgrowth, we found concomitant increases in glucose import and surface glucose transporter 1 (GLUT1) levels, leading to elevated glycolysis, oxidative phosphorylation, and suppression of basal autophagy. Our study demonstrates that oncogene-induced senescence triggered by a combination of metabolic and genotoxic stress acts as an intrinsic barrier to EBV-mediated transformation.
Assuntos
Linfócitos B/virologia , Transformação Celular Viral , Herpesvirus Humano 4/fisiologia , Estresse Fisiológico , Autofagia/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Linfócitos B/ultraestrutura , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Transformação Celular Viral/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Desoxiglucose/farmacologia , Dimetilformamida/farmacologia , Herpesvirus Humano 4/efeitos dos fármacos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Metabolômica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Complexos Multiproteicos/metabolismo , Oncogenes , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.
Assuntos
Heterogeneidade Genética , Linfoma Difuso de Grandes Células B/genética , Adulto , Sequência de Bases , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA , DNA de Neoplasias/genética , Exoma , Expressão Gênica , Variação Genética , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Modelos Moleculares , Dados de Sequência Molecular , Terapia de Alvo Molecular , Mutação , Oncogenes , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Conformação Proteica , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/genéticaRESUMO
The key postulate that one gene encodes one protein has been overhauled with the discovery that one gene can generate multiple RNA transcripts through alternative mRNA processing. In this study, we describe SplicerEX, a novel and uniquely motivated algorithm designed for experimental biologists that (1) detects widespread changes in mRNA isoforms from both conventional and splice sensitive microarray data, (2) automatically categorizes mechanistic changes in mRNA processing, and (3) mitigates known technological artifacts of exon array-based detection of alternative splicing resulting from 5' and 3' signal attenuation, background detection limits, and saturation of probe set signal intensity. In this study, we used SplicerEX to compare conventional and exon-based Affymetrix microarray data in a model of EBV transformation of primary human B cells. We demonstrated superior detection of 3'-located changes in mRNA processing by the Affymetrix U133 GeneChip relative to the Human Exon Array. SplicerEX-identified exon-level changes in the EBV infection model were confirmed by RT-PCR and revealed a novel set of EBV-regulated mRNA isoform changes in caspases 6, 7, and 8. Finally, SplicerEX as compared with MiDAS analysis of publicly available microarray data provided more efficiently categorized mRNA isoform changes with a significantly higher proportion of hits supported by previously annotated alternative processing events. Therefore, SplicerEX provides an important tool for the biologist interested in studying changes in mRNA isoform usage from conventional or splice-sensitive microarray platforms, especially considering the expansive amount of archival microarray data generated over the past decade. SplicerEX is freely available upon request.
Assuntos
Processamento Alternativo/genética , Infecções por Vírus Epstein-Barr/genética , Éxons/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , RNA Mensageiro/genética , Algoritmos , Automação , Linfócitos B/patologia , Linfócitos B/virologia , Biomarcadores/análise , Linhagem Celular Transformada/patologia , Linhagem Celular Transformada/virologia , Células Cultivadas , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/genética , Humanos , Isoformas de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Oncogenic viruses promote cell proliferation through the dramatic reorganization of host transcriptomes. In addition to regulating mRNA abundance, changes in mRNA isoform usage can have a profound impact on the protein output of the transcriptome. Using Epstein-Barr virus (EBV) transformation of primary B cells, we have studied the ability of an oncogenic virus to alter the mRNA isoform profile of its host. Using the algorithm called SplicerEX with two complementary Affymetrix microarray platforms, we uncovered 433 mRNA isoform changes regulated by EBV during B-cell transformation. These changes were largely orthogonal with the 2,163 mRNA abundance changes observed during transformation, such that less than one-third of mRNAs changing at the level of isoform also changed in overall abundance. While we observed no preference for a mechanistic class of mRNA isoform change, we detected a significant shortening of 3' untranslated regions and exclusion of cassette exons in EBV-transformed cells relative to uninfected B cells. Gene ontology analysis of the mRNA isoform changes revealed significant enrichment in nucleic acid binding proteins. We validated several of these isoform changes and were intrigued by those in two mRNAs encoding the proteins XBP1 and TCF4, which have both been shown to bind and activate the promoter of the major EBV lytic trans-activator BZLF1. Our studies indicate that EBV latent infection promotes the usage of mRNA isoforms of XBP1 and TCF4 that restrict BZLF1 activation. Therefore, characterization of global changes in mRNA isoform usage during EBV infection identifies a new mechanism for the maintenance of latent infection.
Assuntos
Transformação Celular Viral/genética , Infecções por Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica , RNA Viral/genética , Latência Viral/genética , Replicação Viral , Linfócitos B/metabolismo , Linfócitos B/virologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Biomarcadores/metabolismo , Western Blotting , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Perfilação da Expressão Gênica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidade , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Isoformas de RNA , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição de Fator Regulador X , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Transativadores/metabolismo , Fator de Transcrição 4 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3'UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3'UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies.
Assuntos
Transformação Celular Viral , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiologia , Transtornos Linfoproliferativos/metabolismo , Transtornos Linfoproliferativos/virologia , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B/virologia , Linhagem Celular Tumoral , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Humanos , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/patologia , MicroRNAs/genética , Latência Viral/genéticaRESUMO
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes that produce mAbs often reactive with microbial or autoantigens. Long-term culture of B-CLL clones would permit the collection and characterization of B-CLL mAbs to study antigen specificity and of B-CLL DNA to investigate molecular mechanisms promoting the disease. However, the derivation of long-term cell lines (eg, by EBV), has not been efficient. We have improved the efficiency of EBV B-CLL transformation of CpG oligonucleotide-stimulated cells by incubating patient peripheral blood mononuclear cells in the presence of an irradiated mouse macrophage cell line, J774A.1. Using this approach, peripheral blood mononuclear cells isolated from 13 of 21 B-CLL patients were transformed as documented by IGHV-D-J sequencing. Four clones grew and retained CD5 expression in culture for 2 to 4 months. However, despite documentation of EBV infection by expression of EBNA2 and LMP1, B-CLL cells died after removal of macrophage feeder cells. Nevertheless, using electrofusion technology, we generated 6 stable hetero-hybridoma cell lines from EBV-transformed B-CLL cells, and these hetero-hybridomas produced immunoglobulin. Thus, we have established enhanced methods of B-CLL culture that will enable broader interrogation of B-CLL cells at the genetic and protein levels.
Assuntos
Processos de Crescimento Celular/fisiologia , Transformação Celular Viral , Células Alimentadoras/citologia , Herpesvirus Humano 4/fisiologia , Leucemia Linfocítica Crônica de Células B/patologia , Macrófagos/citologia , Animais , Linhagem Celular Transformada , Transformação Celular Viral/genética , Transformação Celular Viral/fisiologia , Células Cultivadas , Técnicas de Cocultura , Antígenos Nucleares do Vírus Epstein-Barr/genética , Células Alimentadoras/fisiologia , Herpesvirus Humano 4/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Macrófagos/fisiologia , Camundongos , Regulação para Cima , Proteínas Virais/genéticaRESUMO
Viruses encounter many challenges within host cells in order to replicate their nucleic acid. In the case of DNA viruses, one challenge that must be overcome is recognition of viral DNA structures by the host DNA damage response (DDR) machinery. This is accomplished in elegant and unique ways by different viruses as each has specific needs and sensitivities dependent on its life cycle. In this review, we focus on three DNA tumor viruses and their interactions with the DDR. The viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV) account for nearly all of the virus-associated human cancers worldwide. These viruses have also been excellent models for the study of oncogenic virus-mediated cell transformation. In this review, we will discuss how each of these viruses engage and subvert aspects of the host DDR. The first level of DDR engagement is a result of the genetic linkage between the oncogenic potential of these viruses and their ability to replicate. Namely, the promotion of cells from quiescence into the cell cycle to facilitate virus replication can be sensed through aberrant cellular DNA replication structures which activate the DDR and hinder cell transformation. DNA tumor viruses subvert this growth-suppressive DDR through changes in viral oncoprotein expression which ultimately facilitate virus replication. An additional level of DDR engagement is through direct detection of replicating viral DNA. These interactions parallel those observed in other DNA virus systems in that the need to subvert these intrinsic sensors of aberrant DNA structure in order to replicate must be in place. DNA tumor viruses are no exception. This review will cover the molecular features of DNA tumor virus interactions with the host DDR and the consequences for virus replication.
Assuntos
Dano ao DNA , Vírus de DNA Tumorais/genética , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologia , Replicação Viral/genética , Animais , Dano ao DNA/fisiologia , HumanosRESUMO
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 Knock Out (LPKO) virus-infected cells express EBNA2-activated 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-γ 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 cellular transcription factors. We found a significant overlap between EBNA-LP and YY1 in ChIP-Seq data and confirmed their biochemical association in LCLs by endogenous co-immunoprecipitation. Moreover, we found that the EBNA-LP leucine-rich motifs were required for YY1 interaction in LCLs. Finally, we used Cas9 to knockout YY1 in primary total B cells and naïve B cells prior to EBV infection and found YY1 to be essential for EBV-mediated transformation. We propose that EBNA-LP engages YY1 through conserved leucine-rich motifs to promote EBV transformation of naïve B cells.