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1.
Nucleic Acids Res ; 49(9): 5038-5056, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34009296

RESUMO

ERG family proteins (ERG, FLI1 and FEV) are a subfamily of ETS transcription factors with key roles in physiology and development. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 regulates both transcription and alternative splicing of pre-messenger RNAs. However, whether wild-type ERG family proteins might regulate splicing is unknown. Here, we show that wild-type ERG proteins associate with spliceosomal components, are found on nascent RNAs, and induce alternative splicing when recruited onto a reporter minigene. Transcriptomic analysis revealed that ERG and FLI1 regulate large numbers of alternative spliced exons (ASEs) enriched with RBFOX2 motifs and co-regulated by this splicing factor. ERG and FLI1 are associated with RBFOX2 via their conserved carboxy-terminal domain, which is present in EWS-FLI1. Accordingly, EWS-FLI1 is also associated with RBFOX2 and regulates ASEs enriched in RBFOX2 motifs. However, in contrast to wild-type ERG and FLI1, EWS-FLI1 often antagonizes RBFOX2 effects on exon inclusion. In particular, EWS-FLI1 reduces RBFOX2 binding to the ADD3 pre-mRNA, thus increasing its long isoform, which represses the mesenchymal phenotype of Ewing sarcoma cells. Our findings reveal a RBFOX2-mediated splicing regulatory function of wild-type ERG family proteins, that is altered in EWS-FLI1 and contributes to the Ewing sarcoma cell phenotype.


Assuntos
Processamento Alternativo , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Domínios Proteicos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Regulador Transcricional ERG/química , Regulador Transcricional ERG/metabolismo
2.
Virol J ; 18(1): 99, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001180

RESUMO

BACKGROUND: Sensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines. METHODS: Here, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here. RESULTS: Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/µl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost. CONCLUSIONS: With extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Kit de Reagentes para Diagnóstico , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Humanos , Nasofaringe/virologia , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade
3.
Nat Commun ; 12(1): 1405, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33658502

RESUMO

Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. Here, we describe "Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening" (C19-SPAR-Seq), a multiplexed, scalable, readily automated platform for SARS-CoV-2 detection that is capable of analyzing tens of thousands of patient samples in a single run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employ a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performs with a specificity of 100% and sensitivity of 91% on samples with low viral loads, and a sensitivity of >95% on high viral loads associated with disease onset and peak transmissibility. This study establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/patogenicidade , COVID-19/genética , COVID-19/imunologia , COVID-19/virologia , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga Viral
4.
Cell Rep ; 30(6): 1767-1779.e6, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32049009

RESUMO

EWSR1-FLI1, the chimeric oncogene specific for Ewing sarcoma (EwS), induces a cascade of signaling events leading to cell transformation. However, it remains elusive how genetically homogeneous EwS cells can drive the heterogeneity of transcriptional programs. Here, we combine independent component analysis of single-cell RNA sequencing data from diverse cell types and model systems with time-resolved mapping of EWSR1-FLI1 binding sites and of open chromatin regions to characterize dynamic cellular processes associated with EWSR1-FLI1 activity. We thus define an exquisitely specific and direct enhancer-driven EWSR1-FLI1 program. In EwS tumors, cell proliferation and strong oxidative phosphorylation metabolism are associated with a well-defined range of EWSR1-FLI1 activity. In contrast, a subpopulation of cells from below and above the intermediary EWSR1-FLI1 activity is characterized by increased hypoxia. Overall, our study reveals sources of intratumoral heterogeneity within EwS tumors.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Transcrição Gênica/genética , Linhagem Celular Tumoral , Humanos , Transdução de Sinais
5.
Nat Commun ; 10(1): 4128, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511524

RESUMO

Pediatric malignancies including Ewing sarcoma (EwS) feature a paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome. Here, we demonstrate in EwS how cooperation of dominant oncogenes and regulatory germline variants determine tumor growth, patient survival and drug response. Binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls expression of the transcription factor MYBL2 mediating these phenotypes. Whole-genome and RNA sequencing reveals that variability at this locus is inherited via the germline and is associated with variable inter-tumoral MYBL2 expression. High MYBL2 levels sensitize EwS cells for inhibition of its upstream activating kinase CDK2 in vitro and in vivo, suggesting MYBL2 as a putative biomarker for anti-CDK2-therapy. Collectively, we establish cooperation of somatic mutations and regulatory germline variants as a major determinant of tumor progression and highlight the importance of integrating the regulatory genome in precision medicine.


Assuntos
Mutação em Linhagem Germinativa/genética , Neoplasias/genética , Neoplasias/terapia , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Repetições de Microssatélites/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fenótipo , Polimorfismo Genético , Transativadores , Resultado do Tratamento , Regulação para Cima/genética
6.
Cell Rep ; 21(7): 1737-1745, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29141209

RESUMO

Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/ß and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro. In vivo, reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These results strongly support testing pazopanib/CfT combination therapy in future clinical trials for RTs.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Compostos de Amônio Quaternário/farmacologia , Tumor Rabdoide/metabolismo , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Indazóis , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Proteína SMARCB1/metabolismo
7.
Cell Death Dis ; 8(6): e2895, 2017 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-28640249

RESUMO

Limitless cell proliferation, evasion from apoptosis, dedifferentiation, metastatic spread and therapy resistance: all these properties of a cancer cell contribute to its malignant phenotype and affect patient outcome. MYBL2 (alias B-Myb) is a transcription factor of the MYB transcription factor family and a physiological regulator of cell cycle progression, cell survival and cell differentiation. When deregulated in cancer cells, MYBL2 mediates the deregulation of these properties. In fact, MYBL2 is overexpressed and associated with poor patient outcome in numerous cancer entities. MYBL2 and players of its downstream transcriptional network can be used as prognostic and/or predictive biomarkers as well as potential therapeutic targets to offer less toxic and more specific anti-cancer therapies in future. In this review, we summarize current knowledge on the physiological roles of MYBL2 and highlight the impact of its deregulation on cancer initiation and progression.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Diferenciação Celular , Transativadores/metabolismo , Animais , Ciclo Celular , Proliferação de Células , Sobrevivência Celular , Humanos
8.
Nat Genet ; 47(9): 1073-8, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26214589

RESUMO

Deciphering the ways in which somatic mutations and germline susceptibility variants cooperate to promote cancer is challenging. Ewing sarcoma is characterized by fusions between EWSR1 and members of the ETS gene family, usually EWSR1-FLI1, leading to the generation of oncogenic transcription factors that bind DNA at GGAA motifs. A recent genome-wide association study identified susceptibility variants near EGR2. Here we found that EGR2 knockdown inhibited proliferation, clonogenicity and spheroidal growth in vitro and induced regression of Ewing sarcoma xenografts. Targeted germline deep sequencing of the EGR2 locus in affected subjects and controls identified 291 Ewing-associated SNPs. At rs79965208, the A risk allele connected adjacent GGAA repeats by converting an interspaced GGAT motif into a GGAA motif, thereby increasing the number of consecutive GGAA motifs and thus the EWSR1-FLI1-dependent enhancer activity of this sequence, with epigenetic characteristics of an active regulatory element. EWSR1-FLI1 preferentially bound to the A risk allele, which increased global and allele-specific EGR2 expression. Collectively, our findings establish cooperation between a dominant oncogene and a susceptibility variant that regulates a major driver of Ewing sarcomagenesis.


Assuntos
Neoplasias Ósseas/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/genética , Animais , Sequência de Bases , Neoplasias Ósseas/patologia , Carotenoides/genética , Linhagem Celular Tumoral , Proliferação de Células , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Camundongos SCID , Repetições de Microssatélites , Dados de Sequência Molecular , Transplante de Neoplasias , Oxigenases/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Sarcoma de Ewing/patologia , Carga Tumoral
9.
PLoS One ; 8(8): e73641, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23977391

RESUMO

Human APOBEC3 enzymes deaminate single stranded DNA. At least five can deaminate mitochondrial DNA in the cytoplasm, while three can deaminate viral DNA in the nucleus. However, only one, APOBEC3A, can hypermutate genomic DNA. We analysed the distribution and function of the two APOBEC3A isoforms p1 and p2 in transfected cell lines. Both can translocate to the nucleus and hypermutate CMYC DNA and induce DNA double strand breaks as visualized by the detection of ©H2AX or Chk2. APOBEC3A induced G1 phase cell cycle arrest and triggered several members of the intrinsic apoptosis pathway. Activation of purified human CD4+ T lymphocytes with PHA, IL2 and interferon α resulted in C->T hypermutation of genomic DNA and double stranded breaks suggesting a role for APOBEC3A in pro-inflammatory conditions. As chronic inflammation underlies many diseases including numerous cancers, it is possible that APOBEC3A induction may generate many of the lesions typical of a cancer genome.


Assuntos
Núcleo Celular/metabolismo , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas/metabolismo , Estresse Fisiológico , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Pontos de Checagem do Ciclo Celular , Morte Celular , Citidina Desaminase/genética , Células HeLa , Histonas/metabolismo , Humanos , Ativação Linfocitária , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Proteínas/genética , Edição de RNA/genética , Transdução de Sinais/genética , Uracila-DNA Glicosidase/metabolismo
10.
PLoS One ; 8(6): e63461, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23840298

RESUMO

Deamination of 5-methylcytidine (5MeC) in DNA results in a G:T mismatch unlike cytidine (C) deamination which gives rise to a G:U pair. Deamination of C was generally considered to arise spontaneously. It is now clear that human APOBEC3A (A3A), a polynucleotide cytidine deaminase (PCD) with specificity for single stranded DNA, can extensively deaminate human nuclear DNA. It is shown here that A3A among all human PCDs can deaminate 5-methylcytidine in a variety of single stranded DNA substrates both in vitro and in transfected cells almost as efficiently as cytidine itself. This ability of A3A to accommodate 5-methyl moiety extends to other small and physiologically relevant substituted cytidine bases such as 5-hydroxy and 5-bromocytidine. As 5MeCpG deamination hotspots characterize many genes associated with cancer it is plausible that A3A is a major player in the onset of cancer.


Assuntos
Citidina Desaminase/química , Citidina/análogos & derivados , DNA de Cadeia Simples/química , Proteínas/química , Animais , Sequência de Bases , Linhagem Celular , Ilhas de CpG , Citidina/química , Citidina/metabolismo , Citidina Desaminase/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Desaminação , Genes p53 , Células HEK293 , HIV-1/genética , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Proteínas/metabolismo , Codorniz , Especificidade por Substrato
11.
J Biol Chem ; 287(46): 39182-92, 2012 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-22977230

RESUMO

The human polydeoxynucleotide cytidine deaminases APOBEC3A, APOBEC3C, and APOBEC3H are capable of mutating viral DNA in the nucleus, whereas APOBEC3A alone efficiently edits nuclear DNA. Deamination is rapidly followed by excision of uracil residues and can lead to double-stranded breaks. It is not known to which protein networks these DNA mutators belong. Using a yeast two-hybrid screen, we identified the human homolog of Drosophila Tribbles 3, TRIB3, as an interactor for APOBEC3A and APOBEC3C. The interaction was confirmed by co-affinity purification. Co-transfection of APOBEC3A with a TRIB3 expression vector reduced nuclear DNA editing whereas siRNA knockdown of TRIB3 increased the levels of nuclear DNA editing, indicating that TRIB3 functioned as a repressor of A3A. It also repressed A3A-associated γH2AX positive double-stranded breaks. The interaction results in degradation of A3A in a proteasome-independent manner. TRIB3 has been linked to cancer and via its own interactors and links the A3A DNA mutators to the Rb-BRCA1-ATM network. TRIB3 emerges as an important guardian of genome integrity.


Assuntos
Núcleo Celular/metabolismo , Citidina Desaminase/metabolismo , Citidina/química , DNA/metabolismo , Regulação da Expressão Gênica , Proteínas/metabolismo , Ciclo Celular , Sobrevivência Celular , Citidina Desaminase/fisiologia , Reparo do DNA , Desaminação , Genoma , Células HEK293 , Células HeLa , Humanos , Microscopia Confocal/métodos , Mutagênese , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Técnicas do Sistema de Duplo-Híbrido
12.
J Virol ; 85(15): 7594-602, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21632763

RESUMO

Human APOBEC3 cytidine deaminases target and edit single-stranded DNA, which can be of viral, mitochondrial, or nuclear origin. Retrovirus genomes, such as human immunodeficiency virus (HIV) genomes deficient in the vif gene and the hepatitis B virus genome, are particularly vulnerable. The genomes of some DNA viruses, such as human papillomaviruses, can be edited in vivo and in transfection experiments. Accordingly, herpesviruses should be no exception. This is indeed the case for herpes simplex virus 1 (HSV-1) in tissue culture, where APOBEC3C (A3C) overexpression can reduce virus titers and the particle/PFU ratio ∼10-fold. Nonetheless, A3A, A3G, and AICDA can edit what is presumably a small fraction of HSV genomes in an experimental setting without seriously impacting the viral titer. Hyperediting was found in HSV genomes recovered from 4/8 uncultured buccal lesions. The phenomenon is not restricted to HSV, since hyperedited Epstein-Barr virus (EBV) genomes were readily recovered from 4/5 established cell lines, indicating that episomes are vulnerable to editing. These findings suggest that the widely expressed A3C cytidine deaminase can function as a restriction factor for some human herpesviruses. That the A3C gene is not induced by type I interferons begs the question whether some herpesviruses encode A3C antagonists.


Assuntos
Citosina Desaminase/metabolismo , Genoma Viral , Herpesvirus Humano 1/genética , Herpesvirus Humano 4/genética , Desaminases APOBEC , Animais , Sequência de Bases , Chlorocebus aethiops , Citidina Desaminase , Citosina Desaminase/genética , DNA/genética , Células HeLa , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 4/fisiologia , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , Células Vero , Replicação Viral
13.
Proc Natl Acad Sci U S A ; 108(12): 4858-63, 2011 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-21368204

RESUMO

The human APOBEC3 (A3A-A3H) locus encodes six cytidine deaminases that edit single-stranded DNA, the result being DNA peppered with uridine. Although several cytidine deaminases are clearly restriction factors for retroviruses and hepadnaviruses, it is not known if APOBEC3 enzymes have roles outside of these settings. It is shown here that both human mitochondrial and nuclear DNA are vulnerable to somatic hypermutation by A3 deaminases, with APOBEC3A standing out among them. The degree of editing is much greater in patients lacking the uracil DNA-glycolyase gene, indicating that the observed levels of editing reflect a dynamic composed of A3 editing and DNA catabolism involving uracil DNA-glycolyase. Nonetheless, hyper- and lightly mutated sequences went hand in hand, raising the hypothesis that recurrent low-level mutation by APOBEC3A could catalyze the transition from a healthy to a cancer genome.


Assuntos
Citosina Desaminase/metabolismo , DNA Mitocondrial/metabolismo , Loci Gênicos , Genoma Humano , Mutação , Desaminases APOBEC , Citidina Desaminase , Citosina Desaminase/genética , DNA Mitocondrial/genética , Feminino , Células HeLa , Hepadnaviridae/genética , Hepadnaviridae/metabolismo , Humanos , Masculino , Neoplasias/enzimologia , Neoplasias/genética , Retroviridae/genética , Retroviridae/metabolismo , Uracila-DNA Glicosidase/deficiência , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo
14.
J Virol ; 85(5): 2458-62, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21159878

RESUMO

We sought to examine ADAR-1 editing of measles and influenza virus genomes derived from inactivated seasonal influenza and live attenuated measles virus vaccines grown on chicken cells as the culture substrate. Using highly sensitive 3DI-PCR (R. Suspène et al., Nucleic Acids Res. 36:e72, 2008), it was possible to show that ADAR-1 could hyperdeaminate adenosine residues in both measles virus and influenza virus A genomes. Detailed analysis of the dinucleotide editing context showed preferences for 5'ArA and 5'UrA, which is typical of editing in mammalian cells. The hyperedited mutant frequency, including genomes and antigenomes, was a log greater for influenza virus compared to measles virus, suggesting a greater sensitivity to restriction by ADAR-1.


Assuntos
Adenosina Desaminase/metabolismo , Genoma Viral , Vírus da Influenza A/genética , Influenza Humana/enzimologia , Vírus do Sarampo/genética , Sarampo/enzimologia , Mutação , Adenosina Desaminase/genética , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Humanos , Vacinas contra Influenza/genética , Influenza Humana/virologia , Sarampo/virologia , Vacina contra Sarampo/genética , Dados de Sequência Molecular , Proteínas de Ligação a RNA , Estações do Ano , Vacinas Atenuadas/genética , Células Vero
15.
PLoS Pathog ; 6(5): e1000928, 2010 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-20523896

RESUMO

DNA viruses, retroviruses and hepadnaviruses, such as hepatitis B virus (HBV), are vulnerable to genetic editing of single stranded DNA by host cell APOBEC3 (A3) cytidine deaminases. At least three A3 genes are up regulated by interferon-alpha in human hepatocytes while ectopic expression of activation induced deaminase (AICDA), an A3 paralog, has been noted in a variety of chronic inflammatory syndromes including hepatitis C virus infection. Yet virtually all studies of HBV editing have confined themselves to analyses of virions from culture supernatants or serum where the frequency of edited genomes is generally low (< or = 10(-2)). We decided to look at the nature and frequency of HBV editing in cirrhotic samples taken during removal of a primary hepatocellular carcinoma. Forty-one cirrhotic tissue samples (10 alcoholic, 10 HBV(+), 11 HBV(+)HCV(+) and 10 HCV(+)) as well as 4 normal livers were studied. Compared to normal liver, 5/7 APOBEC3 genes were significantly up regulated in the order: HCV+/-HBV>HBV>alcoholic cirrhosis. A3C and A3D were up regulated for all groups while the interferon inducible A3G was over expressed in virus associated cirrhosis, as was AICDA in approximately 50% of these HBV/HCV samples. While AICDA can indeed edit HBV DNA ex vivo, A3G is the dominant deaminase in vivo with up to 35% of HBV genomes being edited. Despite these highly deleterious mutant spectra, a small fraction of genomes survive and contribute to loss of HBeAg antigenemia and possibly HBsAg immune escape. In conclusion, the cytokine storm associated with chronic inflammatory responses to HBV and HCV clearly up regulates a number of A3 genes with A3G clearly being a major restriction factor for HBV. Although the mutant spectrum resulting from A3 editing is highly deleterious, a very small part, notably the lightly edited genomes, might help the virus evolve and even escape immune responses.


Assuntos
Citidina Desaminase/genética , DNA Viral/genética , Vírus da Hepatite B/genética , Hepatite B/imunologia , Hepatite B/virologia , Desaminase APOBEC-3G , Idoso , Citidina Desaminase/metabolismo , DNA de Cadeia Simples/genética , Evolução Molecular , Feminino , Regulação Viral da Expressão Gênica/imunologia , Genoma Viral , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vacinas contra Hepatite B/genética , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Humanos , Cirrose Hepática/virologia , Masculino , Pessoa de Meia-Idade , Mutação , Regulação para Cima/fisiologia
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